Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one TCR alpha, one TCR beta, and two CD3 epsilon chains.

The stoichiometry of the subunits that comprise the T cell antigen receptor (TCR) complex is not completely known. In particular, it is uncertain whether TCR alpha and TCR beta proteins are present in the TCR complex as one or multiple heterodimeric pairs. In this study we have used mice transgenic for two different TCR alpha and two different TCR beta proteins to determine the number of TCR alpha and TCR beta chains in a single TCR complex. Individual thymocytes and splenic T cells from double TCR transgenic mice simultaneously expressed all four transgenic TCR proteins on their surfaces. Because the individual TCR alpha and individual TCR beta proteins were biochemically distinguishable, we were able to examine association among the transgenic TCR products. We found that each TCR alpha chain paired with each TCR beta chain, but that each TCR complex contained only one TCR alpha and one TCR beta protein. Furthermore, quantitative immunofluorescence revealed that T cells expressed twice as many CD3 epsilon as TCR beta proteins. These findings demonstrate that there are precisely one TCR alpha, one TCR beta, and two CD3 epsilon chains in each TCR/CD3 complex expressed on the surfaces of both thymocytes and mature T cells.

T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus.

Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

Lineage commitment in the thymus: only the most differentiated (TCRhibcl-2hi) subset of CD4+CD8+ thymocytes has selectively terminated CD4 or CD8 synthesis.

Lineage commitment is a developmental process by which individual CD4+CD8+ (double positive, DP) thymocytes make a decision to differentiate into either CD4+ or CD8+ T cells. However, the molecular event(s) that defines lineage commitment is controversial. We have previously proposed that lineage commitment in DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi). In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule. In addition, the present results identify three distinct sub-populations of DP thymocytes that define the developmental progression of the lineage commitment process and demonstrate that lineage commitment is coincident with upregulation of TCR and bcl-2. Thus, this study supports a molecular definition of lineage commitment and uniquely identifies TCRhibcl-2hi DP thymocytes as cells that are already committed to either the CD4 or CD8 T cell lineage.

Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28.

CD4+CD8+ thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCR/self-antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efficiently induce apoptosis of CD4+CD8+ thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4+CD8+ thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.

Early molecular events induced by T cell receptor (TCR) signaling in immature CD4+ CD8+ thymocytes: increased synthesis of TCR-alpha protein is an early response to TCR signaling that compensates for TCR-alpha instability, improves TCR assembly, and parallels other indicators of positive selection.

Differentiation of immature CD4+ CD8+ thymocytes into mature CD4+ or CD8+ T cells occurs within the thymus and is dependent upon expression of antigen receptor complexes (T cell receptor [TCR]) containing clonotypic alpha/beta proteins. We have recently found that CD4+ CD8+ thymocytes express low levels of surface TCR because of limitations placed on TCR assembly by the instability of nascent TCR-alpha proteins within the endoplasmic reticulum (ER) of immature thymocytes. Because TCR-alpha/beta expression increases during development, a molecular mechanism must exist for increasing the number of assembled TCR complexes present in immature CD4+ CD8+ thymocytes that have been signaled to differentiate into mature T cells, although no such mechanism has yet been described. In the current report we have examined the molecular consequences of intracellular signals generated by engagement of surface TCR complexes on immature CD4+ CD8+ thymocytes. Isolated TCR engagement generated signals that increased TCR-alpha RNA levels and increased synthesis of TCR-alpha proteins, which, in turn, significantly increased assembly of complete TCR-alpha/beta complexes in CD4+ CD8+ thymocytes. Increased TCR-alpha protein levels in TCR-signaled CD4+ CD8+ thymocytes was the result of increased synthesis and not increased stability of TCR-alpha proteins, indicating that TCR engagement compensates for, but does not correct, the inherent instability of TCR-alpha proteins in the ER of immature thymocytes. Consistent with the delivery by TCR engagement of a positive selection signal, TCR engagement also increased CD5 expression, decreased RAG-1 expression, and decreased CD4/CD8 coreceptor expression in immature CD4+ CD8+ thymocytes. These data identify amplified TCR-alpha expression as an initial response of immature CD4+ CD8+ thymocytes to TCR-mediated positive selection signals and provide a molecular basis for increased surface TCR density on developing thymocytes undergoing selection events within the thymus.

