ALK7 expression in prolactinoma is associated with reduced prolactin and increased proliferation.

Prolactinoma represents the most frequent hormone-secreting pituitary tumours. These tumours appear in a benign form, but some of them can reach an invasive and aggressive stage through an unknown mechanism. Discovering markers to identify prolactinoma proliferative and invading character is therefore crucial to develop new diagnostic/prognostic strategies. Interestingly, members of the TGFß-Activin/BMP signalling pathways have emerged as important actors of pituitary development and adult function, but their role in prolactinomas remains to be precisely determined. Here, using a heterotopic allograft model derived from a rat prolactinoma, we report that the Activins orphan type I receptor ALK7 is ectopically expressed in prolactinomas-cells. Through immunohistological approaches, we further confirm that normal prolactin-producing cells lack ALK7-expression. Using a series of human tumour samples, we show that ALK7 expression in prolactinomas cells is evolutionary conserved between rat and human. More interestingly, our results highlight that tumours showing a robust expression of ALK7 present an increased proliferation as address by Ki67 expression and retrospective analysis of clinical data from 38 patients, presenting ALK7 as an appealing marker of prolactinoma aggressiveness. Beside this observation, our work pinpoints that the expression of prolactin is highly heterogeneous in prolactinoma cells. We further confirm the contribution of ALK7 in these observations and the existence of highly immunoreactive prolactin cells lacking ALK7 expression. Taken together, our observations suggest that Activin signalling mediated through ALK7 could therefore contribute to the hormonal heterogeneity and increased proliferation of prolactinomas.

Deletion of Menin in craniofacial osteogenic cells in mice elicits development of mandibular ossifying fibroma.

Ossifying fibroma (OF) is a rare benign tumor of the craniofacial bones that can reach considerable and disfiguring dimensions if left untreated. Although the clinicopathological characteristics of OF are well established, the underlying etiology has remained largely unknown. Our work indicates that Men1-a tumor suppressor gene responsible of Multiple endocrine neoplasia type 1-is critical for OF formation and shows that mice with targeted disruption of Men1 in osteoblasts (Men1Runx2Cre) develop multifocal OF in the mandible with a 100% penetrance. Using lineage-tracing analysis, we demonstrate that loss of Men1 arrests stromal osteoprogenitors in OF at the osterix-positive pre-osteoblastic differentiation stage. Analysis of Men1-lacking stromal spindle cells isolated from OF (OF-derived MSCs (OFMSCs)) revealed a downregulation of the cyclin-dependent kinase (CDK) inhibitor Cdkn1a, consistent with an increased proliferation rate. Intriguingly, the re-expression of Men1 in Men1-deficient OFMSCs restored Cdkn1a expression and abrogated cellular proliferation supporting the tumor-suppressive role of Men1 in OF. Although our work presents the first evidence of Men1 in OF development, it further provides the first genetic mouse model of OF that can be used to better understand the molecular pathogenesis of these benign tumors and to potentially develop novel treatment strategies.

Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids.

Liver-resident CD8+ T cells are highly motile cells that patrol the vasculature and provide protection against liver pathogens. A key question is: how can these liver CD8+ T cells be simultaneously present in the circulation and tissue-resident? Because liver-resident T cells do not express CD103 - a key integrin for T cell residence in epithelial tissues - we investigated other candidate adhesion molecules. Using intra-vital imaging we found that CD8+ T cell patrolling in the hepatic sinusoids is dependent upon LFA-1-ICAM-1 interactions. Interestingly, liver-resident CD8+ T cells up-regulate LFA-1 compared to effector-memory cells, presumably to facilitate this behavior. Finally, we found that LFA-1 deficient CD8+ T cells failed to form substantial liver-resident memory populations following Plasmodium or LCMV immunization. Collectively, our results demonstrate that it is adhesion through LFA-1 that allows liver-resident memory CD8+ T cells to patrol and remain in the hepatic sinusoids.

