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The mineralogy and bulk chemical compositions of 15 Kintampo (Late Stone Age) potsherds from the Birimi site on the Gambaga Escarpment and eight samples of local sediment were determined with the intent of characterizing these wares and identifying the material used in their manufacture. Sediment from clay pits still used by potters north of the escarpment contains iron-rich laterite clasts (100 × XFeOt = 100 × FeOt/[FeOt + Al2O3 + SiO2] ≥10). Sedimentary clasts in stream sediments are relatively siliceous and iron-poor (100 × XFeOt < 10). Bulk geochemical data together with the compositions of lithic clasts (laterite, siltstone/sandstone) link the pottery to sediment sources, including escarpment sediments not presently used by Ghanaian potters. Fresh granite clasts found in some of the sherds were not found in the analyzed sediment samples, although some of their distinctive mineralogical constituents (e.g., variably barian alkali feldspar) are present. The analytical data suggest that pots found at Birimi were made locally by mixing escarpment sediment with clay and stream sediment brought in from below the escarpment. This contrasts with present-day practice, whereby the pots themselves are imported. The place where Birimi pottery was made and the outcrop source of aluminous sediment (mudstone with an “escarpment” trace element signature) used in these wares, however, remain unidentified. © 2013 Wiley Periodicals, Inc.

Travertine was one of ancient Egypt's most popular ornamental stones. Upon prolonged exposure to sunlight, the rock's translucent brownish calcite is bleached to nearly white. Ultraviolet and gamma irradiation experiments were undertaken to investigate the source of the brownish colour and the process by which it fades. It was found that the coloration results from the activation of colour centres by natural radioactivity within the rock, and that these colour centres are deactivated by the ultraviolet component of sunlight. The results also demonstrate that the original brownish colour of sun-bleached, travertine objects can be restored by artificial gamma irradiation.

The thermal history of four quartz-rich ochre samples from an Upper Palaeolithic site was studied. This work is based on the changes in thermal activation characteristics (TAC) of the 110 ∘ C TL peak of the quartz inclusions. An isothermal study of a previously unheated sample has highlighted the importance of the duration of annealing on the sensitization of quartz. In fact, the sensitivity change as a function of the duration of annealing is not monotonic. For that reason it seems necessary to consider the “equivalent thermal history” H E rather than an “equivalent temperature”. Isochronal annealing experiments demonstrate that the initial rise of sensitization overestimates the true H E by about 100 ∘ C . Using a geological sample we have thus developed an empirical approach which allows the true H E of artifacts to be determined. Reheating of ochre originally heated in antiquity results in desensitization of the TAC.

We report the first luminescence ages for the archeological and geological sediments forming the substrate of the Birimi archaeological site in the Northern Region of Ghana. The site's significance rests on the fact that it contains a rich collection of artifact assemblages representative of three distinct cultures, and that, on the basis of artifact typology, the earliest assemblage is diagnostic of the Middle Stone Age (MSA) . In situ occurrences of MSA artifacts are found at over 1 m below today's surface. They are overlain by a ceramic-rich complex of a sedentary or semi-sendentary Later Stone Age culture known as the Kintampo. The western half of the site is dominated by the industrial remains of Iron Age smelting activity. Elemental, mineralogical, and sedimentological analysis of the cultural and sub-cultural sedimentary horizons at the site revealed at least three distinct lithostratigraphic units. The quartz sediments are derived from the sandstone of the Gambaga escarpment, mass wasted and accreted fluvially at a rate of 3.2 cm/ka, forming a wide terrace at Birimi. Silts and finer fractions derive from windblown dust, likely from White Volta River and granitic sources to the north. Soil forming processes and wide fluctuations in moisture have progressively reduced the sediments at depth to the resistant quartz and kaolinite, with rich iron oxide coatings, and created two ironstone horizons composed of goethite-cemented quartz nodules. Multiple aliquot green-light stimulated optical ages for 125–150 μm quartz grains yielded ages of 23.6±2.9 and 40.8±11.8 ka for the MSA-bearing sediments, and 58.4±15.3 ka for the base of the terrace. Radiocarbon ages on charcoal from Kintampo-bearing units are 3.36–3.83 ka cal BP, and are supported by thermoluminescence (TL) ages on pottery sherds and burnt house daub fragments of this cultural complex. A 0.4 ka age on sediment from the site's surface confirms that the quartz zeroes well when exposed to natural light. Sediments bearing the Kintampo artifacts, however, yielded ages of 7.8–16.9 ka. These ages were obtained on sediments from large pits, some over 50 cm deep, and they deviate only slightly from the ages expected for naturally aggraded sediments at these depths. We conclude, therefore, that extensive digging of pits by the Kintampo dwellers was followed by rapid refilling, and that the bulk mobilization of the matrix did not permit the sedimentary quartz grains to experience any appreciable zeroing at that time.

