The antigen-inexperienced thymic suppressor cells: a class of lymphocytes in the young chicken thymus that inhibits antibody production and cell-mediated immune responses.

Transfer of thymus cells from young chickens in combination with a light whole body irradiation (360 R) was found to suppress the rejection of skin grafts across strong histocompatibility (B) differences. On the average, the suppressed animals also showed decreased serum hemagglutinin titers against erythrocytes of the skin donor strain and a decreased graft-versus-host (GvH) reactivity against embryos of this strain. The thymic suppressor cells can be obtained from animals that have not experienced the antigen under test. However, after transfer and contact to the antigen (skin graft) they can lead to the formation of specific ("activated") suppressor cells and can mediate in the long run a specific inhibition of the response to this antigen. The suppressive activity is associated with a bursa-dependent cellular subpopulation in the thymus that is different from B lymphocytes, B precursor cells or GvH-reactive T cells. The bursa dependency of the thymic suppressor cell suggests that functionally different lineages of thymic and thymus-derived lymphocytes are derived from different sources of prethymic stem cells. The suppressor cells are predominantly found in the young chicken thymus and already detectable in the 16-day-old embryo, while poor suppressive activity is found in the adult thymus. The suppressive effect can be obtained with thymus cells from either syngeneic or allogeneic donors. Embryonic allogeneic donors provide suppressive cell preparations free of GvH reactivity. The possibility that the thymus suppressor cells mediate self tolerance and "neonatal tolerance" is discussed.     

Age-related changes of thymus--morphological and functional aspects.

The thymus involutes progressively throughout life, beginning at around the sexual maturation. In long-lived BC3F1 hybrid mice, the thymic capacity to induce T cell differentiation begins to decline earlier than the onset of thymic involution, although the magnitude of the decline is different by the subpopulation of T cells. Morphologically, the most active secretory structure seems to be limited exclusively to the neonatal thymus and certain structural changes, reflective of a decline in secretory function, can be detected early in life and they become more pronounced with age. Heterogeneity of thymic epithelial cells is suggested by the facts that age-related and radiation induced decline of immune activities are different in degree by subpopulation of T cells, and the concept is also supported from a morphological viewpoint. It is thus apparent that age-related thymic involution results in decrease of recruitment of fresh capable T cells and increase of old exhausted T cells, eventually bringing about T cell insufficiency in the aged individuals. Such an impaired immune function in the aged mice can be effectively restored by the combined grafting of young bone marrow and newborn thymus, and the thymus is apparently the most limiting factor in the aged. The biological signficance of age-related thymic involution is also discussed.     

AGE-RELATED CHANGES OF THYMUS – MORPHOLOGICAL and FUNCTIONAL ASPECTS –

The thymus involutes progressively throughout life, beginning at around the sexual maturation. In long-lived BG3F₁ hybrid mice, the thymic capacity to induce T cell differentiation begins to decline earlier than the onset of thymic involution, although the magnitude of the decline is different by the subpopulation of T cells. Morphologically, the most active secretory structure seems to be limited exclusively to the neonatal thymus and certain structural changes, reflective of a decline in secretory function, can be detected early in life and they become more pronounced with age. Heterogeneity of thymic epithelial cells is suggested by the facts that age-related and radiation induced decline of immune activites are different in degree by subpopulation of T cells, and the concept is also supported from a morphological viewpoint. It is thus apparent that age-related thymic involution results in decrease of recruitment of fresh capable T cells and increase of old exhausted T cells, eventually bringing about T cell insufficiency in the aged individuals. Such an impaired immune function in the aged mice can be effectively restored by the combined grafting of young bone marrow and newborn thymus, and the thymus is apparently the most limiting factor in the aged. The biological significance of age-related thymic involution is also discussed.     

