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We developed a scoring system that can combine several immunological parameters and express the immune status of individuals as a simple numeral. T cell immune score was obtained by using 5T cell-related parameters: number of T cells, ratio of CD4(+)T cells to CD8(+)T cells, number of naïve T cells, ratio of naïve T cells to memory T cells, and T cell proliferative index (TCPI). TCPI was calculated by using number of T cells and their proliferative activity. We assessed T cell immune score in 103 patients with colorectal cancer and 51 healthy age-matched controls. The results were as follows: (1) T cell-immune score of patients in stages I-IV before surgery was significantly decreased as compared with controls. (2) The number of regulatory T cells in patients in stages I-IV gradually increased with disease progression. (3) T cell immune score was strongly suppressed after surgery, but were recovered to the initial level within a month. (4) Furthermore, restoration of immunological function was attempted in cancer patients by infusion of activated autologous T cells. The effectiveness was confirmed by an increase of TCPI in many cancer patients.

The recruitment of basophils into the sites of allergic inflammation is often observed. However, no definitive evidence has been provided that basophils are crucially involved in the pathogenesis of chronic allergic disorders. Here, we show that basophils are responsible for the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. A single subcutaneous injection of multivalent antigens elicited not only immediate- and late-phase ear swelling but also delayed-onset ear swelling with massive eosinophil infiltration in mice sensitized with antigen-specific IgE. Mast cells were essential for the immediate- and late-phase ear swelling but dispensable for the delayed one. T cells were also dispensable for the latter. Transfer of FcRI-expressing basophils into FcRI-deficient mice restored the development of the delayed-onset allergic inflammation. These findings indicate a novel mechanism of development of chronic allergic inflammation that is induced by basophils through the interaction of antigen, IgE, and FcRI.

The function of the immune system is to maintain body integrity by defending against infections, cancers, autoimmune diseases and inflammation-related chronic diseases. The immune response is known to become defective with aging, leading to decreased longevity and appearance of age-related disease. The most important changes occur in T-cell immunity, and are manifested particularly as altered clonal expansion of cells of limited antigen specificity. The causes of these alterations are multifactorial, and include thymic involution, T-cell subset changes and signal transduction alterations. The clinical consequences of these changes are not well defined, except for their extremely important negative impact on defence against infections, especially by new pathogens, and decreased responses to vaccination. Considering the public health consequences of decreased immune competence in old age, strategies for immune response modulation are desirable to decrease the health burden for the elderly and improve their quality of life.

In order to determine molecules on thymic epithelial cells which play an important role in the process of T cell differentiation, mAb recognizing thymic epithelial cells were made using stromal cells of the embryonic thymus at 15-gestation date. Among many mAb, a specific one was selected in terms of its inhibition of T cell development in an in vitro culture system of the embryonic thymus. cDNA of the protein recognized by one of the mAb was obtained by a panning method. Sequence analysis revealed that the protein was identical to glia maturation factor (GMF)-beta. Northern blot analysis confirmed the expression of GMF-beta mRNA in the thymus and brain. Furthermore, immunoblotting analysis identified the production of GMF-beta protein in the thymus, the brain and a thymic epithelial cell line. GMF-beta protein prepared by a glutathione-S-transferase gene fusion system greatly influenced T cell development in the in vitro culture system of the embryonic thymus in favor of a significant increase of CD4(+) T cells with expression of TCRbeta. These data taken together suggest that GMF-beta protein is produced by thymic epithelial cells and plays a role in T cell development in favor of CD4(+) T cells.

Normal human thymuses at various ages were immunohistologically examined in order to determine whether adult or aged thymus maintained the microenvironment for the T cell development and thymopoiesis was really ongoing. To analyze the thymic microenvironment, two monoclonal antibodies (MoAb) were employed. One is MoAb to IL-1 receptor (IL-1R) recognizing medullary and subcapsular cortical epithelial cells of normal infant human thymus. The other is UH-1 MoAb recognizing thymic epithelial cells within the cortex, which are negative with IL-1R-MoAb. Thymus of subjects over 20 years of age was split into many fragments and dispersed in the fatty tissue. However, the microenvironment of each fragment was composed of both IL-1R positive and UH-1 positive epithelial cells, and the UH-1 positive portion was populated with lymphocytes showing a follicle-like appearance. Lymphocytes in these follicle-like portions were mostly CD4+CD8+ double positive cells and contained many proliferating cells as well as apoptotic cells. Thus these follicle-like portions in adult and aged thymus were considered to be functioning as cortex as in infant thymus. Proliferative activity of thymocytes in the thymic cortex and the follicle-like portions definitely declined with advance of age, while incidence of apoptotic thymocytes increased with aging.