The MHC class II-associated invariant chain: a molecule with multiple roles in MHC class II biosynthesis and antigen presentation to CD4+ T cells.

The MHC class II-associated invariant chain (Ii) plays a central role in the biological function of MHC class II molecules. Ii is a type II membrane glycoprotein that is synthesized as different isoforms that include a major 31 kDa isoform (p31/p33) and a minor 41 kDa isoform (p41) in both, mice and humans. All isoforms share several common regions acting at different steps in the process that lead to functional class II molecule/peptide complexes. In the ER, two C-terminal extracytoplasmic regions of Ii are required for class II assembly: the 153-183 region is involved in the formation of Ii trimers and the 80-104 region mediates binding with class II molecules giving rise to nonamers. Ii association with class II molecules prevents both aggregation of class II dimers and binding with endogenous ER-derived peptides. In addition, two motifs in the cytosolic N-terminal region of Ii direct class II nonamers toward specialized endosomal compartments where peptide loading occurs. In these compartments, Ii undergoes proteolytic degradation leaving only CLIP (residues 80-104) associated with Class II. CLIP modulates loading of class II molecules in endosomes and is removed from the MHC class II groove by monomorphic MHC class II molecules, H2-M or HLA DM, in mouse and human, respectively. The roles of Ii in antigen presentation to MHC class II-restricted T cells and in CD4+ T cell development are discussed in this review.

Suicidal emperipolesis: a process leading to cell-in-cell structures, T cell clearance and immune homeostasis.

"Suicidal emperipolesis" is one of the most recently reported processes leading to cell-in-cell structures that promote cell death. This process was discovered in studies investigating the fate of autoreactive CD8 T cells activated within the liver. Recently, we reported that activated T cells invaded hepatocytes, formed transient cell-in-cell structures, and were rapidly degraded within endosomal/lysosomal compartments by a non-apoptotic pathway. Importantly, pharmacological inhibition of this process caused intrahepatic accumulation of tissue-reactive T cells and breach of immune tolerance. The characterization of the molecular mechanisms of suicidal emperipolesis is still in its infancy, but initial studies suggest this phenomenon is distinct from other reported cell-in-cell structures. As opposed to the formation of other cell-in-cell structures, suicidal emperipolesis takes place in a non-malignant environment, and without obvious pathology. It is therefore the first cell-in-cell structure described to have a role in maintaining homeostasis in normal physiology in higher organisms. T cell emperipolesis within hepatocytes has also been observed by pathologists in a range of chronic human liver pathologies. As T cell-in-hepatocyte structures resulting from suicidal emperipolesis are very transiently observed in normal physiology, their accumulation during liver disease would suggest that severe tissue injury is promoted by, or associated with, defective T cell clearance. In this review, we compare "suicidal emperipolesis" to other processes leading to cell-in-cell structures, and consider its potential biological roles in maintaining immune homeostasis and tolerance in the context of the hepatic environment.

Altered MENIN expression disrupts the MAFA differentiation pathway in insulinoma.

The protein MENIN is the product of the multiple endocrine neoplasia type I (MEN1) gene. Altered MENIN expression is one of the few events that are clearly associated with foregut neuroendocrine tumours (NETs), classical oncogenes or tumour suppressors being not involved. One of the current challenges is to understand how alteration of MENIN expression contributes to the development of these tumours. We hypothesised that MENIN might regulate factors maintaining endocrine-differentiated functions. We chose the insulinoma model, a paradigmatic example of well-differentiated pancreatic NETs, to study whether MENIN interferes with the expression of v-MAF musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), a master glucose-dependent transcription factor in differentiated ß-cells. Immunohistochemical analysis of a series of human insulinomas revealed a correlated decrease in both MENIN and MAFA. Decreased MAFA expression resulting from targeted Men1 ablation was also consistently observed in mouse insulinomas. In vitro analyses using insulinoma cell lines showed that MENIN regulated MAFA protein and mRNA levels, and bound to Mafa promoter sequences. MENIN knockdown concomitantly decreased mRNA expression of both Mafa and ß-cell differentiation markers (Ins1/2, Gck, Slc2a2 and Pdx1) and, in parallel, increased the proliferation rate of tumours as measured by bromodeoxyuridine incorporation. Interestingly, MAFA knockdown alone also increased proliferation rate but did not affect the expression of candidate proliferation genes regulated by MENIN. Finally, MENIN variants with missense mutations detected in patients with MEN1 lost the WT MENIN properties to regulate MAFA. Together, our findings unveil a previously unsuspected MENIN/MAFA connection regarding control of the ß-cell differentiation/proliferation balance, which could contribute to tumorigenesis.