Cellular heterogeneity in the thymus. I. Electrophoretic analysis of lymphoid cell populations from chickens

Micro-cell-electrophoresis has been applied to demonstrate differences and heterogeneities in various lymphocyte populations. The lymphocytes from the blood, spleen, bursa and thymus of young chickens differ significantly in their electrophoretic mobility (EM) pattern. The blood and spleen lymphocytes appear to be homogeneous by this criteria; bursa cells show slight heterogeneity, and thymus lymphocytes contain two clearly distinct subpopulations. One of the two thymocyte subpopulations is absent in neonatally bursectomized and irradiated animals. This bursa-dependent cellular component in the thymus has about the same EM as bursa cells themselves, which may suggest that these cells are, as such, derived from the bursa. The possible role of the bursa-dependent subpopulation is discussed. Within the first months of life, there is a dramatic shift from the predominance of the electrophoretically slower population to its virtually complete disappearence and the subsequent dominance of the other cellular component. Bursectomy plus irradiation provided pure preparations of one of the thymus subpopulations. The other subpopulation could be highly enriched by differential centrifugation in chicken plasma. Both subpopulations are thus separately available for direct immunological tests. The reduction of the EM by neuraminidase show that sialic acid is a major charged component in the cell surface of all lymphocyte preparations tested. The data indicate also that the different EM of the two thymocyte subpopulations is not due to different amounts of neuraminidase labile groups on the cell surface. The high EM of blood lymphocytes, on the other hand, is probably to be explained by a high content of neuraminidase-labile groups, as well as some ribonuclease sensitive material. A substantial reduction of the EM by ribonuclease was observed on blood lymphocytes, but not on lymphocytes from other organs.     

Thymic stromal alterations in mice undergoing a chronic graft-versus-host reaction.

Graft-versus-host induced immunosuppression has previously been shown to be accompanied by severe morphological changes in the thymus; furthermore chronic GvH could become acute by grafting a normal syngeneic thymus, suggesting a functional defect in the autologous thymus. In this work, we monitored the changes occuring in two biologically active thymic stromal fractions during a state of chronic GvH reaction. It was thus observed that a soluble thymic factor (STF), normally found in the reticuloepithelial cells, was lost, and that an insoluble thymic fraction (ITF) found in a double basement membrane surrounding medullary blood vessels, became markedly hypertrophied. These changes are interpreted as being possibly related to the state of immunosuppression by interfering with normal T cell differentiation and traffic through the thymus.     

Characterization of B cells in human thymus.

The surface characteristics of B cells present in the human thymus were investigated. Cytofluorometrical and immunohistological studies, using anti-human IgM or anti-B cell monoclonal antibodies (mAbs; anti-Leu 12Ab, anti-Leu 16Ab, or L26), revealed that a small number of B cells are present in the human thymus. The thymic B cells were detected only in a low-density cell population, whereas in a high-density cell fraction, only T cells were found. In 15 cases, all of which the thymi were histologically normal, the percentages of B cells in the low-density fraction were 0.28% to 50% (6.8% in average), and Leu 1+ (CD5+) B cells in the low-density fraction were 0.1% to 26% (3.5% in average); approximately 50% of the thymic B cells were Leu 1+ B cells. These results indicate that B cells, especially Leu 1 (CD5)+ B cells, are also present in the human thymus, as suggested from our previous reports on mice.     

Bursa-dependent subpopulations of peripheral B lymphocytes in chicken blood

By selective labeling of juvenile chicken bursal cells with colloidal fluorescein isothiocyanate in situ, the emigration rate of bursal lymphocytes to the periphery was estimated at approximately 0.84% and 0.96% of the peripheral blood lymphocyte (PBL) and splenic B cell pool per hour, respectively. Emigrant bursal cells were found primarily in blood and spleen, with very small numbers migrating to thymus, bone marrow, and gut-associated lymphoid tissues. Emigrant bursal cells expressed high levels of both major histocompatibility complex class II antigen and the Ov alloantigen, a phenotype found on a population comprising approximately 4% of bursal cells from which the bursal emigrants may be derived. Surgical bursectomy at 3 weeks of age revealed that peripheral blood B cells could be divided into three distinct populations. Specifically, 60% of the peripheral blood B cells were short lived with a half-life of about 30 h in the blood. These cells accounted for the great majority of emigrants from the bursa to the peripheral blood. Approximately 35 % of PBL B cells had a half-life of 12 days following bursectomy and comprised cells which did not divide in the periphery. Consequently, we propose that physiological differences between this population and the majority of bursal emigrants are established intrabursally. The remaining PBL B cells, whose relative proportion increases with age from about 5 % of PBL B cells at 2-3 weeks of age, are short lived and are being continually produced from (a) post-bursal site(s) of B cell production.     