T cell responses are altered in the aged in a manner usually interpreted as detrimental to host defences against infectious agents and possibly also against cancer. T cell dysregulation may be caused by any or a combination of stem cell deficits, compromised T cell differentiation, inefficient antigen processing and presentation by antigen presenting cells, suboptimal processing of the antigenic signal by T cells or inability of the T cell to respond appropriately thereafter. This review will focus on altered T cell signalling in ageing, encompassing not only alterations in signal transduction by the antigen-specific T cell receptor, but changes in the balance of positive and negative T cell costimulation and the resultant modified cytokine environment, the response to which is itself altered in ageing.

T cell differentiation in the thymus is driven by positive selection through the interaction of alphabeta T cell receptors (TCRs) with self-peptides bound to self-major histocompatibility complex molecules, yet the influence of the peptide sequence on this process remains unknown. To address this issue, we have compared CD4(+) T cell differentiation between two sets of mouse lines in which MHC class II I-A(b) molecules are occupied with either Ealpha chain-derived peptide ((p)Ealpha) or its variant, (p)60K, with one amino acid substitution from leucine to lysine at P5 residue of TCR contacts. Here, we show that despite the comparable expression of I-A(b)-peptide complex in the thymus, this substitution from leucine to lysine affects efficiency of positive selection, resulting in extremely small numbers of CD4(+) T cells to be selected to mature on I-A(b)-(p)60K complex. Furthermore, we show that, although I-A(b)-(p)Ealpha complex selects diverse T cells, T cell repertoire shaped by I-A(b)-(p)60K complex is markedly constrained. Our findings thus suggest that positive selection is both specific and degenerate, depending on the amino acid residues at TCR contacts of the selecting self-peptides.

We performed an immunohistological study using biopsy samples of bone marrow obtained from patients, ranging in age from a newborn baby to 100 years old. Those patients suffering from hematological diseases or diseases that would be capable of affecting hematopoiesis were not included in the present study. The cellularity of the bone marrow did not change significantly with age during the first and the eighth decades, while the oldest group, ranging in age from 80 to 100, revealed significantly low cellularity. Proliferative activity assessed by Ki-67-positive cells was high in the middle-aged group and declined slightly in the elderly group. It was of interest to note that the percentage of apoptosis was relatively low in the young and middle-aged group, but significantly increased in the elderly group. The percentage of T cells did not change greatly between the first and the fifth decades, peaked at the sixth decade and gradually decreased thereafter. The percentage of B cells was about 10% at the first decade, decreased thereafter until the third decade, then increased again showing a peak at the sixth decade, and decreased thereafter. The percentage of CD68-positive cells was high in young patients, and decreased in the adult and elderly patients. The data in the present study suggest that hypocellularity in the bone marrow of elderly people could be ascribed partly to the increase of apoptosis, and might possibly be related to a decrease in the number of lymphocytes and macrophages, which would constitute part of the bone marrow microenvironment supporting hematopoiesis.

We immunohistologically investigated 76 cases of primary colorectal cancer and 13 cases of adenoma to clarify the relationship of MHC class II expression with the grade of differentiation, the density of tumor-infiltrating lymphocytes, and the incidence of metastasis to lymph nodes. MHC class II expression was mostly negative in normal colonic epithelium. In contrast, 32 out of 76 cases (42%) of cancer and five out of 13 cases (38%) of adenoma were positive for MHC class II. MHC class II expression was higher in well-differentiated than in poorly differentiated adenocarcinoma. The density of infiltrating lymphocytes was higher in cancer than in the normal mucosa, and higher in MHC class II-positive tissues than in negative lesions. The incidences of lymphatic invasion and cancer metastasis to lymph nodes were definitely higher in MHC class II-negative cancers than in MHC class II-positive cancers. MHC class II was rarely expressed in metastatic cancer cells of lymph nodes. These results indicated that the loss of MHC class II is correlated with the incidence of metastasis to regional lymph nodes.

Signal transduction pathways of mitogenic plant lectin, concanavalin A (Con A)- and ionomycin (INM)-induced (Ca2+-dependent K+ currents (I(Con A) and I(INM)) have been compared in young and aged T-cell clones by using the nystatin perforated patch-clamp whole-cell recording technique. In young T-cell clones, Con A evoked a long-lasting outward current which is mediated by the activation of the Ca2+-dependent K+ channels. The Ca2+ ionophore, INM, evoked a short-lasting Ca2+-dependent outward K+ current (I(INM)). The protein tyrosine kinase (PTK) inhibitor, herbimycin A (3 x 10(-6) M), but not the G protein blocker, pertussis toxin (PTX, 500 ng ml(-1)), completely prevented the I(Con A), but did not affect the I(INM). In aged T-cell clones, Con A fails to evoke any current response, while INM evokes an outward current which is comparable to that in a young T-cell clone. It is concluded that PTK, but not PTX-sensitive G proteins, plays a critical role in mediation of the signal transduction from Con A stimulation to activation of the Ca2+-dependent K+ channels, and that an impairment of the early signal pathway, perhaps the PTK, might be involved in the mechanism of the age-related decline of the proliferative response of T-lymphocytes to mitogenic stimulation.

