Recipient Thymus Research Articles

In Vivo Stem Cell Function of Interleukin-3-Induced Blast Cells

The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 x 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 x 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: Appearance in the periphery and Vβ utilization in vivo

We have recently demonstrated that bone marrow-resident cells, which are able to repopulate the thymus of irradiated recipient mice (pre-T cells), can be maintained in vitro for at least 2 weeks in the presence of exogenous IL-3. Because this marrow culture system can be applied to the study of early T cell differentiation, it is important to ascertain the extent to which in vitro culture of the pre-T cells might alter the T cell progeny which can develop from them. In previous work, we showed that the progeny of cultured pre-T cells appeared to develop in a kinetically normal fashion within the thymus of recipients and that the acquisition of key developmental markers (IL-2R and CD3) was identical in the progeny of fresh and cultured pre-T cells. Here, we report the results of experiments carried out to characterize the progeny of cultured pre-T cells which were found in the peripheral lymphoid tissues several weeks following intrathymic transfer to irradiated recipients. We found no remarkable differences between the progeny of cultured or fresh marrow cells with respect to the timing of their appearance in the periphery nor their expression of CD4 or CD8. By studying the patterns of utilization of five different Vβ gene products by the T cells derived from fresh or cultured bone marrow, we were able to test the susceptibility of both sets of progeny to both positive and negative selection pressures during their in vivo maturation. These experiments established that the progeny of cultured marrow cells were equally susceptible to TCR repertoire selection, as were the progeny of fresh bone marrow cells, and that the process of in vitro growth did not alter the potential TCR repertoire of the pre-T Cells.

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.

Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.

Although hematopoiesis is known to originate in a population of very primitive cells with both lymphopoietic and myelopoietic potential, a procedure for enumerating such cells has to date not been available. We now describe a quantitative assay for long-term repopulating stem cells with the potential for reconstituting all hematopoietic lineages. This assay has two key features. The first is the use of competitive repopulation conditions that ensure not only the detection of a very primitive class of hematopoietic stem cells but also the survival of lethally irradiated mice transplanted with very low numbers of such cells. The second is the use of a limiting-dilution experimental design to allow stem cell quantitation. The assay involves transplanting limiting numbers of male "test" cells into lethally irradiated syngeneic female recipients together with 1-2 x 10(5) syngeneic female marrow cells whose long-term repopulating ability has been compromised by two previous cycles of marrow transplantation. The proportion of assay recipients whose regenerated hematopoietic tissues are determined to contain greater than or equal to 5% cells of test cell origin (male) greater than or equal to 5 weeks later is then used to calculate the frequency of competitive repopulating units (CRU) in the original male test cell suspension (based on Poisson statistics). Investigation of this assay system has shown that all three potential sources of stem cells (test cells, compromised cells, and the host) can under appropriate circumstances contribute to long-term hematopoietic regeneration, thus establishing both the competitive pressure of hematopoietic stem cells in the cotransplanted compromised population and in the host, and the need to use genetic markers to track the specific contribution of the injected test cells. Analysis of the frequency of CRU in test marrow suspensions that varied widely in their CRU content gave similar values when endpoints of either 5 or 10 weeks posttransplantation were used and when either recipient marrow or thymus was used to identify progeny populations. In addition, repopulation of marrow and thymus was found to be associated in most mice injected with limiting numbers of test cells. These findings are consistent with the conclusion that the assay is highly selective for a very primitive, totipotent, reconstituting hematopoietic stem cell and should therefore be particularly useful in future gene therapy-oriented research as well as for more basic studies of hematopoietic stem cell regulation and differentiation.

Induction of Donor-Specific Unresponsiveness by Intrathymic Islet Transplantation

The application of isolated pancreatic islet transplantation for treatment of diabetes mellitus has been hampered by the vulnerability of islet allografts to immunologic rejection. Rat islet allografts that were transplanted into the thymus of recipients treated with a single injection of anti-lymphocyte serum survived indefinitely. A state of donor-specific unresponsiveness was achieved that permitted survival of a second donor strain islet allograft transplanted to an extrathymic site. Maturation of T cell precursors in a thymic microenvironment that is harboring foreign alloantigen may induce the selective unresponsiveness. This model provides an approach for pancreatic islet transplantation and a potential strategy for specific modification of the peripheral immune repertoire.