Reexpression of oncoprotein MafB in proliferative ß-cells and Men1 insulinomas in mouse.

MafB, a member of the large Maf transcription factor family, is essential for the embryonic and terminal differentiation of pancreatic α- and ß-cells. However, the role of MafB in the control of adult islet-cell proliferation remains unknown. Considering its oncogenic potential in several other tissues, we investigated the possible alteration of its expression in adult mouse ß-cells under different conditions of proliferation. We found that MafB, in general silenced in these cells, was reexpressed in ∼30% of adaptive ß-cells both in gestational female mice and in mice fed with a high-fat diet. Importantly, reactivated MafB expression was also observed in the early ß-cell lesions and insulinomas that developed in ß-cell specific Men1 mutant mice, appearing in >80% of ß-cells in hyperplasic or dysplastic islets from the mutant mice >4 months of age. Moreover, MafB expression could be induced by glucose stimulation in INS-1 rat insulinoma cells. The induction was further reinforced following Men1 knockdown by siRNA. Furthermore, MafB overexpression in cultured ßTC3 cells enhanced cell foci formation both in culture medium and on soft agar, accompanied with the increased expression of Cyclin B1 and D2. Conversely, MafB downregulation by siRNA transfection reduced BrdU incorporation in INS-1E cells. Taken together, our data reveal that Men1 inactivation leads to MafB reexpression in mouse ß-cells in vivo, and provides evidence that deregulated ectopic MafB expression may have a hitherto unknown role in adult ß-cell proliferation and Men1-related tumorigenesis.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010.

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Histometry and the H.O.M.E. concept: an aid to the grading of intra-cervical neoplasia?

The view has been expressed that few quantitative methods are of value to the pathologist in purely diagnostic work. Quantitative systems are perceived as too large for the average reporting room, too time consuming to learn, very expensive to buy and quick to become obsolete. Further, the software supplied usually cannot provide fully automated analysis, and user interaction is often tedious. If measurement techniques have little value in diagnosis they may have a role in assessing the prognosis of tumours. High levels of inter- and intra-observer variation in tumour grading have been reported and quantitative methods have been used to reduce this and more emphasis has been placed on the measurement of changes in tissue architecture, which may help to reduce observer variation. This paper describes such a method based on cell sociology, which has been implemented on a quantitative microscope specifically designed for use in the routine diagnostic pathology environment. The results of a preliminary study on grading cervical intraepithelial neoplasia show a significant difference between all groups (P less than 1 x 10(-5)) and a linear trend for the measurement of Area Disorder (P less than 1 x 10(-5)).

Enhancement of in vivo and in vitro T cell response against measles virus haemagglutinin after its incorporation into liposomes: effect of the phospholipid composition.