Osteopontin (OPN) is an Arg-Gly-Asp-containing phosphoprotein that is secreted by activated T cells. The concentration of serum OPN protein is elevated in autoimmune-prone MRL-lpr mice as well as in patients with systemic lupus erythematosus. Previously, it was shown that OPN induces the polyclonal activation of B cells, resulting in the augmented production of immunoglobulin, indicating that OPN plays some role in the development of autoimmune disease. However, the link between OPN and development of autoimmune disease remains unclear. To analyze the role of OPN in immune system and autoimmune diseases, we have generated two kinds of transgenic mice: one carries the immunoglobulin (Ig) enhancer/SV40 promoter and the other carries the cytomegalovirus enhancer/chicken beta-actin (CAG) promoter. In both groups of transgenic mice, the B1 cell population in peritoneal cavity was markedly increased and titer of IgM and IgG3 antibodies in the serum was considerably higher than that in wild-type mice. Most important, the titer of the IgM class of anti-double-stranded DNA antibody was significantly elevated in transgenic mice. These results strongly suggest that OPN may have an important role in the propagation and differentiation of B1 cells and production of autoantibodies.

The effect of destruction of the anterior hypothalamus (AHT) on the immune system was examined in rats 4-50 weeks after the treatment. The thymic weight significantly increased in the experimental group regardless of age, while a significant decrease was observed in the percentage of splenic T cells as well as their proliferative response to phytohemagglutinin (PHA). The increase of thymic weight was observed as long as 24 weeks and the decrease of splenic T cell number was observed as long as 50 weeks after the operation. Hypophysectomy gave rise to atrophy of both thymus and spleen, while either adrenalectomy or gonadectomy resulted in hypertrophy of both thymus and spleen. The results taken together suggest that development and aging of the immune system are under the balance of positive and negative signals from AHT.

To understand mechanism underlying the age-related impairment of T cell functions, changes in the expression of cell surface receptors were examined in T cells after mitogenic stimulation by flow cytometry and results were compared between young and old mice. Before stimulation, no significant difference was observed in the density of TCR alpha beta, CD3, CD122 (IL-2R beta), IL-2R gamma, CD28 and CD95 (Fas) of T cells between young and old mice. As for CD25(IL-2R alpha) and CTLA-4, relative intensity and percentage of positive cells were higher in old than in young mice, although the levels were low compared with those after stimulation. After mitogenic stimulation, increased expression of the density was observed in CD25, IL-2R beta, IL-2R gamma, CD28, CTLA-4 and CD95 and the magnitude of increase was more pronounced in T cells from young than those from old mice. The expression of TCR alpha beta and CD3 decreased after mitogenic stimulation, and the degree of expression quickly recovered to the initial level in young mice, but not in old mice. Lower expression of TCR, IL-2 receptors and co-stimulatory molecules in T cells from old mice could be responsible for the impaired proliferation after mitogenic stimulation.

An overnight restraint stress was given to young and old mice and its effect was examined in terms of the number and function of T cells and natural killer (NK) cells in spleen and patterns of lung metastasis of B16 melanoma cells. A great decrease was observed in the number and proliferative activity of splenic T cells in old mice after the stress. The decrease in young mice was rather temporary with a quick recovery. The number of NK cells in spleen was not different between young and old mice before giving the stress, but a significant decrease was observed in the old after the stress. NK activity was always much lower in old than in young throughout the experiment. The pattern of metastasis of B16 melanoma cells was different between young and old mice. Metastatic colonies in lungs were larger in number and bigger in size in young mice than in old mice. After the stress, the number increased and the size unchanged in old mice, while the size increased and the number remained unchanged in young mice. It was shown that the same restraint stress resulted in a more serious influence on the immune cells in old than in young mice and gave rise to a differential effect on the pattern of tumor metastasis between young and old mice.

T cells from old mice showed impaired proliferative response to antigenic stimulation. To understand the mechanism underlying the age-related impairment of T cell functions, the signal transduction pathway was examined and compared between T cells from young and old mice, and between T cell clones established from a young and old mouse. The age-related changes in T cells were as follows: (1) reduction in the expression and the activation of protein tyrosine kinases associated with T cell receptor (TCR) after antigenic stimulation; (2) reduced phosphorylation of phospholipase C gamma 1 (PLC gamma 1); (3) reduced production of second messengers such as inositoltrisphosphate (IP3) and diacylglycerol (DAG); and (4) reduced influx of Ca2+ ion. Thus, a T cell clone established from an old mouse showed impaired proliferation by stimulation with anti-CD3 antibody, but was fully activated to the level of a T cell clone from a young mouse by stimulation with phorbol acetate myristate (PMA) plus ionomycin (INM). However, splenic T cells freshly prepared from old mice did not show full recovery by the same treatment. The results indicate that one major blockade in the signal transduction of T cells from old mice is present in the pathway just after TCR, but besides this, the blockade is also present in multiple sites down-stream, which can not be bypassed by stimulation with PMA plus INM.