Cytoplasmic CD3+ surface CD8+ lymphocytes develop as a thymus‐independent lineage in chick‐quail chimeras

We have analyzed the embryonic development of a population of lymphoid cells that express a CD3 antigenic determinant in the cytoplasm but not on the cell surface. Since these cells lack T cell receptor (TcR) molecules, we have provisionally named them TCRO cells. Their development, expansion and distribution was investigated following transplantation of splenic and bursal fragments from chicken embryos into quail embryos. Since quail cells are not recognized by our panel of monoclonal antibodies against chicken TcR1, TcR2, TcR3, CD3, CD4 and CD8 molecules, these antibodies provided reliable markers for donor chick lymphocytes in the tissues of the quail recipients. Transplanted spleen and bursa both generated CD3+ cells, the number of which increased as a function of age. Notably, approximately half of these CD3+ cells expressed surface CD8, but none acquired TcR1 (gamma/delta), TcR2 (alpha/beta) or TcR3 expression. Since TCRO cells normally appear first in the spleen of 8-day chick embryos (E8), their generation in E6 splenic transplants indicated an extrathymic origin. The TCRO cells of chick splenic origin migrated to the spleen, bursa and thymus of the quail recipients. In six of seven chimeras acquiring CT3+ cells in the recipient thymus, these cells were restricted to the medulla and displayed the typical TCRO phenotype: CD3+CD8+TcR1-TcR2-TcR3-. These intrathymic TCRO cells also lacked the CT1 thymocyte antigen. We conclude that the TCRO cells represent a thymus-independent lineage of lymphoid cells that can migrate into a receptive thymus by rarely, if ever, differentiate into conventional T cells.

Quantification of the progenitors for thymic T cells in various organs.

Partial characterization and frequency determination of the progenitors for thymic T cells in various organs were made by transferring cells directly into the thymus (intrathymically, i.t.). B10. Thy-1.1 (H-2b, Thy-1.1) mice were used as the donor, and C57BL/6 (H-2b, Thy-1.2) mice that had been whole body irradiated with 800 rads and reconstituted with 10(7) syngeneic (B6) bone marrow (BM) cells were used as the recipient. BM cells, spleen cells and thymus cells from young adults and fetal liver cells (day 14 of gestation) were treated with anti-Thy-1.1 antibody plus complement, and transferred i.t. The generation of Thy-1.1+ donor type cells in the recipient's thymus was investigated by using flow cytometry. From the time course of generation, it was shown that the progenitor cells in the thymus were distinct from those in other organs. After the transfer of thymic non-T cells, donor-type cells began to generate on the 4th day, the proportion of donor-type T cells increased quickly thereafter, and the progenitors in this organ ceased to produce T cells by day 21. On the other hand, a latent period of about 10 days was required for progenitor cells in the BM, spleen or fetal liver to generate T cells, and T cell producing-activity of the progenitor cells in these organs lasted as long as 7 weeks. The frequency of progenitor cells was analyzed by transferring serial dilutions of anti-Thy-1.1 plus complement-treated cells i.t. and investigating the generation of donor-type T cells in the recipient's thymus on day 11 in the case transferred with thymic cells and on day 21 in other cases. The proportion of negative recipients which did not contain detectable levels of donor-type cells was plotted on a logarithmic scale against the number of cells transferred on a linear scale. The progenitor cell frequencies in BM, spleen, thymus and fetal liver were estimated to be 12.5 x 10(-5), 6.25 x 10(-5), 0.22 x 10(-5) and 0.14 x 10(-5), respectively. The reliability of the frequency determination was supported by the finding that when a limited number (10(3)) of a 1:1 mixture of BM cells from two mutually identifiable donors was transferred i.t., most of the recipients were negative for donor-type T cells and 3 out of 4 positive recipients were seeded by progenitor cells of a single donor.

Functional differentiation and repertoire diversification of T cells derived from single progenitor cells.