Artificial phospholipid bilayer vesicles were tested for their capacity to enhance the priming and the restimulation of mouse T cells against the haemagglutinin (H) glycoprotein of the measles virus in vivo and in vitro. H glycoprotein was purified and incorporated into liposomes made of cholesterol, dicetylphosphate and dilauroylphosphatidylcholine (DLPC) or distearoylphosphatidylcholine (DSPC). H in DLPC or DSPC-liposomes was found to be a potent in vivo stimulator of lymph node T cells harvested from mice immunized with measles virus, whereas H glycoprotein in free form did not elicit any proliferative T cell response. When used to immunize naive mice, only H in DSPC-liposomes was able to prime T cells as evidenced by the capacity of lymph node cells to proliferate in the presence of H in liposomes or measles virus as secondary stimulating agents in vitro. H-specific T cell clones derived from animals immunized with H in DSPC-liposomes were able to recognize H glycoprotein both in free form and incorporated into liposomes in the presence of naive spleen cells as APC. However, compared with the liposome forms, 20-fold more H protein in free form was required to elicit a T cell clone response at a similar level. This liposome immune enhancing effect on the T cell clone recognition of H glycoprotein was also observed when peritoneal exudate cells were used as APC. These data demonstrate that the insertion of a membrane-derived antigen into artificial membranes may be a prerequisite for the priming and stimulation of specific T cells both in vivo and in vitro. In addition, the nature of the phospholipid used to build the liposomes appears to be a critical parameter.

Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival.

Intraperitoneal peptide injection of TCR-transgenic mice or expression of antigen in hepatocytes leads to an accumulation in the liver of specific apoptotic CD8+ T cells expressing activation markers. To determine whether liver cells are capable of directly activating naive CD8+ T cells, we have studied the ability of purified hepatocytes to activate TCR-transgenic CD8+ T cells in vitro. We show that hepatocytes which do not express CD80 and CD86 co-stimulatory molecules are able to induce activation and effective proliferation of specific naive CD8+ T cells in the absence of exogenously added cytokines, a property only shared by professional antigen-presenting cells (APC). Specific T cell proliferation induced by hepatocytes was comparable in magnitude to that seen in response to dendritic cells and was independent of CD4+ T cell help or bystander professional APC co-stimulation. During the first 3 days, the same number of divisions was observed in co-cultures of CD8+ T cells with either hepatocytes or splenocytes. Both APC populations induced expression of early T cell activation markers and specific cytotoxic T lymphocyte (CTL) activity. However, in contrast to T cells activated by splenocytes, T cells activated by hepatocytes lost their cytolytic function after 3 days of co-culture. This correlated with death of activated T cells, suggesting that despite efficient activation, proliferation and transient CTL function, T cells activated by hepatocytes did not survive. Death could be prevented by adding antigen-expressing splenocytes or exogenous IL-2 to the co-culture, indicating that hepatocytes are not involved in direct killing of CD8+ T cells but rather fail to promote survival. Dying cells acquired a CD8(low) TCR(low) B220+ phenotype similar to the one described for apoptotic intrahepatic T cells, suggesting an alternative model to account for the origin of these cells in the liver. The importance of these findings for the understanding of peripheral tolerance and the ability of liver grafts to be accepted is discussed.

Correlation between invariant chain expression level and capability to present antigen to MHC class II-restricted T cells.

In this study we investigated the role of the invariant chain (li) in the presentation of hen egg lysozyme (HEL) and measles virus hemagglutinin (HA) antigens to MHC class II-restricted T hybridoma cells. Fibroblastic cells transfected with Ed or Ak genes, and supertransfected or not with the li gene, were used as antigen-presenting cells (APC). For every APC pair analysed, the amount of exogenous antigen needed to obtain a T-cell response was inversely correlated with the level of li expression. Exogenously provided HEL was efficiently presented by li-supertransfected APC at doses of 10 micrograms/ml or below. In contrast, non-li transfected fibroblastic cells, which express a low level of endogenous li, required at least 10 times more HEL to stimulate most of the T hybridoma cells. Analogous results were also obtained using exogenous HA. Finally, two different experiments suggest that basal li expressed in fibroblastic cells is involved in the presentation of exogenous antigen. In the first one, we showed that li/class II ratio was increased in high-density grown fibroblastic cells and that this increase correlates with the ability of the cells to present exogenous antigen. In the second, treating high-density grown cells with an antisense li oligodeoxynucleotide could impair their ability to present exogenous HEL. We also examined the presentation of endogenously-synthesized HEL or HA after introduction of the antigen into the biosynthetic pathway of the APC by transfection of HEL and HA cDNAs. There was no apparent difference in the capability of high density grown fibroblastic cells, transfected or not with li gene, to present endogenous HEL or HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2Kb in the liver.

