Thymic Maturation Research Articles

Rapidly expanding exophthalmos: an unusual presentation of small cell lung cancer.

A 61-year-old male presented with a rapidly progressive exophthalmos from small cell lung cancer metastatic to the right orbit. His vision in that eye was 20/200, and his intraocular pressure was 36 mmHg. The orbital metastasis responded dramatically to chemotherapy. One week after starting the chemotherapy the patient did not have exophthalmos, his vision was 20/20, and three weeks later the intraocular pressure was 12 mmHg.

Involvement of major histocompatibility complex products in tolerance induction in the thymus.

KJ23a+ T cell clones, which bear the determinant encoded by the V beta 17a T cell receptor gene segment, frequently recognize IE molecules of various murine H-2 haplotypes. In the presence of IE molecules, thymic maturation of KJ23a+ clones is infrequent. We investigated the basis of this phenomenon by blocking expression of IE molecules with monoclonal anti-IE antibodies in organ cultures of fetal thymus and in neonates from the C57BR/cdJ strain (H-2k, V beta 17a homozygous). Our data support the contention that this process results from deletion of clones with anti-IE reactivity, as functional blocking of the IE molecule results in maturation of IE-reactive clones and increased numbers of KJ23a+ mature cells. In addition, we noted that blocking of functional IE expression in this haplotype permitted development of both CD4+/KJ23a+ and CD8+/KJ23a+ T cells. The CD4+ clones isolated from anti-IE-treated animals were frequently reactive against IEk; we could demonstrate no alloreactivity against B cell or B lymphoma stimulators in the CD4- clones. We conclude that clonal deletion events during thymic development may be initiated by T cell precursor interactions with MHC molecules against which the mature clones display no measurable reactivity. Specifically, clones destined to be MHC Class I-reactive may be deleted during development by interactions with MHC Class II molecules.

Molecular and functional properties of novel T cell subsets in C3H-gld/gld and nude mice. Implications for thymic and extrathymic maturation.

Immunological ReviewsVolume 104, Issue 1 p. 121-155 Molecular and Functional Properties of Novel T Cell Subsets in C3H-gld/gld and Nude Mice. Implications for Thymic and Extrathymic Maturation Katsuyuki Yui, Katsuyuki Yui Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorScott Wadsworth, Scott Wadsworth Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorAmy Yellen, Amy Yellen Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorYasuhiro Hashimoto, Yasuhiro Hashimoto Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorYasuo Kokai, Yasuo Kokai Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorMark I. Greene, Mark I. Greene Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this author Katsuyuki Yui, Katsuyuki Yui Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorScott Wadsworth, Scott Wadsworth Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorAmy Yellen, Amy Yellen Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorYasuhiro Hashimoto, Yasuhiro Hashimoto Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorYasuo Kokai, Yasuo Kokai Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this authorMark I. Greene, Mark I. Greene Rm252, John Morgan Bldg., Division of Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th & Hamilton Walk, Philadelphia. PA 19104-6082, U.S.A.Search for more papers by this author First published: August 1988 https://doi.org/10.1111/j.1600-065X.1988.tb00761.xCitations: 19AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume104, Issue1August 1988Pages 121-155 RelatedInformation

Defective thymic education of L3T4+ T helper cell function in graft-vs-host mice.