We report the final results of a multidisciplinary geochronological study of two subfossil mastodon remains and the sediments associated with them, from the East Milford, N.S. mastodon locality discovered in 1991. The mastodons, which were found in a karstic sinkhole system, are the latest and the most complete of several mastodon remains discovered in Nova Scotia, New Brunswick and Maine over the last 170 years. The unconsolidated sediments containing the adult specimen were dated using optical dating (IRSL); the mastodon dental enamel was dated using electron spin resonance (ESR); and bone collagen from both individuals plus associated wood were dated using radiocarbon ( 14 C) dating. Two samples of sediment adhering to the walls of the karstic cavity at the elevation corresponding to the location of the adult specimen yielded statistically indistinguishable IRSL ages of 127±13 (EMM1) and 143±16 ka (EMM2), with a weighted mean of 133±6 ka. This is consistent with pollen data which indicate an interglacial climate. On the basis of its stratigraphic position with respect to overlying till units, the locality is attributed to oxygen isotope stage 5. Three ESR dates on dental enamel (natural prompt, deproteinated prompt, and deproteinated delayed) yielded statistically indistinguishable ages whose weighted mean is 74.9±5.0 ka. This indicates consignment to the geologic record of the mastodon tooth during terminal oxygen isotope stage 5a, when the climatic cooling leading to the Wisconsinan glacial period had already begun. Fission-track analysis of the enamel and dentine cross sections showed minimal U uptake, resulting in no need for early, linear, or late uptake modelling. The direct ESR age indicates that the specimen either became mired in a cavity that was already at least partly infilled with older, probably waterlogged, sediments, or that the sediment's IRSL signal was not completely erased during deposition. The dosimetric ages are consistent with limiting radiocarbon ages on bone collagen (juvenile >45,760 years BP; adult ⩾37,040±1730 BP) and wood (near the adult mastodon >51,000 BP; organic-rich horizon above the karst surface >50,000 BP). These results provide the first direct age on a mastodon fossil from Atlantic Canada, and the first numeric estimate to demonstrate that mammoths were present in this region during pre-Wisconsinan times, specifically during late oxygen isotope stage 5a.

We report direct luminescence ages for the culture-bearing sediments of the Kebaran site of Nahal Hadera V (NHV) in the coastal plain of Israel. Although the site contains, in addition to rich lithic deposits, plentiful mammalian bone, it has proved to be undatable using radiocarbon dating, in spite of the fact that the cultural context places the time of occupation well within the range of radiocarbon dating. In contrast, luminescence dating of the site sediments proved successful. Luminescence ages were determined using the single aliquot additive-dose (SAA) method, applied to sand-sized quartz extracts to determine past equivalent doses (De). Dose rates (R) were calculated using thick source alpha counting for the uranium (U) and thorium (Th) concentrations and x-ray fluorescence analysis for the potassium (K20) concentration. Of the five samples collected at the site, four represent cultural and subcultural deposits and the fifth represents the geological substrate for the archaeological deposit, a quartz-rich, carbonate-cemented dune sand known as aeolianite or kurkar. The luminescence age of the kurkar is 42.7 ± 6.3 ka. Human occupation of the site occurred between 21.3 ka and 14.0 ka ago, during the Last Glacial Maximum. © 2003 Wiley Periodicals, Inc.

Most inland eolian dunes associated with rivers on the Georgia Coastal Plain probably date to glacial periods. Direct dating of the dune sand by optically stimulated luminescence dating, combined with limiting ages from radiocarbon dating, shows that dunes formed during isotope stage 2, stage 3, the transition between stages 4 and 5, and isotope stage 6 or earlier. Most of the dates indicate dune activity between 30,000 and 15,000 years ago. Holocene activity included limited and local reworking of the crests of some thick dunes along streams that flow from the Piedmont.