Identification of CD8 as a peanut agglutinin (PNA) receptor molecule on immature thymocytes.

Differentiation of most T lymphocytes occurs within the thymus and is characterized by variable expression of CD4/CD8 coreceptor molecules, increased surface density of T cell antigen receptor (TCR) alpha beta proteins, and decreased expression of glycan chains recognized by the galactose-specific lectin peanut agglutinin (PNA). Although appreciated for several decades that PNA agglutination is useful for the physical separation of immature and mature thymocyte sub-populations, the identity of specific PNA-binding glycoproteins expressed on immature thymocytes remains to be determined. In the current report, we studied the expression of PNA-specific glycans on immature and mature T cells and used lectin affinity chromatography and immunoprecipitation techniques to characterize PNA-binding glycoproteins on thymocytes. Our data demonstrate that PNA-specific glycans are localized on a relatively small subset of thymocyte surface proteins, several of which were specifically identified, including CD43, CD45, and suprisingly, CD8 molecules. CD8 alpha and CD8 alpha' proteins bound to PNA in the absence of CD8 beta expression showing that O-glycans on CD8 beta glycoproteins are not necessary for PNA binding and that glycosylation of CD8 alpha and CD8 alpha' proteins proceeds effectively in the absence of CD8 beta. Finally, we demonstrate that PNA binding of CD8 is developmentally regulated by sialic acid addition as CD8 proteins from mature T cells bound to PNA only after sialidase treatment. These studies identify CD8 as a PNA receptor molecule on immature thymocytes and show that PNA binding of CD8 on immature and mature T cells is developmentally regulated by sialic acid modification.

Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4(+)CD8(+) thymocytes.

Thymocyte maturation is governed by antigen-T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen-TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.

Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.

Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells.

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.

Primary non-Hodgkin's lymphoma of the CNS treated with BVAM or CHOD/BVAM chemotherapy before radiotherapy.

PURPOSE: To assess whether chemotherapy that includes drugs that cross the blood-brain barrier improves survival in primary CNS non-Hodgkin's lymphoma (PCNSL) when combined with radiotherapy. PATIENTS AND METHODS: Thirty-four patients, with no evidence of human immunodeficiency virus type 1 (HIV-1) infection, were treated with the related chemotherapy regimens of carmustine (BCNU), vincristine, cytarabine, and methotrexate (BVAM; 12 patients), cyclophosphamide, doxorubicin, vincristine, and dexamethasone (CHOD)/BVAM (17 patients) and intensified CHOD/BVAM (five patients) between 1986 and 1994. The median age was 60 years (range, 16 to 73) and 47% had a performance status of 3 or 4 (Eastern Cooperative Oncology Group [ECOG]/World Health Organization [WHO]). Ten patients were treated with BVAM chemotherapy between 1986 and 1989, and subsequently 17 patients were treated with CHOD/BVAM (cytarabine 3 g/m2). Twenty of these 27 patients received whole-brain radiotherapy (craniospinal in four). RESULTS: The complete response (CR) rate at the completion of chemotherapy was 63% for BVAM and 67% for CHOD/BVAM; more neutropenia occurred with CHOD/BVAM. The 5-year actuarial probability of survival of all 34 patients was 33% (95% confidence interval [CI], 14% to 52%), with so far only one recurrence after 2 years. Using multivariate analysis, age (P = .0005) and number of tumors at diagnosis (P = .0358) were prognostic factors. All five patients aged > or = 70 years died during or shortly after chemotherapy. Performance status was not an independent variable. CONCLUSION: The BVAM or CHOD/BVAM regimens can be delivered despite neutropenia without significant treatment delay or dose reduction in patients less than 70 years of age. Further intensification of this type of chemotherapy is probably not possible with patients of this age, many of whom have a poor performance status.