Our study investigates the effect of a prior graft-vs-host (GVH) reaction on the subsequent ability of irradiated, bone marrow-re-populated mice to develop T cell function. The results indicate that such GVH-bone marrow transplanted (BMT) mice do not generate CTL responses to trinitrophenyl-modified syngeneic cells (TNP-self), but do generate strong CTL activity to H-2 alloantigens. This selective deficiency in TNP-self CTL response potential appeared as early as 10 days after GVH, and required both L3T4+ and Lyt-2+ donor T cells. The in vitro addition of either soluble Th factors or L3T4-enriched spleen cells from normal mice circumvented the defect in the TNP-self response in GVH-BMT mice. These results indicate that T effector function was not defective, and instead suggest a Th defect. Cell depletion and antibody-blocking, as well as IL-2 production experiments, indicate that the Th defect was selective for L3T4+ Th population and not for Lyt-2+ Th population. This defect in L3T4 Th function is not accounted for by the approximate twofold reduction in L3T4 cell numbers in GVH-BMT mice, because IL-2 production and CTL generation to L3T4-dependent Ag were at least eightfold below control levels. Rather, defective L3T4 Th function appears to be the consequence of a GVH-induced defect in thymic maturation because the defect was corrected in vivo by a neonatal parental thymus graft before irradiation and bone marrow transplantation. This system may be useful for elucidating the role of the thymus in the maturation of Th cells. Our findings raise the possibility that impaired development of T cell function occurring in marrow grafted patients who have undergone a GVH reaction could be partly due to a GVH-induced thymic defect.

Correlated expression of surface antigens in human thymocytes. Evidence of class I HLA modulation in thymic maturation.

The correlated expression of several surface antigens (CD 1, class I HLA, CD3) was examined in human unfractionated thymocytes or selected subsets by using single or double-color flow cytometry. Prethymocyte subpopulations expressed high levels of HLA. A high proportion of cortical cells expressed low levels of either HLA or CD 3 antigens. Most of these HLA+ cortical cells corresponded to the more immature cells and did not express HLA-B loci products. CD 1 a and CD 3 antigens were expressed in a high percentage of cells and the levels of expression of each antigen in individual cells were inversely correlated. These data and the contour of double-color histograms are suggestive of the existence of a single pathway of thymocyte differentiation in which class I HLA expression is switched off around the time of the initiation of CD 3 (and Ti?) expression. We suggest that the anti-major histocompatibility complex (MHC) specificity of the unselected Ti receptor may be incompatible with the expression of MHC products on the cell membrane. At this stage, CD 1 antigens, whose expression is inversely correlated with that of HLA, may fulfill the role of MHC antigens. The latter can be re-expressed later on, once the anti-MHC specificities of the Ti receptors have been selected against. Studies on in vitro modulation of HLA molecules by interferon-alpha did not reveal any correlation to the expression of CD 1 or CD 3 antigens.

Expression of MHC class I and class I-like gene products on the cell membrane of mature and immature T cells

Beta2-microglobulin (β2m) constitutes the 12 kD light chain of the MHC-encoded 43kD glycoprotein HLA-ABC molecules, which are expressed on most nucleated cells. The T6 (CDla) antigen is expressed on thymocytes in reciprocal manner to HLA-ABC, having a similar structure to the HLA-ABC molecule. We have determined the expression of β2m, HLA-ABC and the T6 antigen on the cell-surface of CD1a + and CD1a − thymocytes (defined by NA1/34 expression), and have shown that despite immunophenotypic differences between these two stages of thymic maturation, the quantity of β2m-associated molecules expressed was not significantly different. The nature of the heavy (α) chain associated with β2m, however, differed since HLA-ABC had largely replaced T6 on the surface of CD1a − thymocytes. We also determined the expression of β2m and HLA-ABC on the surface of peripheral blood CD4 + and CD8 + T- cells, and showed that the CD8 + population expressed higher levels of these antigens than CD4 + T- cells, with no detectable excess β2m over HLA-ABC for either subpopulation. In addition, thymocytes expressed fewer β2m-associated determinants than peripheral blood T-cells. These results indicate an increase in the expression of β2m-associated molecules with differentiation from thymic to peripheral T-cell, with a further increase in such molecules expressed on the CD8 + compared to the CD4 + peripheral T-cell subpopulation.