Wistar rats were subjected to dietary restriction and daily physical exercise for 21 months, starting from 2 months of age. At the age of 23 months, the rats were sacrificed and examined for various indices including immunological functions. Body weight was almost the same between rats fed ad libitum (Group A) and those (Group B) fed 80% of the diet consumed by Group A. The body weights of Groups A and B were significantly heavier than that of rats (Group C) fed 60% of the diet consumed by Group A and those (Group D) which were subjected to physical exercise and fed 80% of the diet consumed by Group A. Highly proliferative response of T cells to mitogens was observed in 5 out of 13 rats in group D. Some enhancement of B cell proliferation was also observed in the same rats. Present results suggested that enduring physical exercise together with dietary restriction can retard the age-related decrement of immunological functions.

Two T-cell clones were established from young and old C57BL/6 mice, respectively. The proliferative response to anti-CD3 stimulation was significantly greater in the young (YT5) than in the old (OT13) T cell clone. However, a similar high response was observed in both T-cell clones upon stimulation with phorbol myristate acetate (PMA) and ionomycin (INM). The calcium-dependent K+ current (IK(Ca)) was recorded with the patch-clamp method in these T-cell clones. With anti-CD3 stimulation, the amplitude of the outward K+ current was significantly lower in OT13 than in YT5 cells. With stimulation with PMA and INM, however, no significant difference in IK(Ca) was obtained between the two types of cells. The level of proliferative response of T-cells to mitogens was well reflected by the amplitude of IK(Ca). Some abnormality in the early pathway of signal transduction, which led to the intracellular Ca2+ influx, appears to be responsible for the impaired proliferation of the old T-cell clone.

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG2a and had kappa light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G1, G2 and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characterize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis. The nuclear protein(s) detected by Th-10a monoclonal antibody was mostly 95 kDa and also 83 kDa polypeptide. The results also indicated that the 95 kDa nuclear protein was phosphorylated in vivo.

Twenty-six week-old BDF1 mice were gonadectomized and grafted with thymus from irradiated (8.5 Gy) newborn, 6-week-old, or 26-week-old mice. One month later, grafted thymuses were recovered and examined in terms of thymocyte numbers, subpopulations and proliferative responses to Concananavlin A (Con A). The growth of the irradiated thymus was significantly higher in gonadectomized (Gx) than in sham-operated (Sham) mice and the magnitude of thymic growth was apparently age-dependent, as it was greater for newborns than for older mice. Con A response of thymocytes was also significantly higher in Gx mice than in Sham mice, and the magnitude of the response declined with advancing age of the thymus donors. Flow cytometric analysis revealed that a significant increase in the percentage of CD4+CD8- was observed in thymus grafts showing high Con A responses. However, this effect of Gx on the thymus graft was dependent on age of the thymus donor. Namely, newborn thymus grafts could grow equally well in both Gx and Sham recipients, whereas thymus grafts from 6- and 26-week-old mice could grow well only in Gx, but not in Sham recipients. The number of thymocytes was comparable in thymus grafts from 6- and 26-week-old mice, but the proliferative response to Con A was higher in the former than in the latter graft. Collectively, Gx appeared to promote immigration of thymocyte precursors into the thymus and to enhance proliferation and differentiation of thymocytes towards CD4+CD8- T cells, in an age-related manner.

A 5- to 10-fold decline was observed in the proliferative activity of T cells stimulated with anti-CD3 mAb between young and old mice. However, the number of CD3 molecules on the T cell surface was almost comparable between young and old T cells. The formation of the second messenger such as inositol triphosphate (IP3) and diacylglycerol (DAG) after mitogenic stimulation decreased in old T cells as compared with young ones. The activity of phospholipase C (PLC), which is responsible for the liberation of IP3 and DAG from phosphatidylinositol-4,5-bisphosphate (PIP2) was not different between young and old T cells. The content of PIP2 in the membrane was also comparable between young and old T cells. These findings have suggested that the age-related decline in the proliferative activity of T cells could be due to impairment of intracellular signal transduction, probably in the pathway somewhere between TCR and PLC.

We have established a set of transgenic mouse lines in which the HLA-DRA gene was expressed in different cell types. In one line (DR alpha-24), DR alpha E beta b molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow-derived antigen-presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DR alpha-30) was found on thymic medullary and cortical epithelial cells but not on bone marrow-derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DR alpha-24 and DR alpha-30 was performed. In DR alpha-30, T cells expressing TcR V beta 5 and V beta 11 were eliminated to comparable levels to those in DR alpha-24, suggesting that expression of the DR alpha E beta b molecules on thymic epithelial cells are sufficient for clonal deletion of the self-superantigen-reactive T cells. In addition, CD4+ T cells from DR alpha-30 as well as those from DR alpha-24 were tolerant to DR alpha-derived peptide/I-Ab complex expressed on spleen cells from DR alpha-24 even in the presence of exogenous interleukin-2. These observations suggest that expression of the DR alpha chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DR alpha-derived peptide bound to I-Ab molecules, probably via clonal deletion of the self-reactive T cells.