Thymic epithelial injury in graft-versus-host reactions following adrenalectomy.

In four separate experiments 140 adults A(H-2a) x C57BL/6(H-2b) F1hybrid mice were surgically adrenalectomized and divided into three experimental groups. Seventy-one additional adult F1hybrids (AXC57BL/6) which had not been adrenalectomized were divided into three similar groups. In Group 1 (GvH group), GvH reactions were induced by the injection of 50 x 106 pooled parental lymphoid cells intravenously. The second group (syngeneic group) received 50 x 106 pooled F1 hybrid lymphoid cells intravenously. The third group (uninoculated group) received no lymphoid inoculum. At regular intervals the animals were killed, autopsied, and histologically studied. Visceral alterations of GvH reaction were recorded in the thymus, lymph nodes, spleen, and liver in the GvH groups; none was present in the other groups. The thymuses in the nonadrenalectomized GvH group underwent prompt involution characterized by size reduction and cortical lymphoid cell depletion. These changes were not apparent in the GvH adrenalectomized group. Both GvH groups, however, demonstrated an effacement of the medulla, lymphocyte incursion into the medulla, lymphocyte emperipolesis of medullary epithelial cells, gradual disappearance of Hassall's corpuscles, epithelial cell injury, and an ingress of macrophages laden with nuclear and cellular debris. This study suggests that the stress and corticosteroid response which accompany a GvH reaction account for the reduction in the thymic size and cortical lymphoid cell mass. The medullary alterations, therefore, would appear to be initiated by the GvH reaction per se.     

Immunofluorescent detection of surface antigens specific to T and B lymphocytes in the chicken

Rabbit antisera specific for chicken T and B cells as judged by surface immunofluorescent staining have been raised. Specificity was established by the staining of thymus and bursal cell suspension and by the effects of thymectomy and bursectomy on the staining of peripheralized lymphocytes. Furthermore, double labeling experiments showed that anti-T and anti-B sera reacted with different populations of blood lymphocytes. Comparable numbers of cells in blood and spleen stained for B and light chain determinants. No evidence for “null cells” was obtained. There was little change in the percentage of cells staining in the various lymphoid organs from 4 days to 12 months of age. The thymus contained approximately 7 % B cells, although no T cells were demonstrable in the bursa. One antiserum showed only thymocyte specific antibodies not reacting with peripheral T cells. The specific B and T markers seem to be acquired during differentiation within the appropriate central lymphoid organ. Demonstrable surface immunoglobulins appear later in ontogeny than the B antigens. The majority of cells bearing the B marker in bone marrow were large cells lacking surface light chain determinants.     

Thymic export in aged sheep: a continuous role for the thymus throughout pre- and postnatal life

A diverse repertoire among peripheral T cells is established in early life by thymic export when the naive T cell pool is first formed. In contrast, during adult life the thymus has been thought to play only a minor role in T cell homeostasis. As individuals age there is an increasing proportion of peripheral T cells bearing a memory phenotype, as well as a corresponding decrease in the number of naive T cells. The change in the composition of the peripheral T cell pool with age is thought to occur as a result of reduced or completely curtailed thymic export following thymic involution at puberty together with the antigen-driven expansion of naive T cells in the periphery. We examined thymic export throughout life in fetal, neonatal and aged sheep. We found that the thymus in adult animals showed an efficiency of production and export on a per gram basis equivalent to that observed for much younger animals, and continued to export substantial numbers of T cells long after puberty. The data demonstrate that naive T cells constantly enter the peripheral T cell pool at the same rate throughout fetal, neonatal and adult life, and that one in every 50 T cells in the peripheral lymphoid tissues of aged sheep had emigrated from the thymus in the previous 24 h. The data suggest that restoration by the thymus of a normal peripheral T cell repertoire in chronic T cell-depleting conditions should be possible in adult patients, provided the thymus is not damaged by disease or therapy.     