Changes in the cholinergic system of lymphocytes due to mitogenic stimulation

A significant increase in acetylcholinesterase (AChE) activity and muscarinic-type cholinergic receptor (mAChR) expression was demonstrated in normal human peripheral blood lymphocytes as an early response to various stimuli. AChE activity of lymphocytes from T-cell acute lymphoid leukemia patients, and that of leukemic cell lines was enhanced or increased only slightly, in good accordance with their significantly decreased response to mitogenic stimuli. The higher initial AChE activity of cultured leukemic cell lines probably reflects their permanently activated state. AChE activity and mAChR expression in these cases could only be increased by a differentiation-inducing agent. Based on these observations, the possible role of the cholinergic modulation in thymic lymphocyte maturation is assumed.

T cell rearranging gene gamma: diversity and mRNA expression in fresh cells from T cell acute lymphoblastic leukemia

Rearrangement and in most cases expression of the T cell rearranging genes gamma (TRG gamma) and T cell antigen receptor beta chain (TCR beta) genes were studied in 19 cases of T cell acute malignancies where the surface phenotype is representative of the different stages of thymic maturation. TCR alpha gene transcription was also studied. TRG gamma and TCR beta genes were found to be rearranged in all but one case. The TRG gamma rearrangement pattern seen in most cases is compatible with biallelic rearrangement by loop excision involving the J gamma 2 regions. The sizes of all but two rearranged bands were identical to those of the rearranged bands seen in polyclonal T lymphocytes also studied in this work. One identical-sized band was found in 11 of the 18 rearranged cases. The expression of TRG gamma mRNA (transcripts of 1.6 kilobases [kb]) was highly variable from case to case and did not correlate with the stage of differentiation of the malignant cells, the expression of the molecules CD4 and CD8, the expression and size of the transcripts of the TCR beta genes, and the transcription of TCR alpha genes. In one CD3 + case, strong expression of the TRG gamma transcripts coexisted with the exclusive presence of TCR beta mRNA of 1.0 kb. The cells from this case did not react with anti-Ti antibody and exhibited no natural killer activity. These findings are suggestive of a malignancy that may express the recently isolated CD3-TRG gamma complex.

T Cell Rearranging Gene γ: Diversity and mRNA Expression in Fresh Cells From T Cell Acute Lymphoblastic Leukemia

Rearrangement and in most cases expression of the T cell rearranging genes gamma (TRG gamma) and T cell antigen receptor beta chain (TCR beta) genes were studied in 19 cases of T cell acute malignancies where the surface phenotype is representative of the different stages of thymic maturation. TCR alpha gene transcription was also studied. TRG gamma and TCR beta genes were found to be rearranged in all but one case. The TRG gamma rearrangement pattern seen in most cases is compatible with biallelic rearrangement by loop excision involving the J gamma 2 regions. The sizes of all but two rearranged bands were identical to those of the rearranged bands seen in polyclonal T lymphocytes also studied in this work. One identical-sized band was found in 11 of the 18 rearranged cases. The expression of TRG gamma mRNA (transcripts of 1.6 kilobases [kb]) was highly variable from case to case and did not correlate with the stage of differentiation of the malignant cells, the expression of the molecules CD4 and CD8, the expression and size of the transcripts of the TCR beta genes, and the transcription of TCR alpha genes. In one CD3 + case, strong expression of the TRG gamma transcripts coexisted with the exclusive presence of TCR beta mRNA of 1.0 kb. The cells from this case did not react with anti-Ti antibody and exhibited no natural killer activity. These findings are suggestive of a malignancy that may express the recently isolated CD3-TRG gamma complex.

CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes.

In this study data are presented indicating that the molecule identified by the recently described CD43 cluster of monoclonal antibodies (mAb; Oxford, 1986) is the human analogue to the one originally described in rat as leukocyte sialoglycoprotein (LSGP). This conclusion is based on several criteria. Both molecular mass (105 kDa) and cellular distribution are similar to the antigens previously described in the rat with the mAb W3/13 and subsequently in humans with L10 mAb. Sialidase treatment of the molecule immunoprecipitated by 84-3C1 mAb (CD43) resulted in a decreased electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. L10 mAb inhibited the binding of 84-3C1 mAb to the cell membrane suggesting that both mAb recognized the same molecule. Moreover, the presence in the CD3-4-8- thymic populations of the sialidase-sensitive epitope recognized by mAb 84-3C1 suggests that there is no simple correlation between the thymic maturation and the degree of sialylation.