We have established a strain of transgenic mice in which the HLA-DRA gene was integrated into the X-chromosome and the xenogeneic mixed isotype molecule, DR alpha E beta b, was expressed in a cell type-specific manner, although the transgenic DRA gene contained only 268 base pairs of the 5'-flanking region. The DR alpha E beta b molecules expressed in the transgenic mice functioned as major histocompatibility complex (MHC) class II to select T-cell repertoire, and to stimulate mixed lymphocyte reaction. In female transgenic mice homozygous for HLA-DRA (DR alpha-B6-F-homo) and male transgenic mice (DR alpha-B6-M), DR alpha E beta b molecules were expressed in almost all of the MHC class II Ab-positive cells. In contrast, the expression of DR alpha E beta b molecules in female transgenic mice hemizygous for HLA-DRA (DR alpha-B6-F-hemi) was found only in part of the Ab positive cells, and the proportion of cells expressing the DR alpha E beta b molecules varied due to random inactivation of one of the X-chromosomes. Clonal deletions of the T cells and mature thymocytes bearing Tcrb-V5 and Tcrb-V11, which are eliminated from the peripheral repertoire in mice expressing self-superantigen and MHC class II E molecules, were incomplete in DR alpha-B6-F-hemi as compared with those in DR alpha-B6-F-homo, and were correlated with the proportion of DR alpha E beta b-positive spleen cells. These observations suggested that the number of bone marrow-derived cells expressing DR alpha E beta b molecules was critical for clonal deletions of Tcrb-V5+ and Tcrb-V11+ T cells in the thymus.

We previously demonstrated that the liver may be a major site of extrathymic T-cell differentiation in mice. In the present study, the ontogeny and subsequent development of such T cells in the liver and other organs were investigated. This study was possible because these T cells have T-cell receptors (TcR) of intermediate intensity (i.e. intermediate TcR cells) and constitutively express a high level of interleukin-2 receptor beta chain (IL-2R beta). Therefore the two-colour staining for CD3 (or alpha beta TcR) and IL-2R beta identifies even a small proportion of intermediate TcR cells. The total numbers of mononuclear cells obtained from the liver, thymus and spleen varied from foetal to adult life. Especially in the liver, many haematopoietic cells were present in the parenchymal space at the foetal stage. There were no lymphocytes in the sinusoidal lumen at this period. In contrast, lymphocytes appeared in the hepatic sinusoids after birth and increased with ageing. Phenotypic analysis revealed that intermediate TcR cells appeared in the liver and spleen on Day 4 after birth. Bright TcR cells of thymic origin were also present in the peripheral organs on Day 4. Thereafter, intermediate TcR cells increased in the liver, whereas bright TcR cells increased in the periphery as a function of age. Similarly, thymectomized and congenitally athymic mice had mainly intermediate TcR cells in the liver and, to some extent, periphery. It is concluded that intermediate TcR cells, possibly of extrathymic origin, are generated only after birth and expand with ageing.

A monoclonal antibody Th-5 has been produced against mouse immature thymic lymphocytes and employed to study the process of T cell differentiation in the thymus. Immunohistologically, Th-5 positive thymic T lymphocytes were first found at Day 12 of gestation. They increased in number as well as staining intensity until Day 18 of gestation and decreased thereafter. Th-5 antigen expression was not seen in lymphoid cells in the fetal liver. In the newborn thymus, lymphocytes in the subcapsular layer were still strongly positive, while other cortical lymphocytes became moderately positive for Th-5. Th-5 positiveness was more pronounced in the medulla than in the cortex in the thymus of young adult mice. The staining pattern of Th-5 in the thymus was apparently different from those with other T cell markers (Thy-1, CD3, CD4, CD5, CD8) including J11d, Pgp-1, IL-2R, and 3A10 (TCR gamma delta). Flow cytometric analyses showed that the expression of Th-5 was mostly associated with the Thy-1 antigen. However, the fluorescent intensity of Th-5 gradually declined with ontogenic development of the thymus, and the molecular size of the antigen was approximately 100 kDa, which is different from Thy-1 antigen (25-30 kDa). Considering these findings, the strong expression of Th-5 could be one of the markers of immature thymic T lymphocytes in the early phase of the ontogenic development.

