kappa+lambda+ dual receptor B cells are present in the human peripheral repertoire.

It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. The kappa+lambda+ cells were sorted, immortalized by Epstein-Barr virus, and five independent clones were characterized in detail. All clones express both kappa and lambda on the cell surface and produce immunoglobulin M that contain both kappa and lambda chains in the same molecule, i.e., hybrid antibodies. Sequencing of the L chains revealed in three out of five clones evidence for somatic mutations. It is interesting to note that among a panel of single receptor B cell clones we identified two lambda+ clones that carried a productively rearranged kappa, which was inactivated by a stop codon generated by somatic mutation. These findings indicate that dual receptor B lymphocytes can be found among mature antigen-selected B cells and suggest that somatic mutation can contribute to increase the degree of isotypic exclusion by inactivating a passenger, nonselected L chain.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion.

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein.

Selection by two powerful antigens may account for the presence of the major population of human peripheral gamma/delta T cells.

V gamma 9/V delta 2 cells represent a fraction of human gamma/delta cells that is expanded after birth in the periphery, carries markers of activated cells, and becomes a major population in peripheral blood. We found that these cells do not comprise a single population but actually represent two nested sets, the smaller of which, specific for Mycobacterium tuberculosis-pulsed antigen-presenting cells (APC), is contained in a larger set specific for an antigen found on the Molt-4 lymphoma. The larger set, representing 40-80% of all blood gamma/delta cells, is comprised of cells bearing the V gamma 9/C gamma 1 chain. Cells in the smaller, included set have an additional requirement for V delta 2 (and probably for certain permissive junctional regions, since a very small percentage of V gamma 9/V delta 2 cells do not react against mycobacteria-pulsed APC). Optimal stimulation by mycobacteria is dependent on the presence of APC, and is not restricted by classical major histocompatibility complex molecules. Some of the V gamma 9/V delta 2 mycobacteria-specific clones are also stimulated by APC pulsed with different bacteria, such as Listeria monocytogenes and Escherichia coli, indicating that the population includes several different patterns of reactivity. These data establish a relationship in humans between specificity and V gamma/V delta gene usage, and offer an explanation for the peripheral expansion of these gamma/delta cells.

Integration and sensory experience-dependent survival of newly-generated neurons in the accessory olfactory bulb of female mice.

Newborn neurons generated by proliferative progenitors in the adult subventricular zone (SVZ) integrate into the olfactory bulb circuitry of mammals. Survival of these newly-formed cells is regulated by the olfactory input. The presence of new neurons in the accessory olfactory bulb (AOB) has already been demonstrated in some mammalian species, albeit their neurochemical profile and functional integration into AOB circuits are still to be investigated. To unravel whether the mouse AOB represents a site of adult constitutive neurogenesis and whether this process can be modulated by extrinsic factors, we have used multiple in vivo approaches. These included fate mapping of bromodeoxyuridine-labelled cells, lineage tracing of SVZ-derived enhanced green fluorescent protein-positive engrafted cells and neurogenesis quantification in the AOB, in both sexes, as well as in females alone after exposure to male-soiled bedding or its derived volatiles. Here, we show that a subpopulation of SVZ-derived neuroblasts acquires proper neurochemical profiles of mature AOB interneurons. Moreover, 3D reconstruction of long-term survived engrafted neuroblasts in the AOB confirms these cells show features of fully integrated neurons. Finally, exposure to male-soiled bedding, but not to its volatile compounds, significantly increases the number of new neurons in the AOB, but not in the main olfactory bulb of female mice. These data show SVZ-derived neuroblasts differentiate into new functionally integrated neurons in the AOB of young and adult mice. Survival of these cells seems to be regulated by an experience-specific mechanism mediated by pheromones.

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.

Apelin-induced cardioprotection against ischaemia/reperfusion injury: roles of epidermal growth factor and Src.

AIM: Apelin, the ligand of the G-protein-coupled receptor (GPCR) APJ, exerts a post-conditioning-like protection against ischaemia/reperfusion injury through activation of PI3K-Akt-NO signalling. The pathway connecting APJ to PI3K is still unknown. As other GPCR ligands act through transactivation of epidermal growth factor receptor (EGFR) via a matrix metalloproteinase (MMP) or Src kinase, we investigated whether EGFR transactivation is involved in the following three features of apelin-induced cardioprotection: limitation of infarct size, suppression of contracture and improvement of post-ischaemic contractile recovery. METHOD: Isolated rat hearts underwent 30 min of global ischaemia and 2 h of reperfusion. Apelin (0.5 µm) was infused during the first 20 min of reperfusion. EGFR, MMP or Src was inhibited to study the pathway connecting APJ to PI3K. Key components of RISK pathway, namely PI3K, guanylyl cyclase or mitochondrial K+ -ATP channels, were also inhibited. Apelin-induced EGFR and phosphatase and tensing homolog (PTEN) phosphorylation were assessed. Left ventricular pressure and infarct size were measured. RESULTS: Apelin-induced reductions in infarct size and myocardial contracture were prevented by the inhibition of EGFR, Src, MMP or RISK pathway. The involvement of EGFR was confirmed by its phosphorylation. However, neither direct EGFR nor MMP inhibition affected apelin-induced improvement of early post-ischaemic contractile recovery, which was suppressed by Src and RISK inhibitors only. Apelin also increased PTEN phosphorylation, which was removed by Src inhibition. CONCLUSION: While EGFR and MMP limit infarct size and contracture, Src or RISK pathway inhibition suppresses the three features of cardioprotection. Src does not only transactivate EGFR, but also inhibits PTEN by phosphorylation thus playing a crucial role in apelin-induced cardioprotection.