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2Kb (Kb) under the control of the sheep metallothionein promoter (Met-Kb mice). To follow the fate of Kb-specific T cells, and to prevent any aberrant thymic expression of the Kb transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a Kb-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although Kb-specific CD8+ T cells were produced in the thymus grafts of these manipulated Met-Kb mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8+ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen-presenting cells, activated the Kb-specific CD8+ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.

Antigen-specific primary activation of CD8+ T cells within the liver.

It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8(+) T cells specific for H-2K(b) were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-K(b) mice expressing H-2K(b) in the liver. Activated CD8(+) cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8(+) cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8(+) T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8(+) T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis.

Cryotherapy of liver tumours--a practical guide.

The use of cryotherapy for the treatment of some unresectable liver tumours has been clearly established as a therapeutic option. Intra-operative ultrasound has enhanced the process by enabling the surgeon to identify hepatic lesions and to allow visualisation of the freezing process to ensure that the cryolesion will include the tumour mass. The purpose of this paper is to provide a practical guide to surgeons who wish to perform cryotherapy of liver tumours. Patient selection and anaesthetic considerations are important. The surgeon should be able to deal with the complications of cryotherapy, particularly the intra-operative haemorrhage which may arise from cracking of the hepatic parenchyma as the iceball thaws. Follow-up is based on tumour marker assay and imaging of the liver and repeat cryotherapy can be considered for selected cases.

Invariant chain expression similarly controls presentation of endogenously synthesized and exogenous antigens by MHC class II molecules.

I-Ak- and I-Ed-transfected L fibroblasts were supertransfected with cDNA coding for hen egg lysozyme (HEL) or measles virus hemagglutinin (HA). Well-defined cell culture conditions allowed us to obtain L cells with either no detectable endogenous Ii mRNA or a high level of endogenous Ii mRNA induced by serum starvation. Cells supertransfected with mouse Ii chain gene stably expressing a high level of Ii chain were also used as APC in parallel experiments. Class II presentation of endogenously secreted HEL or an ER-retained form of HEL to the HEL-specific I-Ak-restricted 3A9 T cell hybridoma was found to be strongly enhanced in cell transfectants expressing Ii chain. Similar results were obtained with the presentation of transmembrane HA to the HA-specific I-Ed-restricted TH5.143 T cell hybridoma. These findings correlate with those obtained with the presentation of exogenous HEL and HA. In addition, as reported to be the case for exogenous antigen, expression of a large amount of endogenous HA by the APC supplants the requirement for Ii chain expression in the enhancement of antigen presentation. These data demonstrate that presentation by MHC class II molecules of a given antigen, either exogenously provided or endogenously synthesized, is controlled in a similar manner by the Ii chain.

Inhibition by chloroquine of the class II major histocompatibility complex-restricted presentation of endogenous antigens varies according to the cellular origin of the antigen-presenting cells, the nature of the T-cell epitope, and the responding T cell.