Three thymic epithelial cell lines (TEC1C5, TEC1-4, and TEC2-3) were established from the thymus of newborn C57BL/6 mice. TEC1C5 was revealed to be an interleukin (IL)-1 producing cell line. TEC1-4 produced a cofactor to promote proliferation of double negative (CD4-8-) thymic lymphocytes by the presence of IL-1. Production of the same cofactor was also seen in TEC2-3, but only when it was cultured by the presence of indomethacin. The chemical analysis of the TEC1-4 culture supernatant by ion-exchange column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the factor was approximately 35 kDa in molecular weight. The present study revealed that a factor produced by TEC1-4 acted as a cofactor to promote the proliferation of immature T cells stimulated by IL-1 and various mitogens and was considered to be a new one in terms of molecular weight.

This report describes the development of hyperplasia of both the thymus and the peripheral T-cell system with advancing age in the Buffalo rat. Buffalo/Mna rats do not show age-related thymic involution, but rather develop thymic hyperplasia with advancing age. This thymic growth is expansile and there is no infiltration of the surrounding tissues. Because the enlarging thymus occupies the thoracic cavity, most of the rats die of respiratory failure by the age of 24 months. Thymic enlargement is due to primary hyperplasia of cortical epithelial cells and the large number of proliferating lymphocytes. The hyperplastic epithelial cells are bizarre in shape and strongly positive when stained with Th-3 monoclonal antibody (MoAb), anti-thymosin antibody and anti-EGF antibody, but negative with Th-4 MoAb. The patterns of distribution of CD-5+, CD-4+ and CD-8+ lymphocytes within the hyperplastic thymus are similar to those seen in young rats of other species. The high level of T-cell emigration from the thymus to the periphery appears to persist throughout life, since the percentage of normal splenic T-cells also increase with advancing age and exceed 70% of the total by 24 months of age. This thymic enlargement with abnormal hyperplasia of cortical epithelial cells can be prevented by hypophysectomy.

The incidence of focal lymphoid infiltrates in the renal interstitium was examined in autopsy cases of young and old subjects, and the infiltrating lymphocytes were immunohistologically characterized by a panel of monoclonal antibodies. Histologically, 198 and 227 autopsy cases over 60 years of age (87.2%) were shown to have mononuclear cell infiltrates of varying degree in the renal interstitium, whether or not these were accompanied by progressive arteriosclerotic changes. Above all, severely infiltrating foci in the renal interstitium were frequently found in the elderly over 70 years of age overlapping arterio-artherolosclerotic changes. In contrast, in the 54 younger control subjects under 49 years of age, the incidence of such a lesion was less (5.6%). An immunohistologic study revealed that the infiltrating mononuclear cells were predominantly composed of CD4+ cells, whereas CD22+ B cells were apparently lesser in number. Moreover, a considerable proportion of T cells was activated as judged by IL-2 receptor expression. From these findings, we now propose that susceptibility to the development of interstitial renal lesions in the elderly involves the cellular immune response, and may be related to an age-associated disturbance in regulatory T-cell function.

Bone marrow cells obtained from B10.Thy-1.1 mice (H-2b, Thy-1.1) were injected directly into the thymus of C57BL/6 mice (H-2b,Thy 1.2) of various ages. Thymocyte precursors in the injected donor-bone marrow cells could proliferate in the thymic microenvironment in the following manner: first, preferentially proliferating into the subcapsular cortex; and second, spreading to the whole layer of the cortex, a portion of them gradually moving into the medulla. The proliferation of donor-type thymocytes was most pronounced when intrathymic injection of bone marrow cells (ITB) was performed in newborn mice and especially prominent in week-old mice; it took approximately ten weeks for donor-type thymocytes to finish the whole course of proliferation, differentiation, and emigration to the periphery. When ITB was performed in mice 4 weeks of age and older, the proliferation of donor-type thymocytes was retarded at onset, less pronounced in magnitude, and disappeared earlier. Emigration of donor-type T cells from the thymus to the peripheral lymphoid tissues occurred most rapidly when ITB was performed in newborn mice, and these T cells continued to reside thereafter in the peripheral lymphoid tissues. However, when ITB was performed in mice 4 weeks of age and older, the number of emigrated T cells in the spleen decreased (about a tenth of that in newborn mice) and, moreover, these T cells resided only transiently in the spleen. It was suggested that T cells emigrating from the thymus of mice from newborn to 2 weeks of age are long-lived, whereas those from the thymus in mice 4 weeks of age and older are short-lived. However, when 4-week-old young adult mice were treated by irradiation or hydrocortisone, the thymic capacity was enhanced in terms of proliferation and peripheralization of thymocytes, and emigrated T cells became long-lived.