The Host Component of the Popliteal Lymph Node Graft-versus-Host Reaction

We have examined the cellular changes taking place in rat popliteal lymph nodes undergoing a graft-versus-host (GvH) reaction. Examination of immunoperoxidase-stained lymph node sections, using a panel of mouse monoclonal antibodies directed against different rat lymphoid cell subsets, revealed a disorganization of the lymph node architecture with disappearance of the follicles, and an intermingling of T and B cells, so that no distinct T- and B-cell areas were visible any more. Since the GvH nodes showed a preferential accumulation of host B cells over host T cells (particularly over the W 3/25+ T helper cell subset), we also investigated the requirements for host B cell activation. The popliteal lymph node GvH reaction was induced in (PVG X DA)F1 rats by the injection of PVG cells into one foot and by DA cells into the other foot, and then immunoglobulin kappa allotype marked PVG B cells from athymic donors were injected intravenously. The allotype marked B cells proliferated vigorously in response to the DA T cells, but much less in response to the PVG T cells. These results indicate that the massive B-cell activation taking place in GvH reactions may require an alloantigen incompatibility between donor T cells and host B cells, and argue against non-specific mitogenic induction of the B cells.     

The thymus is a site of mast cell development in chicken embryos

Thymic mast cells were studied by light and transmission electron microscopy in chicken embryos during organogenesis. Mast cells made their first appearance at day 15. At days 16 and 17, there was a burst of mast cell development with a peak of 278 ± 54 cells/mm² at day 16. Then, mast cell density decreased until hatching. During the whole embryonic period, about 80% of mast cells localized to the thymic medulla. In the cortex, they were less numerous, and some rare mast cells could be identified in the capsule and septa. Thymic mast cells could be recognized in association with hematopoietic foci, but frequently they grew independently from areas of hematopoiesis and appeared as single cells interspersed among thymocytes, thymic epithelial cells, and interdigitating cells. They were often recognized in close relationship with the scanty and delicate extracellular matrix of the developing gland. Viewed by electron microscopy, mast cells were relatively small cells, with a few secretory granules. Exocytosis was never seen, but, notably, granules emptied in a piecemeal degranulation fashion. This study demonstrates that the chicken thymus is a site of mast cell development during embryogenesis. The high mast cell density we found suggests a possible role for these cells during thymus organogenesis.     

The ontogeny of B cells in the thymus of normal, CD3 epsilon knockout (KO), RAG-2 KO and IL-7 transgenic mice.

The ontogeny of thymic B cells was determined by three-color flow cytometry and the presence or absence of B cell progenitors confirmed by cell culture experiments. In the thymus of young normal mice, CD117(+), B220(low) pro- and pre-B cells are present but disappear with age. B220(low), CD5(+), B-1 B cells are present in the thymus of older animals following the appearance of similar cells in the peritoneal cavity and blood. In CD3 epsilon gene-deleted mice, the phenotypic progression and number of thymic B cells remains unaltered, showing that blocking T cell development does not automatically result in an increase of thymic B lymphopoiesis. Pro-B cells in RAG-2 knockout mice are found in the fetal and neonatal blood, spleen and thymus, but with increasing age are only found in the bone marrow. B lymphopoiesis in adult IL-7 transgenic mice is dramatically altered with CD117(+) pro- and pre-B cells present in spleen, lymph node and blood. In the thymus of adult IL-7 transgenic mice, the fraction of CD117(+) thymic B cells is significantly increased. These results show that in the steady state, the phenotype of thymic B cells is critically dependent on both mouse age and the phenotype of circulating B cells.     

B cells in epithelial and perivascular compartments of human adult thymus

The thymus is the site of T-cell differentiation. However, the relatively recent observation that B cells are also present in the human thymus has prompted studies to determine the origin and function of these B cells. Our studies show that phenotypically distinguishable B cell populations are located within both the thymic medulla and the thymic perivascular space and that cellular trafficking occurs between these compartments, including B cells trafficking from the periphery. The numbers of thymic B cells increase with age, correlating with increases in lymphocyte-rich regions of thymic perivascular space that are prominent between ages 10 and 50 years. B cells within both thymic epithelial and perivascular compartments contain mutated immunoglobulin VH sequences characteristic of post-germinal center B cells, suggesting that the B cells that most often give rise to thymic B-cell lymphomas may originate from either the thymic medulla or perivascular space.     