Using the classic thermoluminescence (TL) dating technique for fired ceramic materials, we have obtained the first direct ages for five ceramic artifacts recovered from BfDa-1, a prehistoric archaeological site located in St. Croix, southcentral Nova Scotia. BfDa-1 is the only archaeological site in Nova Scotia to produce the full range of ceramic styles associated with the Woodland or Ceramic Period (c. 1050 B.C. to A.D. 1500); thus our new chronology can be used to revise the current local and regional ceramic chronologies. The five sherds from the St. Croix site yielded absolute gas of 1.15 ± 0.15 ka to 2.62 ± 0.29 ka (1 ka = 1000 years). The oldest of these TL ages was obtained on a sherd decorated with a pseudoscallop shell design and is in excellent agreement with a 14C date of 2500 ± 120 B.P. associated with Early Ceramic artifacts at this site. The other TL ages are in agreement with the relative ceramic chronology deduced from their decorative attributes. All five TL dates are in the correct depth-age order. This demonstrates that the recent excavations explored a part of the site which remained undisturbed in spite of extensive agricultural and horticultural activities in the vicinity in recent centuries. The dates also indicate a high degree of compression for the cultural remains at the site. They suggest a sediment deposition rate of 1.3 cm/100 yr during the early stages of the occupation period (2000–2600 yr B.P.), and a decrease in the deposition rate to 0.5 cm/100 yr during the last 2000 years. These dates provide the first successful application of TL dating to ceramics of the Minas Basin area. They confirm an initial hypothesis based on the stylistic attributes of ceramics recovered from BfDa-1, that human occupation at the site must have been of long duration. The excellent quality of the analytical data indicates that the TL dating technique should be well suited to the study of ceramic technology in other parts of the province, and that local mineralogy is suitable for optical dating of sediments at the site and elsewhere in Nova Scotia. © 1997 John Wiley & Sons, Inc.

Currently, retrospective biophysical radiation dosimetry lacks a technique that is sensitive, non-invasive, and portable. This has made reliable cause and effect relationships between radiation exposure and its outcomes in humans difficult to establish. Since optical technology is amenable to miniaturization, a search for optically stimulated luminescence in dental enamel was begun. The first successful detection of time dependent optically stimulated luminescence from gamma irradiated enamel was accomplished. This luminescence is absent in enamel that is not irradiated or that was heated following irradiation. Thermoluminescence observations were made concurrently with the optical measurements which clarified the role of the organic component of enamel.

Most studies of T cells have focused on those that respond to foreign peptides. However, other specialized populations of T cells exist that recognize lipid antigens and make up a substantial component of the human immune system. These lipid reactive T cells recognize antigens presented by antigen presentation molecules from the CD1 family. Four CD1 molecules exist (CD1a, CD1b, CD1c and CD1d), and each is capable of presenting a unique repertoire of lipids antigens to T cells. Much of what we have learned about lipid reactive T cells stems from studies of CD1d restricted NKT cells as these are present is both mice and humans and can be detected using CD1d/α-GalCer tetramers. In contrast, our understanding of the biology of CD1a, CD1b, CD1c restricted T cells is relatively limited. However, the recent generation of CD1a, CD1b and CD1c tetramers is helping with the identification and characterisation of these CD1-restricted T cells. We have produced CD1 tetramers loaded with mammalian self-lipids or lipid antigens from Mycobacterium tuberculosis. In conjunction, with a tetramer-based enrichment method, we have successfully identified both autoreactive and microbial lipid antigen specific T cells from healthy human blood. We reveal the phenotypic characteristics of these CD1-restricted T cells and used CD1 mutagenesis to provide new insight into TCR recognition of CD1-lipid antigen complexes. Collectively, these studies will serve as a basis for future studies of lipid reactive T cells in health and disease.