Radiation effects on regeneration and T-cell-inducing function of the thymus were studied in three sets of experiments. When TXB mice were grafted with 1-week-old thymus which had been previously irradiated at various doses, an exponential decrease was observed in the morphological regeneration of the thymus grafts and in their T-cell-inducing function at doses of 600 R and over, showing about 10% that of the control at 1500 R. When in situ thymus of adult mice was locally irradiated, the radiation effect on T-cell-inducing function was less pronounced as compared with the first experiment; i.e., about 40% of the control at 1797 R. When in situ thymus of 1-day-old newborn mice was locally irradiated, regeneration potential of 1-day-old newborn thymus was highly resistant to radiation exposure and no effect on immunological functions was observed even by local irradiation of 2000 R.

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)

The anti-SRBC antibody response of normal young adult mice (about 3 months old) was specifically suppressed, when the spleen cells derived from the syngeneic donor mice that had been previously primed with a high dose of SRBC were adoptively transferred at the time of antigenic challenge. The suppression was antigen-specific and was mediated by a fraction of the immune spleen cells which appeared to belong to either B cells or their progeny antibody-forming cells. The differences were observed in the properties of the suppressor cells in terms of the period after the priming and the radiosensitivity; i.e., immune spleen cells taken from the mice which had been immunized with SRBC 5 days earlier were able to suppress only 19S PFC, whereas those taken from the mice which had been immunized 14 days earlier were able to suppress both the 19S and 7S PFC responses, and the former cells were susceptible to 400 R X-rays, but not the latter. These two types of suppressor cells appeared to play an important role in the regulation of the sequential change of 19S and 7S antibodies during the primary immune response.

The immigration route of thymocyte precursors into the thymic microenvironment was examined in various experiments using two strains of mice (B10.Thy-1.1 and C57BL/6) that were identical in H-2 and different in Thy-1 locus. The experiment of thymus grafting revealed that there were two types of thymocyte precursors; one immigrated into the cortex and vigorously proliferated and the other directly immigrated into the medulla. Such a direct immigration of host-type cells into the medulla of the grafted thymus was not observed, when thymus was grafted into young adult nude mice having no T cells. When bone marrow cells were iv injected into intact mice, the direct immigration of donor-type cells was observed only in the cortex, not in the medulla. In parabiotic mice, the immigration of partner's cells into the medulla was observed independently before the proliferation of partner's cell in the cortex. These findings taken together indicate that peripheral T cells directly immigrate into and recirculate through the thymic medulla.

Thymic lobes of B6C3F1 mice ranging in age from 1 day to 11 weeks were implanted under the kidney capsule of T cell deprived syngeneic young adult TXB mice, and the capacity of the thymus grafts to influence the maturation of T cells was assessed at 6 and 12 weeks after the implantation in terms of (a) regenerative activities of the grafted thymus, (2) splenic T cell dependent anti-SRBC response, and (c) mitogenic reactivity of spleen and lymph node cells to T cell specific mitogens. The results revealed that: (1) thymic tissues from 1 week old donors were most efficient in restoring the immune potential of adult TXB mice; (2) a decline in mitogenic reactivities of spleen and lymph node cells was observed in recipients of thymus grafts from donors of 1 month and older; and a decline of splenic helper T cell function was observed in recipients of thymus grafts from 11 weeks old donors. The significance of this early decline in the thymic effect on T cell differentiation is discussed.

The thymus of long-lived BC3F mice involutes progressively throughout life, beginning at 6 weeks of age. This is manifested by the loss of cortical lymphoid mass and by the degenerative changes in epithelial cells. The purpose of this study was to determine to what extent age-related degenerative changes of the thymus affect its capacity to influence the maturation of thymic-derived (T) cells. Accordingly, thymic lobes of mice ranging in age from 1 day to 33 months were implanted under the kidney capsule of T cell-deprived syngeneic young adult TXB mice, and the emergence of T cells was assessed kinetically by various morphologic and functional indices which may be reflective of different T cell subpopulations. They are: a) histology of the thymsu graft, b) lymphocyte repopulation of the T cell-dependent areas of lymph nodes, c) total number of splenic lymphocytes carrying theta antigens (theta-+), d) T cell-dependent humoral immune response and e) proliferative response of splenic cells to plant lectins, phytohemagglutinin (PHA) and succinyl-concanavalin A (s-Con A), and allogeneic lymphocytes. The results revealed that the T cell-transforming influence of thymic tissues generally decreases with increasing age. The difference in the patterns of recovery of the various indices of thymus-grafted TXB mice suggests that the extent to which T cells can mature is dependent upon the degree of involution the thymic tissue has undergone with age. In particular thymic tissues lose the capacity to influence the following functions with advancing age: 1) lymphocyte repopulation of the T cell-dependent areas of lymph nodes; 2) mitogenic reactivity of splenic cells to T cell-specific mitogens (PHA and s-Con A): 3) number of splenic theta-+ lymphocytes and splenic T cell helper function; and 4) mitogenic reactivity of splenic T cells to allogeneic lymphocytes.