Chloroquine treatment of antigen-presenting cells (APC) was explored as a tool to investigate the processing pathway for major histocompatibility complex (MHC) class II-restricted presentation of the endogenous secreted hen egg lysozyme (HEL) and transmembrane measles virus haemagglutinin (HA). A 72-hr pretreatment of the APC with 25 microM chloroquine blocked the presentation of the HEL(52-61) T-cell epitope generated from endogenous HEL to the I-Ak-restricted 3A9 T-cell hybridoma by MHC class II-transfected L cells expressing the invariant chain (Ii). The presentation of exogenously added HEL peptides was not affected. Under the same conditions, no inhibition of the presentation of HEL(106-116) to the I-Ed-restricted G28 high-avidity T-cell hybridoma, nor of HA when synthesized by L cells, was observed. When B-lymphoid APC were used, inhibition was observed in every case with a low number of B APC pretreated for 48 hr with chloroquine prior to the T-cell stimulation test. Moreover, addition of chloroquine to untreated B APC during the T-cell stimulation assay was sufficient to inhibit completely the presentation of HEL(106-116) to the B10.D24.42 low avidity T-cell hybridoma. Altogether these studies suggest that an apparent resistance of endogenous Ag presentation to chloroquine inhibition may not necessarily indicate the existence of a non-endosomal pathway but may be due to the nature of the T-cell epitope, to the use of 'non-professional' APC such as L cells, to the use of T cells of high avidity, and to high amounts of pre-existing MHC class II-peptide complexes expressed by the APC. We demonstrate here that, at least in conventional APC such as B cells, class II-restricted presentation of both endogenous secreted HEL and transmembrane HA involves an endosomal pathway.

Expression of two T cell receptor alpha chains on the surface of normal murine T cells.

We have previously reported that a subset of T cells in T cell receptor (TCR)-transgenic mice may express two different alpha chains on their surface. The expression of two functional alpha chains has also been demonstrated for human peripheral blood T cells. In this report, we show that a proportion of normal murine lymph node T cells express two functional alpha chains on their surface. The extrapolated frequency of these cells present in the normal repertoire ranges from 7-21%, with an average of 15%. Our analysis of a small number of antigen-specific T cell clones suggests that the frequency of antigen-responsive cells expressing two surface alpha chains is relatively low. This raises the possibility that dual alpha chain T cells may have a selective disadvantage in responding to specific antigen.

Major histocompatibility complex class II-restricted presentation of secreted and endoplasmic reticulum resident antigens requires the invariant chains and is sensitive to lysosomotropic agents.

We have tested the involvement of the invariant chains (Ii) p31 and p41 in the presentation of peptides derived from hen egg lysozyme (HEL) constructs targeted to different intracellular compartments within transfected fibroblasts. The endogenous HEL constructs were either present in the cytosol (HELc), secreted (HELs), or linked to the mammalian (KDEL C-terminal sequence that causes retention of HEL in the endoplasmic reticulum (ER)/pre-Golgi recycling compartment (HELr). Using Ii-negative antigen-presenting cells, the presentation of HELr to a HEL 46-61 specific T cell hybridoma was far less efficient than the presentation of the HELs. High levels of Ii expression enhanced drastically the presentation of the HEL 46-61 determinant derived from both HELr and HELs. HELr and HELs presentation was fully sensitive to lysosomotropic agents such as chloroquine, indicating that the formation of complexes between major histocompatibility complex (MHC) class II molecules and determinants derived from endogenous antigens entering the secretory pathway is taking place in an acidic compartment. The degradation and dissociation of Ii might be a prerequisite for the efficient presentation of endogenously derived determinants by MHC class II molecules, as for the presentation of most exogenous antigens. All our results are compatible with the notion that endogenous molecules being translocated into the lumen of the ER could be presented by class II molecules through a processing pathway involving an acidic compartment in which Ii chains dissociate from class II molecules.

Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling of naïve lymphocyte populations provides unique insights into the immune response. The clonal nature of immune responses, necessitating clonal expansion to achieve a sufficiently large number of Ag-reactive effector cells, combined with the dependence of lymphocyte differentiation on cell division, underlie the usefulness of CFSE in understanding the factors that regulate responses both in vitro and in vivo. We have combined CFSE labelling with Ag receptor transgenic models, using seven channel flow cytometry to track the correlation between cell division and a number of other parameters, such as surface expression of activation markers, cytokine receptors and homing receptors, cytokine production, cytotoxic activity and indicators of apoptosis. Our data have allowed us to classify and understand immune responses in novel ways, suggesting many further avenues of enquiry and indicating previously unrecognized relationships between cell division and eventual cell fate.