Murine T-cell receptor genes.

The recent identification of the molecules which govern T cell specificity and the genes which encode them (as reviewed in references 1–4) has now provided a strong structural basis from which to study the phenomena of T-cell recognition and differentiation. Major issues which are now approachable include the precise nature of the T cell receptor-MHC-antigen interactions and the purposes and mechanics of thymic maturation. A third nagging question has to do with the role (or absence of a role) of the γ chain in any of the above processes. After a brief review of current molecular genetic data, we will discuss our approaches to some of these issues.

Enhanced mitogenic reactivity of human T cells after passage through the thymus. Response to phytohemagglutinin and mercuric chloride.

Lymphocytes in blood samples taken from the thymic, brachiocephalic and internal jugular vein of children undergoing thoracic surgery were compared regarding their phenotypic markers and their proliferative response to mercuric chloride (HgCl2) and phytohemagglutinin (PHA). Of these two mitogens, PHA stimulated the DNA synthesis of the lymphocytes from the thymic vein to a significantly higher degree than those taken from the brachiocephalic or internal jugular vein. The response to HgCl2 resulted in the highest stimulation of the DNA synthesis in cells from the thymic and the brachiocephalic veins, though the differences were not significant. The higher reactivity to PHA and the tendency to higher reactivity to HgCl2 of lymphocytes taken from efferent thymic blood might be due to a higher frequency of T cells. This, however, could not be verified by immunohistochemical staining, as Leu4-positive cells occurred in the same proportion in all three venous samples. We therefore suggest that the passage through the thymus of circulating T cells may increase their immune reactivity by the influence of thymic maturation hormones.

Correlation of cell surface antigen expression on human thymocytes by multi-color flow cytometric analysis: implications for differentiation.

Thymocytes undergo a complex series of phenotypic and genotypic changes during maturation in the thymus. This dynamic process involves qualitative and quantitative changes in the expression of certain cell surface differentiation antigens. In this study, we have directly examined the relationship of T cell differentiation antigen expression on normal human thymocytes by using multi-color immunofluorescence and multi-parameter flow cytometric analysis. The results from these studies have provided new insights into the complexity of antigen expression during thymic maturation and suggest that the CD3/T cell antigen receptor complex is expressed early in the development of thymocytes. Direct quantitative measurements of antigen expression by using multi-parameter flow cytometric analysis also suggest quantitative co-regulation of certain antigens (e.g., CD3 and CD5) during thymic maturation.

Characterization of a monoclonal antibody, RTE-21, that binds to keratohyalin granule-associated proteins in epithelial cells of human skin and thymus

The role of skin and thymic epithelium in the promotion of T-cell activation and maturation is currently an area of great interest. In thymus. epithelium is located in the cortex, medulla, and in medullary epithelial swirls called Hassall's bodies. During studies of antigens shared by skin and thymic epithelial cells, we produced a murine monoclonal antibody (RTE-21) raised against the rat thymic epithelial cells line, IT26R21, that identified an antigen present in terminally differentiated epithelium of normal human skin and thymus. In indirect immunofluorescence studies, antibody RTE-21 identified cytoplasmic granules located in the stratum granulosum of normal human skin. Hassall's bodies of human thymus, and in a subset of cells of the IT26R21 rat thymic epithelial cell line. Moreover, the granular reactivity pattern of antibody RTE-21 in the stratum granulosum could be extracted by 1 M potassium phosphate (1 M KPO 4). A 1 M KPO 4 extract of epidermis containing keratohyalin granule proteins was dialyzed against distilled water, solubilized in reduced sample buffer and subjected to polyacrylamide gel electrophoresis and Western immunoblot analysis. In this assay, antibody RTE-21 recognized proteins of 70, 36, 34, and 30 kDa. Using antibody RTE-21 and indirect immunofluorescence, we demonstrated that keratohyalin granules in human thumus were localized exclusively to Hassall's bodies. These data support the notion that human thymic Hassall's bodies result from terminal differentiation of medullary thymic epithelium. Thus, antibody RTE-21 should be an important probe for the study of skin and thymic epithelial cell maturation in vitro and in vivo.