Maternal deprivation and early handling affect density of calcium binding protein-containing neurons in selected brain regions and emotional behavior in periadolescent rats.

Adverse early life experiences can induce neurochemical changes that may underlie modifications in hypothalamic-pituitary-adrenal axis responsiveness, emotionality and cognition. Here, we investigated the expression of the calcium binding proteins (CBPs) calretinin, calbindin and parvalbumin, which identify subpopulations of GABAergic neurons and serve important functional roles by buffering intracellular calcium levels, following brief (early handling) and long (maternal deprivation) periods of maternal separation, as compared with non-handled controls. CBP-expressing neurons were analyzed in brain regions related to stress and anxiety. Emotionality was assessed in parallel using the social interaction test. Analyses were carried out at periadolescence, an important phase for the development of brain areas involved in stress responses. Our results indicate that density of CBP-immunoreactive neurons decreases in the paraventricular region of deprived rats but increases in the hippocampus and lateral amygdala of both early-handled and deprived rats when compared with controls. Emotionality is reduced in both early-handled and deprived animals. In conclusion, early handling and deprivation led to neurochemical and behavioral changes linked to stress-sensitive brain regions. These data suggest that the effects of early experiences on CBP containing neurons might contribute to the functional changes of neuronal circuits involved in emotional response.

Glypican-2 levels in cerebrospinal fluid predict the status of adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is a remarkable form of brain plasticity through which new neurons are generated throughout life. Despite its important roles in cognition and emotion and its modulation in various preclinical disease models, the functional importance of adult hippocampal neurogenesis in human health has not been revealed because of a lack of tools for monitoring adult neurogenesis in vivo. Therefore, we performed an unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model, and we identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons. Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain. Its expression is increased by physiological stimuli that increase hippocampal neurogenesis and decreased in transgenic models in which neurogenesis is selectively ablated. Changes in neurogenesis also result in changes in Gpc2 protein level in cerebrospinal fluid (CSF). Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time. Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.

Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo.

Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.

Clonal predominance, but preservation of a polyclonal reservoir, in the normal alpha beta T-cell repertoire.

We recently demonstrated that the peripheral gamma delta T-cell repertoire becomes oligoclonal with increasing age. Although this junctional homogeneity should not severely affect the ability of gamma delta T cells to respond to foreign antigens, we reasoned that a similar oligoclonal repertoire of alpha beta T cells would lead to a profound impairment of the MHC-restricted response. We used heteroduplex analysis in this research to study the clonal complexity of the peripheral alpha beta T-cell repertoire in human subjects and supply evidence for the presence of alpha beta clonal expansions. Clonal predominance in the alpha beta T-cell repertoire of normal subjects was not simply related to age, since the PBL of young donors also showed clonal expansions and did not always correlate with a numeric increase in the corresponding V beta family. However, the type of alpha beta expansion appears to be strikingly different from the gamma delta expansions. In the case of alpha beta T cells, even in the presence of clonal dominance, evidence for a residual polyclonal background was found in all the donors tested, irrespective of age. The observation that true oligoclonality is exceptionally rare among alpha beta T lymphocytes could mean that maintenance of a highly diversified reservoir of TCR is primary for these cells throughout life.

An alternative approach to the assessment of gamma delta T-cell clonality in celiac disease intestinal lesions through cDNA heteroduplex analysis of T-cell receptor VJ junctions.