CD4+/CD8- thymocytes dominate the fetal thymus treated with a combination of anti-T cell receptor-beta and anti-CD4 antibodies.

Two major phenotypic changes characterize the development of a mature thymocyte from its CD4+/CD8+ (double positive or DP) precursor: the loss of expression of either CD4 or CD8 and the increase in the level of surface TCR. The specific surface interactions responsible for these changes are unknown, but studies using the fetal thymus as an experimental system have provided clues by identifying conditions that alter these maturational events. Development to the CD4+/CD8-/TCR-alpha beta high (single positive) phenotype is inhibited when thymocytes in fetal organ culture are exposed to antibodies directed against the CD4 molecule, the CD3 complex or the TCR-alpha/beta heterodimer. We show in this study, however, that treatment of fetal thymic lobes with a combination of anti-CD4 and anti-TCR-beta antibodies results in a marked increase in the proportion of mature CD4+/CD8-/TCR-alpha beta+ thymocytes and a decrease in the proportion of DP thymocytes. Although treatment of lobes with a combination of anti-CD4 and anti-CD3 epsilon antibodies also depletes cultures of DP thymocytes, the CD4+/CD8-/TCR+ population does not develop. Our results are consistent with the hypothesis that coengagement of CD4 and TCR biases development to the CD4 single positive phenotype and with observations that TCR engagement and CD3 engagement have different developmental consequences.

Cerebral contusional tears as a marker of child abuse--detection by cranial sonography.

A series of 6 infants subjected to child abuse is presented in whom contusional tears of subcortical white matter were detected during life by intracranial sonography. The sonographic appearances of this highly pathognomonic marker of shaking injury are described for the first time and their significance discussed. On the basis of our experience we suggest that high resolution cranial sonography is an extremely valuable part of the diagnostic work up in cases of suspected non-accidental injury.

Third ventricular cysts and membranes unsuspected on conventional CT and MRI.

Three cases are presented of symptomatic cysts or membranes within the third ventricle interfering with CSF flow and presenting as non-communicating triventricular hydrocephalus. None was visible on conventional CT or MRI, two being discovered at neuroendoscopy and one only with a specific MRI sequence designed to show CSF partitioning.

Spinal arteriovenous malformations in children.

Three cases of spinal AVM in children are presented, each illustrating a different clinical presentation and anatomical variant. The natural history, treatment, and pathophysiology of these cases is discussed with a survey of the literature.

Carotico-cavernous fistula after trans-sphenoidal hypophysectomy.

A case is presented of a 48-year-old woman with an iatrogenic carotico-cavernous fistula consequent upon a transphenoidal hypophysectomy. Successful closure of the fistula was achieved by interventional radiology. The causes and management of vascular injuries during trans-sphenoidal pituitary surgery are reviewed and discussed.

Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals.

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4(+)CD8(+) thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4(+)CD8(+) thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.

Management and outcome of severe head injuries in the Trent region 1985-90.

In a five year period, 39 children (29 boys, 10 girls) aged 2 months to 13 years (mean 7.8 years) were studied who had suffered a major head injury (29 road traffic accidents, six falls, and four non-accidental injury). The injury had been assessed clinically and by cranial computed tomography or cranial ultrasound (in a single baby of 2 months). Initial Glasgow coma scores for all subjects ranged from 3-11 (mean 5.5), intact survivors 5-11 (7.4), minor handicap 4-11 (6.1), major handicap 3-6 (4.3), fatalities 3-6 (4.1). All were treated with sedation, paralysis, hyperventilation (arterial carbon dioxide tension 3.0-3.5 kPa), intracranial pressure monitoring and moderate body surface hypothermia to 32 degrees C. Nine children died and 30 survived (nine intact, 13 minor disability, and eight major disability). The worst cerebral perfusion pressure was over 40 mm Hg in all but one survivor, and less than 40 mm Hg in seven of nine fatalities. Severe hypocapnia both in the first 24 hours and overall was correlated with poor outcomes (dead or major disability), as were bilateral contusions or diffuse axonal injury.