Population movement and fate of autoreactive V beta 6+ T cells in Mls-1a mice.

In order to demonstrate the precise fail-safe mechanisms involved in the prevention of autoreactive T-cell functions, we analysed the movement of the population of self-reactive V beta 6+ cells in Mls-1a mice. T cells bearing V beta 6 T-cell receptor (TcR) could be detected in the thymus at birth. They increased in number during the next few days, then decreased and disappeared by 1 week after birth. These cells are autoreactive and capable of eliciting a syngeneic graft-versus-host reaction (GVHR). The autoreactive V beta 6+ cells in the thymus on day 3 were abolished by a previous injection of Mls-expressing syngeneic adult spleen cells, showing that the tolerance-inducing antigens had probably not yet developed in newborn mice. These autoreactive V beta 6+ cells escaping clonal deletion may leave the thymus and become appreciable as their percentages rise in the periphery in mice thymectomized 3 days after birth (d3-ThX). However, the 'autoreactive' T cells seemed to be neither cell cycling nor proliferating even after exogenous antigenic stimulation. The proportion of these peripheralized V beta 6+ cells in an 'anergy' state decreased gradually to a half-life of about 50 days in adults, in contrast to the complete deletion in a few days of V beta 6hi cells in the developing thymus. On the other hand, in weanlings the percentage of V beta 6+ T cells was reduced to a half-life of less than 20 days, probably because of the diluting out of these cells by the physiological expansion of the irrelevant T-cell population and probably by an increase of body fluid by a factor of 10. In contrast, V beta 8+ T cells, Mls-1a-unrelated, maintained a constant proportion, as in non-thymectomized mice. Thus, T-cell repertoire shaping may not always be achieved in the thymus, but may be completed after the cells leave the thymus a few days after birth in a developmentally programmed process.

Involvement of IL-7 in the development of gamma delta T cells in the thymus.

The effect of IL-7 on the growth of thymic T lymphocytes was investigated by adding recombinant IL-7 into cell suspension cultures and submersion organ cultures (SOC) of murine fetal thymuses (FT) and newborn thymuses (NBT). FT and NBT were obtained from C57BL/6 mice at day 15 of gestational age and at day 3 after birth, respectively. In both cell suspension cultures and SOC, addition of IL-7 highly improved the cell recovery. In cell suspension cultures, addition of IL-7 resulted in the growth of gamma delta T-cells from FT-cells, whereas the same cytokine promoted the growth of both alpha beta and gamma delta T-cells from NBT-cells. These results may indicate that this cytokine is able to support the proliferation of T-cells of both alpha beta and gamma delta lineages. In marked contrast, in SOC, addition of IL-7 resulted in the growth of gamma delta T-cells not only in FT but also in NBT, despite the fact that the SOC of NBT without exogenous cytokine exclusively promoted the growth of alpha beta T-cells. A similar effect was also seen when IL-2 was added to NBT-SOC, though the skewing to gamma delta lineage was not so strong as in the case of IL-7. In addition, we found that IL-7 mRNA is expressed in the day 15 FT at a much higher level than in the adult thymus. These results strongly suggested that the production of a large amount of IL-7 synthesized in the FT is one of the major factors leading to the generation of gamma delta T-cells in FT.

Neonatal tolerance induction in the thymus to MHC-class-II-associated antigens. V. Thymus medulla and the site for deletional signaling achievement in Mls tolerance.

Intravenous (i.v.) injection of Mls-1a peritoneal cavity (PerC) cells from (BALB/c x AKR)F1 (Mls-1b/a, H-2d/k) mice into newborn BALB/c (Mls-1b, H-2d) mice induced thymus cell tolerance by one week of age, accompanied by V beta 6+ cell elimination. The tolerant state is associated with intrathymic chimerism with MHC-class II(IA+) cells, confluent in the medulla and scattered in the cortex. To clarify the anatomical site for the deletional signaling, we injected Mls-1a PerC cells directly into the thymus lobe of BALB/c mice on the day of birth. Thus induced tolerant state was limited to the injected lobe and there was no penetration to the contralateral lobe. The tolerant state lasted less than 2 weeks, by which time donor-derived Ia+ cell had disappeared from the thymus. Thus, PerC cells seem to have little self-renewing ability. One week after the intrathymic injection of a small amount (0.3 microliter) of PerC cell suspension in several different sites, the thymus lobes were removed without killing the mice and serial cryostat sections were cut and stained immuno-histochemically for analysis of the donor cell distribution. Four weeks later, the functional activities of peripheral T cells in the spleens of the treated mice were tested. These experiments revealed that inoculated cells lodging in the medulla, but not in the cortex, induced tolerance to the Mls determinants. Target thymocytes for negative signaling are probably located in the medulla/juxta-medullary area in the thymus. Data are discussed in relation to Mls-bearing stroma cells in the thymus.

A murine thymic stromal cell line which may support the differentiation of CD4-8- thymocytes into CD4+8- alpha beta T cell receptor positive T cells.

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4-8- (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4+CD8- cells were maintained further while the CD4+8+ cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor alpha beta. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of alpha beta lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.

Intrathymic induction of neonatal tolerance to Mls-1a determinant: clonal deletion and clonal anergy by haematolymphoid cells.