We have investigated the clonality of the gamma delta T lymphocytes infiltrating the intestinal mucosa of CD patients and control subjects by means of a simple and powerful method based on the heteroduplex analysis of the TCR VJ junctions. Each V-specific TCR chain, amplified either from fresh biopsy material or intestinal T-cell-line cDNA, is denatured and renatured to allow the random reshuffling of the various strands carrying different junctional sequences, coamplified in the same reaction. The mismatched chains (heteroduplexes) are separated from the matched ones (homoduplexes) through polyacrylamide gel electrophoresis, and whenever one or more T-cell clones are emerging over the polyclonal background, discrete bands are visible by ethidium-bromide staining. Through this method, we have estimated the diversity of the V delta 1-3 chains and a newly described V gene (V delta 8) whose homologue in mice is abundantly expressed in gamma delta iLs. We demonstrate that the well-documented expansion of V gamma 1+ gamma delta lymphocytes in the jejunum of CD patients is polyclonal. Overall, the heteroduplex analysis on fresh intestinal and peripheral blood lymphocytes from both healthy and affected subjects shows a polyclonal pattern of all the V delta+ subsets. In contrast, most intestinal T-cell lines produce oligoclonal patterns, suggesting a dramatic in vitro selection effect. The cell expansion in culture is generally not required for the TCR heteroduplex analysis, which can therefore be applied to rapidly monitor the T-cell response in a variety of physiologic and autoimmune reactions, substituting the standard approach of TCR cloning and multiple VJ sequencing.

Dual receptor T-cells. Implications for alloreactivity and autoimmunity.

Using monoclonal antibodies to human V alpha, we have estimated that up to one-third of mature T-cells express two V alpha chains as part of two functional and independent T-cell receptors. Cells belonging to this dual TCR subset may be specific for a broader range of antigens than cells with a single receptor. We discuss the possibility that dual receptor T-cells may be involved in alloreactivity and autoimmunity.

Cytotoxic T-lymphocyte responses in melanoma through in vitro stimulation with the Melan-A peptide analogue A27L: a qualitative analysis.

Modifications in tumour antigen-derived epitopes that stabilize the major histocompatibility complex (MHC)-peptide complex result in enhanced stimulatory capacity and improved immunogenicity of the altered peptide. These epitope analogues are attractive candidates for the development of peptide-based vaccine trials. Any modification, however, in tumour antigens may induce T-cell responses that could either fail to react against the naturally occurring peptides or represent only a subset of the total antigen-specific repertoire. In the present study, we performed a critical analysis of the ability of cytotoxic T-lymphocyte (CTL) clones, derived from two melanoma patients through stimulation with the A27L peptide analogue, to cross-react with the naturally processed Melan-A/MART-1 (Melan-A) peptides in terms of T-cell receptor (TCR) affinity, functional avidity and fine antigen specificity. We found that all the A27L-specific clones analysed possessed a very low avidity for the natural Melan-A peptides, and that their binding affinity for human leukocyte antigen (HLA) tetramers complexed with both the modified and the natural Melan-A peptides did not strictly correlate with their functional avidity. We also observed that these clones were able to cross-recognize both natural Melan-A peptides in one patient, but only one peptide in the second patient. We discuss the capability of the A27L peptide analogue to stimulate all the available Melan-A-specific repertoire.

α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors.

Emerging evidence suggests that new cells, including neurons, can be generated within the adult hypothalamus, suggesting the existence of a local neural stem/progenitor cell niche. Here, we identify α-tanycytes as key components of a hypothalamic niche in the adult mouse. Long-term lineage tracing in vivo using a GLAST::CreER(T2) conditional driver indicates that α-tanycytes are self-renewing cells that constitutively give rise to new tanycytes, astrocytes and sparse numbers of neurons. In vitro studies demonstrate that α-tanycytes, but not ß-tanycytes or parenchymal cells, are neurospherogenic. Distinct subpopulations of α-tanycytes exist, amongst which only GFAP-positive dorsal α2-tanycytes possess stem-like neurospherogenic activity. Fgf-10 and Fgf-18 are expressed specifically within ventral tanycyte subpopulations; α-tanycytes require fibroblast growth factor signalling to maintain their proliferation ex vivo and elevated fibroblast growth factor levels lead to enhanced proliferation of α-tanycytes in vivo. Our results suggest that α-tanycytes form the critical component of a hypothalamic stem cell niche, and that local fibroblast growth factor signalling governs their proliferation.

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes.

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both kappa and lambda light chains productively rearranged, but fail to express the x chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the lambda chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.

Sexually dimorphic neurogenesis is topographically matched with the anterior accessory olfactory bulb of the adult rat.

The accessory olfactory bulb (AOB) is a sexually dimorphic structure of the vomeronasal system, which plays a role in the control of sexual behaviors. In adult rats, we have demonstrated previously that the migrating neuroblasts of the subependymal layer (SEL) directed to the main olfactory bulb (MOB) also reach the AOB. To tackle the relation between sexual dimorphism and targeted cell migration, we quantified the neo-neurogenesis in the AOB of adult rats of both sexes. Our results confirm a morphological sexual dimorphism in the AOB granular layer volumes. We showed that the number of newly generated cells reaching the AOB in both sexes was considerable, even if lower than those directed to the MOB. Moreover, we demonstrated that the rate of neurogenesis in the anterior AOB of the two sexes was significantly different.