Two hybridoma cell lines secreting monoclonal antibodies against stromal tissues of mouse thymus were produced using the spleen cells of BALB/C mice immunized with newborn thymic homogenate of C57BL/6 mice emulsified in Freund's complete adjuvant. The monoclonal antibody Th-3 reacted with stromal cells in the thymic cortex and the monoclonal antibody Th-4 reacted with stromal cells in the thymic medulla. The stromal cells revealed by Th-3 showed a meshwork structure in the cortex, and formed a monolayered border at the cortical surface and around the vasculature. Each mesh of this structure was connected to each other, forming a complex labyrinth and being open toward the medullary area. Neither lymphoid cells nor any cells in any other organs were reacted with this Th-3 antibody. However, the reactivity of Th-3 with the thymic cortical stromal cells was observed not only in C57BL/6 mice which had been used as source of antigen, but also in other strains of mice such as C3H and BALB/C. Immunoelectron microscopy revealed that Th-3 monoclonal antibody was reactive with some component, diffusely present in the cytoplasm of cortical epithelial cells. The pattern of Th-3 positive meshwork in the thymic cortex was quite similar to that stained by either anti-IA or anti-IE antibody, but the Th-3 positive reaction was not inhibited by these anti-IA and anti-IE antibodies. Thymic nurse cells prepared by the method of Wekerle were positive for Th-3 antibody. On the contrary, Th-4 reacted only with epithelial cells in the thymic medulla. It was suggested that Th-3 monoclonal antibody detected some antigen specific to so called thymic nurse cells.

The incidence of mononuclear cell infiltration in the adrenal cortex was examined in autopsy cases of young and old subjects, and the infiltrating mononuclear cells were immunohistologically characterized by monoclonal antibodies. Histologically, 110 of 174 autopsy cases of persons greater than 60 years (63.2%) were shown to have mononuclear cell infiltration of varying degree within the adrenal cortex, whereas such a lesion was observed in lesser incidence (7.4%) in the 54 younger, control subjects aged less than 49 years. In addition, severely infiltrating lesions in the adrenal cortex were found frequently in the elderly greater than 70 years. Immunohistochemical study revealed that the infiltrating mononuclear cells were mainly composed of CD3+ T cells. The major proportion of CD3+ T cells expressed CD4, whereas CD8+ T cells were less in number. Moreover, a considerable proportion of CD4+ T cells was activated as judged by interleukin 2 receptor expression. These findings indicate that T lymphocytes infiltration in aged human adrenal cortex may represent a pre-clinical manifestation of organ-specific autoimmune adrenalitis which is based on autoimmunity associated with ageing process.

Splenic T cells of C57BL/6 mice, ranging in age from newborn to 24 months old, were examined by two-colour flow cytometry using monoclonal antibodies (moAbs) to Thy-1, Lyt-1, Lyt-2 and L3T4. Thy-1+ cells in the spleen increased gradually after the birth, and reached a plateau around at 3 months of age, but did not show a significant age-related decline even at 24 months of age. Lyt subpopulations of T cells (Lyt-1+2+, Lyt-1+2-, Lyt-1-2+) showed a proportional increase until 1 month of age, but the onset of their imbalance was observed as early as 3 months of age. The percentage of L3T4+ subpopulation stayed at a relatively constant level throughout life (approx. 55%). At the individual cell level, Lyt-2 antigen was most vulnerable to aging and its membrane surface density showed a prominent decrease in 24-month-old T cells. An apparent decline was observed in the mitogen reactivity and cell-mediated cytolytic T lymphocyte (CTL) activity of old T cells or their subpopulations which were purified by the cell sorter.

The regeneration and persistence of host- and donor-derived T cells were examined in the thymus as well as the spleen of mouse radiation bone marrow chimeras of two semiallogeneic combinations (F1----P, P----F1) with different Thy-1 markers on T cells of donor and host origins. An unexpectedly large number of host-type T cells were recovered from the spleens of F1----P chimeras, amounting to as high as 45 and 25% of total T cells at 6 and 14 weeks after bone marrow transplantation (BMT), respectively. To the contrary, the residual host-type T cells in the spleens of P----F1 chimeras disappeared quickly, resulting in less than 0.1% of total T cells at 6 weeks after BMT. It was also revealed that the number of host-type T cells in the spleens of F1----P chimeras decreased in proportion to increase of radiation dose given to the recipients.

Bone marrow chimeras were produced between various combinations of young and old mice using either C57BL/6 mice only or a combination of C57BL/6 and B10.Thy-1.1 mice. The wet weight of the thymus and the number of thymocytes and splenic T cells of donor origin were assessed at appropriate intervals after the bone marrow transplantation. It was revealed that the old bone marrow was inferior to young in terms of the capacity to repopulate the thymus and splenic T cells. Moreover, some age-related qualitative changes appeared to occur in the thymocyte progenitors, as the composition of Lyt phenotype of donor-type T cells in the spleen was different between chimeras produced with young bone marrow and those with old.