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.

Human T cell antigen expression during the early stages of fetal thymic maturation.

Human thymus tissue was examined from 7 wk of gestation through birth for the expression of antigens reacting with a panel of anti-T cell monoclonal antibodies. Additionally, the reactivities of reagents against the transferrin receptor, against leukocytes, against low m. w. keratins, and against major histocompatibility complex antigens were studied on human fetal thymic tissue. Frozen tissue sections were evaluated by using indirect immunofluorescence assays. At 7 wk of gestation, no lymphoid cells were identified within the epithelial thymic rudiment; however, lymphoid cells reacting with both antibody 3A1, a pan T cell marker, and antibody T200, a pan leukocyte reagent, were identified in perithymic mesenchyme. After lymphoid colonization of the thymic rudiment at 10 wk of fetal gestation, fetal thymic tissue reacted with antibodies T1, T4, and T8. At 12 wk of gestation, antibodies T3, T6, A1G3 (anti-p80, a marker of mature thymocytes), and 35.1 (anti-E rosette receptor) all reacted with thymic tissue. Our findings indicate that T cell antigens were acquired sequentially on thymocytes at discrete stages during the first trimester of human fetal development. The 3A1 antigen was present on fetal lymphocytes before lymphoid cell colonization of thymic epithelium, suggesting that passage through the thymus was not required for the expression of the 3A1 antigen by T cell precursors. The appearance of mature T cell antigens, T3 and p80, on thymocytes by 12 wk of gestation implies that the T cell antigen repertoire may be established in the thymus during the first trimester. Thus, a critical period of T cell maturation appears to occur between 7 and 12 wk of human fetal gestation.

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts.

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.

Interleukin 2-dependent natural killer (NK) cell lines from patients with primary T cell immunodeficiencies.

Bulk cultured cell lines with natural killer (NK) activity were derived by in vitro culture with interleukin 2-containing conditioned medium (IL 2-CM) of peripheral blood mononuclear leukocytes (PBL) from patients with primary T cell deficiencies. Lines were developed from three patients with severe combined immunodeficiency (SCID) and one patient with Nezelof's syndrome and contained several populations of cells with distinct phenotypes. All lines contained a cell population expressing the Leu-5 (50K) (sheep red blood cell receptor), 3A1 (40K), and OKT10 antigens, but lacking the pan T cell antigens Leu-1 (67K) and Leu-4 (19K) as well as the markers of T cell subsets Leu-2a (32K) and Leu-3a (56K). These cells failed to express the Leu-7 antigen and only weakly expressed OKM1. In addition, one line contained a population of Leu-5+, 3A1+, OKT10+, Leu-2a+, Leu 1-, and Leu 4- cells. Three of the lines also contained populations with classic T cell (Leu-1 and-Leu 4+) phenotypes. The lines were enriched in NK activity compared with the PBL from which they were derived. Their growth was strictly dependent on IL 2-CM. Highly purified IL 2, lacking any other detectable protein contaminants or lymphokine activities, was capable of supporting the growth of the Leu-5+, 3A1+ "null" cell populations from these lines without alteration in their functional activity or phenotype. Thus, studies of in vitro expanded cell lines from patients with severe disorders of T cell function and thymic involution indicate that this "null" cell population does not require thymic maturation to develop its effector function. This "null" cell population can be maintained in vitro in the presence of IL 2. This finding is analogous to the data obtained from study of NK cells in athymic (nude) mice.