Preheating is the simplest energetically justifiable approach taken to ensure that the charges yielding luminescence have the same activation energies in natural and irradiated aliquots. Luminescence depletion due to preheating of Holocene and Quaternary sediments with natural and artificial doses was examined and found to behave as expected. Preheating was found to have the following undesirable effects. In aliquots with very high doses, preheating lowered the onset of saturation by 20 to 40%. This effect is thought to be due to thermal quenching of recombination centres. In optically zeroed quartz and feldspar aliquots, preheating caused the luminescence signal to increase. In aliquots with geologically very low adsorbed doses, preheating to 230 degrees C caused dose response growth curves to become nonlinear. This effect can be explained in terms of second-order kinetic behaviour. A procedure is suggested which corrects for the undesirable thermal effects in young samples. When verified against thermoluminescence measurements, this procedure yielded correct optical dose estimates for sediments

TCR-beta gene rearrangement or expression is necessary and sufficient for the progression of early alpha beta thymocyte differentiation from the CD3-CD4-CD8- triple negative (TN)3 to the CD4+CD8+ double positive stage. The onset of TCR-beta rearrangement is currently thought to occur gradually. Some thymocytes were reported to be rearranged at the earliest (CD44+CD25-) TN stage, whereas other thymocytes did not initiate TCR-beta rearrangement until the latest (CD44-CD25-) TN stage. Here, we have isolated subsets of TN thymocytes on the basis of surface expression of CD44 and CD25, with c-kit as an additional marker. We present a revised model of early T cell development in which TCR-beta and TCR-gamma rearrangements occur abruptly, at the CD44lowCD25+ c-kitlowTN stage. A high level of c-kit expression defines pro-T cells which have not yet rearranged their TCR genes. Germ-line TCR-beta transcripts, and transcripts of recombination activating genes (RAG)-1 and 2, are detected before TCR-beta gene rearrangement. Analyses of TN thymocytes of RAG-1 mutant mice, and of various TCR mutant and TCR transgenic RAG-1 mutant mice, indicate the existence of a control point at the CD44-CD25+TN stage at which cells expressing a productively rearranged TCR-beta chain are selected for further differentiation.

We determined the suitability of optically stimulated recombination luminescence in quartz for low-level dosimetry of recent events such as radiation accidents. Minimum detectable doses were determined for both fired and unfired quartz. Using a single aliquot technique, good detection of doses on the order of 0.01 Gy in fired quartz was achieved. In unfired quartz, the minimum dose detected was an order of magnitude larger. Using single aliquot optically stimulated regeneration of a natural quartz sample, a dose of 0.18 +/- 0.02 Gy was measured and was in good agreement with the dose determined by multiple-activation thermoluminescence. Delayed measurements of radiation doses on the order of 1 Gy were shown to be feasible in an emergency response situation when measured up to 7 wk following the irradiation event, using a regeneration-ratio technique. Our conclusions are based on observations of 100 degrees C isothermal decay of the luminescence, high-temperature preheating experiments, thermal fading after prolonged room temperature storage, and observations of the relationship between post-annealing residual luminescence and the TL glow curve. We were able to correlate the optically stimulated recombination luminescence of quartz extracted from building tile with the sample's 278 degrees C TL emission.

In many instances, geologically distinct obsidian flows located within even a relatively small geographic area can be uniquely identified by their chemical composition. This happens to be true for several obsidian sources from central Oregon. Internally each obsidian locality is chemically homogeneous, but the obsidian rocks from different collection sites exhibit chemical differences. Based on the geochemical variations and on K/ Ar dating of the end members of the chemical differentiation trend, these differences are related to the fractionation of a single Late Miocene magma chamber, dated at 6.5 Ma. By understanding the underlying causes of the chemical differences, constraints are disclosed that will govern the possible chemical variations of other, as yet unidentified but related obsidian flows. These can be useful for identifying the possible natural sources of obsidian artifacts which do not match known obsidian sources, and for suggesting possible geographic areas where these as yet undiscovered obsidian flows may be found. © 1993 John Wiley & Sons, Inc.

We have subdivided mouse CD4-CD8-CD3- triple-negative (TN) thymocytes into four subsets based upon expression of CD44 and CD25, including CD44+CD25-, CD44+CD25+, CD44-CD25+ and CD44-CD25-. Characterization of these cells revealed several features distinct to each subset, in particular the expression of high levels of c-kit (the receptor for stem cell factor) by CD44+CD25-TN and CD44+CD25+TN but not by CD44-CD25+TN and CD44-CD25-TN. The CD44+CD25+TN subset also included the IL-7 and stem cell factor-responsive cells, whereas only minimal responsiveness was observed by the CD44- populations. These subsets also showed differential cytokine production potential (CD44+CD25- > CD44+CD25+ > CD44-CD25+ > CD44-CD25-) after stimulation with calcium ionophore, PMA and IL-1. The repopulation potential of these subsets in 2-deoxyguanosine-treated fetal thymic lobes supports the following maturation sequence: CD44+CD25- -->CD44+CD25+ -->CD44-CD25+ -->CD44-CD25-. Furthermore, the sequence of progression from CD44+CD25+ to CD44-CD25+ cells was confirmed by their TCR beta-chain gene configuration. The former population exhibits germ-line TCR beta-chain configuration, whereas the latter subset shows a rearranged pattern.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.