Functions of T cells derived from single progenitor cells were investigated. B10. Thy-1.1 recipient mice were either whole body-irradiated and marrow reconstituted or thymus-shielded, irradiated and marrow reconstituted, and limited numbers (3 x 10(3) or 6 x 10(3] of a 1:1 mixture of bone marrow cells from C57BL/6 and B6.Lyt-2.1 mice were transferred intrathymically (i.t.). Donor-type (Thy-1.2+) cells of the thymus of a small portion of recipients which expressed the phenotype of either Ly-2.2 or Ly-2.1 but not both were regarded to be a clone of T cells derived from a single progenitor cell, and such clones were assayed for polyclonal helper (Th) and polyclonal cytolytic (CTL) activities as well as alloantigen-specific proliferative (mixed lymphocyte reaction; MLR) and CTL activities. Clones taken 4 weeks after transfer (4-week-old clones) which were generated in the thymus of whole body-irradiated recipients showed polyclonal CTL but not polyclonal Th activity, whereas 4-week-old clones generated in the thymus of thymus-shielded recipients showed both polyclonal CTL and Th activities. Similarly, 4-week-old clones generated in whole body-irradiated recipients responded with CTL to alloantigens when induced in the presence of T cell growth factors but not with MLR, whereas 4-week-old clones generated in thymus-shielded recipients showed both MLR and CTL to alloantigens. Repertoire diversification of 4-week-old clones, however, was incomplete, since clones generated in whole body-irradiated recipient did not necessarily respond with CTL to all alloantigens examined, and those generated in thymus-shielded recipients did not necessarily respond with MLR to all the antigens. On the other hand, T cell clones were shown to fully mature by 7 weeks after transfer in terms of cell function as well as repertoire diversification.

Dysfunction of irradiated thymus for the development of helper T cells.

The development of cytotoxic T cells and helper T cells in an intact or irradiated thymus was investigated. C57BL/6 (H-2b, Thy-1.2) mice were whole body-irradiated, or were irradiated with shielding over either the thymus or right leg and tail, and were transferred with 1.5 X 10(7) bone marrow cells from B10.Thy-1.1 mice (H-2b, Thy-1.1). At various days after reconstitution, thymus cells from the recipient mice were harvested and a peanut agglutinin low-binding population was isolated. This population was further treated with anti-Thy-1.2 plus complement to remove host-derived cells and was assayed for the frequency of cytotoxic T cell precursors (CTLp) and for the activity of helper T cells (Th). In the thymus of thymus-shielded and irradiated mice, Th activity reached normal control level by day 25, whereas CTLp frequency remained at a very low level during these days. In the thymus of whole body-irradiated mice, generation of CTLp was highly accelerated while that of Th was retarded, the period required for reconstitution being 25 days and more than 42 days for CTLp and Th, respectively. Preferential development of CTLp was also seen in right leg- and tail-shielded (L-T-shielded) and irradiated recipients. Histological observation indicated that Ia+ nonlymphoid cells were well preserved in the thymus of thymus-shielded and irradiated recipients, whereas in L-T-shielded and irradiated recipients, such cells in the medulla were markedly reduced in number. These results suggest strongly that the generation of Th but not CTLp is dependent on radiosensitive thymic component(s), and that such components may represent Ia+ cells themselves in the medulla or some microenvironment related to Ia+ cells.

A human autoreactive T cell line specific for minor histocompatibility antigen(s) isolated from a bone marrow-grafted patient.

A human autoreactive T cell line named Bur-1 has been obtained from a woman 4 mo after an allogeneic bone marrow transplantation (BMT) from one of her HLA-identical brothers. The phenotype of the cell line is 100% T11+ and over 90% T4+, and the karyotype confirms its donor (male) origin. These donor T cells proliferate specifically in the presence of donor's peripheral blood monocytes (PBM) but not recipient's cells, and they kill specifically donor's but not recipient's Epstein-Barr virus (EBV)-induced lymphoblastoid cell lines (LCL). PBM from another HLA-identical brother and from several unrelated donors also stimulate Bur-1 cells, and EBV-induced LCL from the same donors are killed in cytotoxicity assays. All of these donors share HLA-DR5 or HLA-DRw11 (the major split of HLA-DR5) with Bur-1 cells. However, some but not all of the PBM sharing HLA-DR5 with Bur-1 cells are recognized. Therefore, in contrast with the previously described autoreactive T cells, Bur-1 cells are not directed against self-MHC antigens but rather recognize autologous minor histocompatibility (mH) antigens in the context of autologous HLA class II molecules. Because both male and female cells can be recognized, the reacting minor antigen could not be the male-specific HY antigen. It is suggested that autoreactivity against mH antigens can be observed in bone marrow-grafted patients due to the education of bone marrow donor precursors in the recipient thymus not allowing tolerance to autologous (donor) mH antigens not shared by the recipient.