CD23 expression in mediastinal large B-cell lymphomas

AIMS: Mediastinal large B-cell lymphoma (MLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) in the WHO classification with peculiar features, such as female prevalence, young patient age and bulky presentation. It shows a B-cell phenotype with variable expression of surface immunoglobulin, negative CD21 and CD10 and positive CD30 in a large number of cases. An origin from activated thymic B cells has been suggested in several studies. A subpopulation of large, dendritic cells (asteroid cells) strongly expressing CD23 has been identified amongst thymic B cells and these could represent the normal cellular counterpart for this type of primary mediastinal large cell lymphoma. METHODS AND RESULTS: To explore this possibility, we immunostained 24 cases of primary mediastinal lymphomas and 100 cases of non-mediastinal, nodal and extranodal, DLBCLs for CD23 in routinely processed paraffin-embedded tissues. CONCLUSIONS: Our results show that a vast majority (70%) of mediastinal lymphomas strongly express CD23 whilst the same antigen is expressed in only 15% of non-mediastinal nodal DLBCLs and 9% of non-mediastinal extranodal DLBCLs. These results support the hypothesis that most cases of MLBCL arise from activated dendritic thymic B cells. We also suggest that CD23 should be included in the panel of antibodies currently used to characterize this subtype of DLBCL.     

A novel cell surface molecule on cortical epithelial cells of the human thymus

A novel cell surface molecule, DN4, defined by an mAb raised against human thymic epithelial cells, showed a specificity for epithelial cells of the thymic cortex. This antigen was not expressed at detectable levels on any other types of tissues in the human body except for the thymus and bone marrow. Immunohistochemical analysis revealed that the reactivity of anti-DN4 mAb was restricted to the thymic cortex, and the antigen-expressing cells were arranged in a reticular network with long processes between thymocytes. The cellular nature of DN4-positive cells was identified as cortical epithelial cells, as DN4 was expressed in a subpopulation of freshly prepared thymic stromal cells which contain a large amount of keratin and expression of DN4 was strictly confined to the cortical area within the thymus on immunohistochemical analysis of frozen tissues. Immunofluorescence and flow cytometric analysis revealed that a subpopulation of bone marrow cells was also positive for DN4 (20%). The large blasts of normal bone marrow cells were clearly labeled with anti-DN4 mAb, in contrast to small-sized bone marrow cells. This finding suggests that DN4 seems to be transiently expressed in certain blastic stages during the differentiation of bone marrow cells.

Immunoprecipitation of ¹²⁵I-labeled cell lysates from THP-1 and U937 cell lines with anti-DN4 mAb yielded a single chain glycoprotein with an approximate size of 80–85 kd. There was a reduction in apparent molecular weight of approximately 40 kd in the immunoprecipitation of cell lysates after endoglycosidase F treatment. Thus, DN4 seemed to have a considerable amount of carbohydrate group.

DN4 appears to be a novel cortical epithelial cell antigen of the human thymus, and although the role of this molecule has not been well established experimentally, the possibility can be suggested that the DN4 molecule might be involved in the positive selection of thymocytes which occurs predominantly in the thymic cortical area.     

Immunological properties of thymus cell subpopulations: rat thymic dendritic cells are potent accessory cells and stimulators in a mixed leukocyte culture

Rat thymus cells were fractionated by centrifugation on a discontinuous bovine serum albumin gradient into two subpopulations: one of high density that accounted for>90% of the recovered cells, and a minor low-density subpopulation containing 4 to 10% of the total cells. The high-density subpopulation consisted mainly of uniform small-sized thymocyteS, whereas the low-density subpopulation contained mostly larger-sized cells. High-density thymus cells did not function either as stimulators in a mixed leukocyte reaction or as accessory cells required for T-cell response to mitogens, Con A and sodium periodate, as determined by ³H-thymidine incorporation. Dense thymus cells also responded poorly to allogeneic and to mitogenic stimulation, even when accessory cells were added. In contrast, the low-density thymus cells responded well to allogeneic stimulation and to both mitogens. In addition, low-density thymus cells possessed stimulatory activity in mixed leukocyte cultures, as well as accessory activity for mitogenic responses. Both activities were found to reside in dendritic cells that were purified extensively (70-90% of the preparation) with good yield. When tested as accessory cells for T-cell responses to periodate, thymic dendritic cells were as potent as lymph node dendritic cells on a per cell basis. A small number of thymic dendritic cells was able to cause marked enhancement in T-lymphocyte proliferation in response to stimulation. By immunofluorescence thymic dendritic cells were shown to be Ia-positive, but Thy 1.1-negative.     