NKT cells represent a unique T cell lineage that recognize glycolipid antigens in the context of the non-classical MHC class I molecule CD1d. NKT cells are potent producers of immunoregulatory cytokines, and have been implicated in several different autoimmune diseases in mice and humans, including Type 1 diabetes, experimental autoimmune encephalomyelitis--a mouse model for multiple sclerosis, systemic lupus erythematosus, and scleroderma. This review will cover the evidence for an involvement for NKT cells in these autoimmune diseases, and discuss the potential for therapeutic manipulation of these cells as a means of preventing autoimmune disease in the clinic.

NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.

As a result of failed induction of T cell tolerance to pancreatic B cells, non-obese diabetic (NOD) mice develop spontaneous autoimmune insulin-dependent diabetes mellitus (IDDM). The thymic stroma, which plays a crucial role in thymic T cell maturation, undergoes extensive premature disorganization in NOD mice, so it is of interest to examine NOD T cell development. In this study, both major and minor developmental populations of thymocytes of NOD/Lt mice were studied and compared to those of BALB/c, C57BL/6 and CBA mice by multiparameter flow cytometry (FACS). These results are described in detail and reveal that most thymocyte subsets were normally represented, including alphaTcR-CD4-CD8- (triple negative; TN), alphabetaTcR-CD4+CD8- and alphabetaTcR-CD4-CD8+ (immature single positive; ISP), alphabetaTcR-/lowCD4+CD8+ (double positive; DP) and alphabetaTcR+CD4+CD8- and alphabetaTcR+CD4-CD8+ (mature single positive; SP) as well as gammadelta T cells. However, NOD mice exhibited a marked deficiency of thymic alphabetaTcR+CD4-CD8- (alphabeta+DN) T cells. alphabeta+DN T cells, which are included among NK1+ T cells in C57BL/6 mice, produce large amounts of IL-4 on primary stimulation. Given the potential significance of NKT cells in immunoregulation, it is possible that the scarcity of these cells in NOD mice plays a role in the pathogenesis of IDDM.

IL-12 has been implicated in the maturation and activation of peripheral T lymphocytes and NK cells. In the present study we have investigated the potential role of IL-12 in intrathymic T cell development. Treatment of mouse fetal thymic organ culture with IL-12 caused a significant reduction in size and cell number compared with the untreated controls. Flow cytometric analysis of the thymocytes recovered from these lobes showed differential effects on individual thymocyte subsets, most but not all of which were significantly decreased. In contrast, however, we observed an increase in both the percentage and cell number of alpha beta TCR+CD4-CD8+ thymocytes. This effect could be neutralized with an anti-IL-12 antibody, demonstrating the specificity of the influence of IL-12 in the fetal thymic organ culture system. IL-12 caused proliferation of isolated thymocyte subsets, particularly CD3+CD4-CD8+ cells in the presence of IL-2 and IL-4. Additionally, IL-12 induced significant proliferation of early CD3-CD4-CD8- triple negative CD44+CD25+ pro-T cells in combination with stem cell factor. We show that both the p35 and p40 chains of IL-12 are produced in the fetal and adult mouse thymus and that thymic stromal cells are a potential source of this cytokine.