T cell receptor V delta 2-C alpha transcripts are present in the thymus but virtually absent in the periphery.

To investigate whether the V delta 2-(D)-J alpha gene configuration, characteristically associated with the major subset of acute lymphoblastic leukemias in humans, might have a physiologic role in T cell ontogeny, we have looked for V delta 2-C alpha transcripts in the thymus and peripheral blood of normal donors. Here we show by PCR analysis that these transcripts are virtually absent in the PBMC, whereas they are present in fetal and postnatal thymus. Interestingly, over 80% of 43 V delta 2-C alpha cDNAs randomly isolated from one postnatal thymus appeared to maintain an open reading frame. This suggests that in the thymus the V delta 2-C alpha products might be exposed to selective pressure. Furthermore, in two of three thymuses tested for J alpha usage, it was found overrepresented in a J alpha element (J alpha 58) located 2 kb downstream to a pseudo-J (J alpha 61), known to be a hot spot of recombination in alpha beta committed cells. A possible alternative pathway to alpha beta T cell differentiation via a V delta 2-J alpha intermediate is discussed.

A novel SH3-containing human gene family preferentially expressed in the central nervous system.

The Src-homology-3 domain (SH3) is an evolutionarily conserved, 50- to 60-amino-acid module carried by intracellular proteins involved in the transduction of signals for cell polarization, motility, enzymatic activation, and transcriptional regulation. The SH3 drives protein-protein interactions through binding to proline-rich ligands. This function relies on the conserved secondary structure, whereas the SH3 primary structure is highly diverse. Taking advantage of the fact that the few conserved amino acids are clustered near the N- and C-terminal ends, we designed degenerate oligonucleotides spanning these two regions and screened by PCR a variety of normal and tumor tissues for the expression of SH3-containing transcripts. Using this strategy, we have identified a novel SH3-containing human gene family of six related transcripts that map to four different chromosomes. The SH3 domain lies at the C-terminal end and shows 56-50% amino acid homology to the C-terminal SH3 of Sem-5/Drk/GRB2. The N-terminal segment of this novel SH3GL (from SH3-containing Grb2-like) gene family does not resemble any known protein. Three of these transcripts are in-frame and show a peculiar tissue distribution: SH3GL2 is preferentially expressed in the brain, SH3GL3 in brain and testis, and SH3GL1 is ubiquitous.

Increased frequency of RAG-expressing, CD4(+)CD3(low) peripheral T lymphocytes in patients with defective responses to DNA damage.

Accumulating evidence indicates that peripheral lymphocyte variants with altered antigen receptor expression may be capable of expressing recombination-activating genes (RAG). We and others recently observed functional RAG gene products in mature T cells with defective TCR expression (MacMahan and Fink, Immunity 1998. 9: 637 - 647; Lantelme et al., J. Immunol., 2000. 164: 3455 - 3459). Here, the association between TCR expression and RAG activity was assessed further in lymphocytes from patients with defective responses to DNA damage. We show that T cells with altered TCR surface expression are present in increased numbers in these patients and that they express RAG genes. The finding of RAG gene expression by TCR variants suggests the possibility that secondary V(D)J rearrangements could be induced in these cells to rescue their defective phenotype and cellular function. Moreover, as V(D)J recombination has been implicated in chromosome translocations involving antigen receptor genes, we discuss a possible relationship between altered TCR expression, RAG activity and the frequent lymphoma-specific translocations observed in these patients.

Kinetics of GATA-3 gene expression in early polarizing and committed human T cells.

Different transcription factors have been shown to control the transition of naive T cells into T helper 1 (Th1)/Th2 subsets. The T-cell-specific transcription factor GATA-3 is known to be selectively expressed in murine developing Th2 cells and to exert a positive action on Th2-specific cytokine production. Investigating GATA-3 gene regulation in human T cells we have found that naive T cells highly express GATA-3, and during early T2 or T1 polarization, respectively, they either maintain or quickly down-regulate expression. In developing T2 cells, as well as in committed Th2 cell lines and clones, we found a positive correlation among GATA-3, interleukin (IL)-5 and IL-4 gene expression kinetics, supporting the positive action of GATA-3 on Th2-specific cytokine production. A possible relationship between GATA-3 gene expression and the down-regulation of the IL-12 receptor (beta2-chain; IL-12Rbeta2) gene was evident only in the early phases of T2 polarization (within 24 hr), and not demonstrated at later times. During T-cell commitment the presence of IL-4 in the culture was essential to maintain or enhance GATA-3 transcription, while IL-12 was not necessary for full repression of GATA-3. Finally, we showed selective GATA-3 up-regulation in human Th2 cell lines and clones and the maintainance of a low basal level of GATA-3 expression in Th1 cells upon activation.