Newborn BALB/c (Mls-1b) mice were intravenously injected with either bone marrow cells (BMC) or peritoneal exudate cells (PEC) from Mls semi-allogeneic (BALB/c x AKR)F1 mice. Thymic cells of these mice, obtained 7 days after the injection, were found to be unresponsive to the superantigen Mls-1a, as determined by graft-versus-host reactivity. On Day 7, deletion of T cells expressing the V beta 6 element at high levels (V beta 6hi) was observed in thymic cells of mice receiving PEC. In mice given BMC, it took 2 weeks until the proportion of V beta 6hi T cells began to decline, and a longer period was required for complete disappearance of V beta 6hi T cells. These results may indicate that although both BMC and PEC contain cells mediating tolerance, a component(s) of cells responsible for clonal deletion is deficient in BMC. Immunohistological investigation showed that on Day 7 donor type B cells were present in the thymus of mice that received PEC but absent from mice that received BMC, whereas cells expressing donor type class I as well as class II antigens were seen in both recipients. The presence of donor type B cells could be observed 8 weeks after injection of BMC. By this time, the deletion of V beta 6hi T cells was completed. These results indicate, collectively, that the tolerance of both anergy type and deletion type occurs in the naturally developing thymus, and suggest that the presence of B cells in the thymus might be required for clonal deletion.

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. IV. Significance of intrathymic chimerism of blood-born Ia+ cells in Mls tolerance.

The significance of thymus cell chimerism in the induction and maintenance of tolerance was investigated. Mls-1b BALB/c mice were neonatally tolerized by the intravenous administration of either bone marrow (BM) cells or peritoneal cavity (PerC) cells from Mls-1b/a (BALB/c x AKR) F1 mice. Tolerance was long-lasting in the BM cell group, but transient in the PerC cell group, probably because PerC cells lack hemopoietic stem cells required for a continuous supply of tolerance-inducing cells. The degree of anti-Mls-1a responsiveness of these BALB/c thymus cells was correlated with the degree of intrathymic distribution of the inoculated F1 cells. The effect of BM cell inoculation, resulting in a year-long deletion of Mls-1a-reactive V beta 6-bearing T cells is in marked contrast to that of PerC cell inoculation which causes only a transient loss of V beta 6+ mature thymocytes (for about 1 week after birth). This functional profile of the tolerant state correlates well with the degree and persistence of the intrathymic presence of F1 type Ia+ cells. The long-lasting presence of donor-derived cells throughout the thymus tissue in the BM cell group is also in marked contrast to the early disappearance of Ia+ cells (within 2-3 weeks) from the cortex and then from the medulla in the PerC cell group, although these Ia+ cells were once spread throughout the thymus tissue 4 days after the tolerance-inducing cell inoculation. Taken together with a failure to induce consistent unresponsiveness to Mls-1a determinants in Mls-1b thymocytes regenerating in Mls-1a-thymic epithelial environments, all the above data indicate that intrathymic chimerism caused by hemopoietic stem cell-derived MHC-class II-bearing cells is a requisite for the induction and maintenance of unresponsiveness by means of clonal deletion in experimentally as well as naturally induced tolerance to Mls determinants.

Demonstration of clonal proliferation of T lymphocytes in early neoplastic disease. Studies with probes for the beta-chain of the T cell receptor and human T cell lymphotropic virus type I.

This study demonstrates that the detection of rearrangement of the T-cell receptor gene and adult T cell leukemia virus or human T cell lymphotropic virus type I (ATLV/HTLV-I) integration in the genome are sensitive and practical methods for the diagnosis and characterization of cutaneous T cell neoplasms. Biopsy specimens obtained from small skin nodules containing a dense infiltration of lymphocytes were analyzed by Southern blotting with the use of probes for both the beta-chain of the T-cell receptor gene and the ATLV/HTLV-I genome. DNA samples from one patient with early, chronic adult T cell lymphoma or leukemia, revealed the monoclonal proliferation of lymphocytes. For another patient with early cutaneous T cell lymphoma analysis by Southern blotting with the beta-chain probe of DNA from three separate lesions revealed identical rearranged bands, indicating not only the monoclonal proliferation of T cells in each lesion but also the clonal origin of each lesion. In contrast, analysis by Southern blotting of DNA samples extracted from three nodules from a patient with lymphomatoid papulosis showed no rearranged band and therefore no clonal proliferation. DNA extracted from blood lymphocytes of these patients failed to hybridize with the probes described above as a rearranged band. These results indicate that analysis of DNA extracted from skin lesions may have diagnostic value in determining monoclonal proliferation of lymphocytes in patients with early stage cutaneous lymphomas.

A case of a lymphoproliferative disorder: usefulness of DNA analysis in diagnosis.

We describe a patient in whom a suppressor/cytotoxic T cell lymphoma was suspected on the basis of histology and immunohistochemistry. DNA analysis of specimens from three different lesions, using restriction enzymes Eco RI and Bam HI, showed the same rearrangement of the T cell receptor beta-chain genes. We consider this technique to be of value in the early diagnosis of malignant lymphoma when differential diagnosis from other lymphoproliferative disorders is difficult with conventional methods.