We have developed a panel of rat mAbs against dibutyryl cAMP-activated 5C2 cells. In this panel, one mAb, 1G10, recognized murine B7. Another mAb designated 2D10 did not bind to murine B7 but could recognize a surface molecule expressed only on dibutyryl cAMP-activated 5C2 mouse B lymphoma cells or on LPS-stimulated splenic B cells. This new molecule is referred to as early T cell costimulatory molecule-1 (ETC-1). From both activated 5C2 cells and splenic B cells, mAb 2D10 immunoprecipitated a 59- to 60-kDa protein, which was different from the 47- to 55-kDa murine B7 protein precipitated from the same cell populations. FACS analysis showed that, in contrast to B7, the expression of ETC-1 on 5C2 cells was induced by lower concentrations of dibutyryl cAMP and displayed a faster kinetics. LPS-stimulated splenic B cells expressed relatively low levels of B7 and much higher levels of ETC-1. Importantly, in an Ag presentation assay using activated 5C2 cells as APC, the secretion of IL-2 by C8A3 T hybrids was partially inhibited by mAb 2D10 alone and completely blocked by combination use of mAbs 2D10 and 1G10 in a dose-dependent and synergistic fashion. In a one-way primary MLR, mAb 2D10 alone at 0.1 to 1 microgram/ml inhibited T cell proliferation by 19 to 56%. However, an additive blocking effect (up to 76%) was observed when two mAbs were added in combination. Thus, our data have demonstrated that a novel T cell costimulatory molecule is present on activated murine B cells, which, in cooperation with B7, may play a critical role in optimal T cell activation.

We have previously characterized a novel mouse thymocyte marker, defined as thymic shared Ag-1 (TSA-1), present on both immature thymocytes and a subset of thymic medullary epithelial cells. MTS 35, a mAb specific for TSA-1, alters T cell differentiation when added to fetal thymic organ cultures, suggesting TSA-1 may be important for T cell development in the thymus. In this study, we describe the isolation of a cDNA encoding TSA-1 using transient expression of COS-7 cells and selection with MTS 35. The predicted amino acid sequence of this cDNA encodes a 15 to 17-kDa protein and the expressed protein is linked to the membrane via a phosphatidylinositol moiety. TSA-1 is transcriptionally active at various levels in all organs examined, suggesting that its role is not solely intrathymic. TSA-1 shares amino acid sequence homology to the mouse Ly6 multigene family, epidermal growth factor-like receptors, and to cobra venom neurotoxin. The Tsa-1 locus is located on chromosome 15 linked to Ly6 on the mouse genome. We also examined the effects of MTS 35 in fetal thymic organ cultures repopulated with two subsets of thymocytes representing defined stages of T cell development. Our results suggest that TSA-1 may play a role during positive selection and the transition from CD4+CD8+ thymocytes to the mature CD4+CD8- and CD4-CD8+ subsets.

Previously, we detailed the characterization of thymic shared Ag-1, a unique marker of immature thymocytes and isolated thymic stromal cells, defined by the mAb MTS 35. In this study, the functional relevance of this molecule to thymopoiesis was investigated by the addition of purified MTS 35 to fetal thymus organ culture. It down-regulated thymic shared Ag-1 expression and dramatically reduced thymocyte cell yield through inhibition of alpha beta-TcR+ T cell differentiation, post CD3-CD4-CD8- triple negative thymocytes. These effects were specific for the mAb MTS 35, because controls, which include both isotype-matched and other lymphostromal mAb, showed no similar effects. These results demonstrate that thymic shared Ag-1 is a functionally important marker of early thymocyte differentiation, particularly with regard to the alpha beta-TcR lineage.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.

The effects of IL-1, IL-2, and a panel of monoclonal antibodies to thymic stroma and/or thymocytes, on T-cell differentiation in murine fetal thymus organ cultures were followed. Day-14 fetal thymic lobes were cultured for up to 12 days in the presence of IL-1 and/or IL-2 at concentrations of 100 U/ml. Development of all the major subpopulations defined by CD4 and CD8 expression was inhibited by IL-2, however the degree of inhibition was greatest for CD4+CD8- and CD4+CD8+ subsets. IL-1 alone caused only minor shifts in the subpopulations, but when added together with IL-2 the inhibitory effects of IL-2 were markedly enhanced. Analyses of subsets of CD4-CD8- cells demonstrated that the inhibition was most dramatic at the IL-2R positive and subsequent stages of CD4-CD8- differentiation. Interestingly, the putative precursors of IL-2R+CD4-CD8- increased in the presence of IL-2. In preliminary studies the organ culture system was used to examine the effects of a panel of antibodies to thymocytes and/or thymic stromal cells. Out of 14 antibodies tested, MTS 35 and MTS 37 have caused relative increases in the CD8 and CD4 single-positives, respectively. Both antibodies also induced increases in the percentages of CD4-CD8- cells and decreases in the percentages of CD4+CD8+ cells.