Biosynthesis of glycosaminoglycans by epithelial and lymphocytic components of murine thymus.

Interactions between the epithelial and lymphocytic components of the thymus are required for T cell maturation, yet the molecular bases for these interactions remain elusive. In the development and function of other endodermally derived organs, glycosaminoglycan-containing proteins are known to play a critical role. In contrast, virtually nothing is known about the macromolecules that are major constituents of thymic interstitial spaces. For these reasons, we undertook metabolic labeling studies in vitro with D-(6-3H)glucosamine and 35SO4(-2) to begin to characterize systematically the relative amounts and types of glycosaminoglycans made by enriched subpopulations of cells within the thymus. Hydrocortisone, which depletes the thymus of 90% of its lymphocytes, was used both to enrich for epithelium-derived glycoconjugates and to determine if significant alterations in glycoconjugate metabolism accompany drug-induced premature thymic involution. Results indicate: 1) glycosaminoglycans account for a substantial proportion of the total glycoconjugates synthesized by both thymocytes and epithelium; 2) Glycosaminoglycans show a tissue-specific distribution. Hyaluronic acid is the major glycosaminoglycan synthesized by thymic epithelium, whereas it accounts for less than 15% of the total glycosaminoglycans made by thymocytes; 3) Similar proportions of sulfated glycosaminoglycans are made by thymic epithelium and thymocytes. Chondroitin sulfates predominate (75 to 90%) over heparan sulfates (10 to 25%). Chondroitin sulfates from both nonstimulated thymocytes and epithelium are nearly exclusively sulfated at the 4-position of their N-acetylgalactosamine residues; 4) The major high m.w. glycoconjugate of thymocytes, however, is nonsulfated and is resistant to pronase, hyaluronidase, chondroitinase ABC, nitrous acid, keratanase, and neuraminidase; 5) Although hydrocortisone treatment causes a dramatic inhibitory effect on the incorporation of radioactivity into smaller oligosaccharide side-chains by "cortisone-resistant" thymocytes, the drug exerts negligible effects on the metabolism of glycoconjugates by epithelium. These data, which quantify and categorize the complex arrays of glycoconjugates synthesized by the major cell types of the thymus, establish the necessary foundations for further investigations into the functional roles of these glycoconjugates in thymic epithelium-induced maturation of T lymphocytes.

Demonstration of abnormalities in expression of thymic epithelial surface antigens in severe cellular immunodeficiency diseases.

Thymic epithelium from three patients with severe cellular immunodeficiency diseases were compared with age-matched normal thymic epithelium using three markers of human thymic epithelium and antibodies against thymosin alpha 1, thymopoietin, and thymosin beta 4. We have previously shown that normal thymic epithelium reacts with antibodies against GQ gangliosides (antibody A2B5) and binds tetanus toxin (TT). In addition, some areas of normal thymic epithelium express human Thy-1 antigen. We found thymic epithelium in patients with severe cellular immunodeficiency diseases to be different from normal subjects. Two children with severe combined immunodeficiency disease (SCID) had thymic epithelium that bound anti-GQ ganglioside antibody but, unlike in normals, did not bind TT. The patient with severe cellular immunodeficiency and normal serum immunoglobulins (Nezelof syndrome) had thymic epithelium that bound TT but, unlike normal thymic epithelium, did not react with anti-GQ ganglioside antibody. Thymic epithelium from both SCID and Nezelof syndrome patients contained thymosin alpha 1, thymopoietin, and thymosin beta 4 and expressed human Thy-1 antigen. In contrast to SCID thymus rudiments, Nezelof thymus contained numerous (though fewer than normal) lymphocytes with mature T cell surface antigens. Thus, using these probes of human thymic epithelium, we have demonstrated heterogeneous defects in thymic epithelial surface marker expression in severe primary cellular immunodeficiency diseases. These defects presumably reflect abnormalities of in vivo thymic epithelial maturation.