Intra- and extrathymic B cells in physiologic and pathologic conditions

Summary Normal thymuses and thymuses with lymphofollicular hyperplasia have been examined immunohistologically using immunoenzymatic single and double labelling methods and a panel of monoclonal antibodies against B lymphocyte differentiation antigens (CD19-, CD20-, CD21-, CD22-, CD23- and CD37ag) and human immunoglobulins (IgM, IgD) for the presence and localisation of B lymphocytes and cells expressing B cell differentiation antigens. The numerous hyperplastic lymph follicles which occur in the pathological condition of lymphofollicular hyperplasia of the thymus were found to originate in the extrathymic compartment of the interlobular septal space. This area was found to be blown up by the growing lymph follicles with exactly the same cellular composition as their counterparts in the peripheral lymphatic tissue. Some of the B lymphocytes expressing the immunophenotype of follicular mantle zone lymphocytes which were detected in the thymic medulla probably infiltrated through discontinuities of the border between the perivascular space and the thymic medulla. Apart from this primarily extrathymic B cell compartment, B lymphocytes and cells expressing B cell antigens were found within the thymus medulla of normal control thymuses of different ages from fetal to adult life. These cells were detected as a small subpopulation in normal fetal, juvenile and adult thymuses. Morphologically they could be subdivided into small, round lymphoid cells accounting for less than 1% of medullary lymphoid cells, and into a larger variant, asteroidally shaped because of short cytoplasmic processes. These asteroid cells were even more infrequent than the lymphoid variant. Immunophenotype (CD19ag+, CD20ag+, CD22ag+, CD37ag+, IgM+, IgD+) and morphology of the first cell type led to the conclusion that the lymphoid cells were in fact B lymphocytes. They were scattered throughout the medulla of fetal and juvenile and adult thymuses alike. The second, the asteroid cell type, constantly expressed CD20ag and inconstantly IgM, CD22ag and CD37ag; furthermore, CD23ag was detected in a subset of the asteroid cells either restricted to the perinuclear zone or expressed in the entire cytoplasma and on the plasma membrane. The asteroid cells were located in the corticomedullary region of the fetal thymuses but were randomly distributed with a tendency to Hassall's corpuscles in juvenile and adult thymuses. They often formed rosettes with non-B lymphocytes. It can be concluded that a small number of B cells and asteroid cells of still uncertain origin, but expressing B cell antigens, are constitutive elements of the fetal and adult thymic medulla. It can be assumed that the asteroid cell might represent a novel type of thymic accessory cell and that the rosetting of non-B lymphocytes around this asteroid cell might simulate or in fact be the earliest B cell interaction of maturing T cells.Abbreviation mAb(s) monoclonal antibody(-ies) - CDxxag antigen defined by the mAb cluster xx - CDxx(mAb) mAb of the cluster xx This study was supported by the Land Baden-Württemberg (Förderung der AIDS Forschung)Dedicated to Prof. Dr. V. Becker on the occasion of his 65th birthday     

Cellular heterogeneity in the thymus. II. Distribution patterns of different lymphocyte populations in chickens

Lymphocyte preparations from chicken blood, spleen, bursa, as well as fractionated thymus cell preparations, were labeled in vitro with ⁵¹Cr and reinjected into various groups of recipients. The relative distribution patterns of labeled cells in numerous recipient organs remained practically unchanged after different time intervals, but were significantly different for the various cell preparations. The distribution patterns provided additional evidence for the existence of different lymphocyte subpopulations in the thymus of young chickens, one of which is bursa-dependent. Thymus cell preparations fractionated by differential centrifugation showed significant differences in their accumulation in the spleen and liver. The analogous lymphocyte preparations from neonatally bursectomized birds showed no such differences. Cells from 6-month old donors gave results similar to those obtained with cells from young bursectomized birds, suggesting that the bursa-dependent subpopulation in the thymus disappears after involution of the bursa. An unexpected finding was the rather large accumulation of blood lymphocytes in the thymus of the recipients. This observation stands in contrast to the concept of a blood-thymus barrier.