Progenitor cells in the thymus: Most thymus-homing progenitor cells in the adult mouse thymus bear Pgp-1 glycoprotein but not interleukin-2 receptor on their cell surface

PgP-1-positive and interleukin-2-receptor (IL-2R)-positive cells are both minor (< 5%) subpopulations within the adult thymus. A thymocyte population enriched for these two cell types, obtained by killing the bulk of thymocytes with anti-Thy-1 antibody and complement, contains thymus-homing progenitor cells which can transiently repopulate the thymus of irradiated recipients. Using two-color immunofluorescence, we demonstrate that the Pgp-1 + and IL-2R + cells present in this enriched population represent largely nonoverlapping subsets, although some cells do express both markers. We also show by depletion of these two cell types that the bulk of the thymus-homing progenitors present in this enriched population are found in the Pgp-1 + population, and not in the IL-2R + population. We discuss the relationship between the thymus-homing progenitors in this depleted thymus subpopulation and the thymus-homing progenitors present in the thymus as a whole.

Two subpopulations of stem cells for T cell lineage.

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.

Endocrine control of thymic serum factor production in young-adult and old mice

The influence of different endocrinological manipulations on the blood concentration of serum thymic factor (FTS) was studied in young-adult and old mice. Among the experimentally induced endocrinopathies in youth, hypothyroidism and diabetes caused strong reductions of FTS levels, which were restored to normal by the appropriate hormonal substitutive therapy. Removal of adrenals or gonads has no significant effect on FTS level. Old mice, which show undetectable levels of FTS and low levels of thyroxine, can regain the capacity to produce FTS, provided they are treated with thyroxine. The variations of FTS blood levels in the course of endocrinological manipulations were due to a direct or indirect effect exerted on the recipient thymus. Hormonal treatment of thymectomized mice did not induce any FTS-like activity in their sera, nor did hormones interfere in vitro with the bioassay used to test for FTS. These data suggest that the neuroendocrine balance modulates the synthesis and/or the release of FTS from the thymus during the whole life of the organism and that the decline of FTS production with advancing age is largely dependent on age-associated endocrinological imbalances.

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation.

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.

Clonal abortion of bone marrow T cell precursors: T cells acquire specific antigen reactivity prethymically.

Lethally irradiated (900 R) mice were reconstituted with bone marrow cells from syngeneic donors that had been tolerized 2 to 3 wk earlier to either DNP or TNP compounds. Five weeks after reconstitution, these animals were tested for their ability to mount a delayed hypersensitivity (DH) response to the tolerizing haptens. Recipient mice were specifically tolerant to the hapten that was used to induce tolerance in the marrow donor. Mixing experiments in which mice were reconstituted with marrow from DNP-tolerant and TNP-tolerant donors showed no indication of active suppression or effective antigen carry-over in this system. This observation held true even in experiments in which mice were reconstituted with a mixture of marrow from tolerant and normal donors at a ratio of 5:1. Thus the reduced responsiveness in recipient mice seemed to be due to the functional elimination of hapten-responsive T cell precursor (pre-T) clones. Recipient unresponsiveness was also shown to be MHC restricted. Maintenance of unresponsiveness appeared to be due to the restricted access of regenerating pre-T cell clones to the maturational influence of the recipient's thymus.

FUNCTION AND MIGRATION OF SUPPRESSOR LYMPHOCYTES FROM CYCLOSPORINE-TREATED HEART GRAFT RECIPIENTS

(LEW × BN)F1 cardiac allografts survive more than 100 days in LEW recipients treated with cyclosporine 15 mg/kg i.m. for 7 days only. Transfer of thymocytes (TL) or splenocytes (SL) from these hosts into untreated syngeneic rats significantly prolongs the survival of (LEW × BN)F1 test cardiac allografts (P< 0.001). In the present study the subset of T lymphocytes responsible for this effect and its migration patterns have been defined. T helper lymphocytes (Th) and T cytotoxic/suppressor cells (Tc/s) were separated from thymus and spleen of cyclosporine-treated recipients using monoclonal antibodies W3/25 and MRC OX8, respectively. The cell subsets were adoptively transferred into syngeneic recipients. TL from cyclosporine-treated, grafted rats significantly improved test graft survival to about 12–14 days, irrespective of the subpopulation transferred. Unseparated SL as well as T lymphocytes and Tc/s cells also prolonged test graft survival (P < 0.001); transfer of The or serum was ineffectual. The presence of monoclonal antibody on the surface of the transferred lymphocytes inhibited their function in vivo. Thus, test grafts following transfer of unseparated SL and Tc/s coated with monoclonal antibody were rejected acutely, in contrast to uncoated cells or cells coated with antibody and then treated with pronase. We studied the migration patterns of 3H-adenosine labelled SL, Th, and Tc/s adoptively transferred into normal LEW rats. SL and Tc/s, both active in transfer of unresponsiveness from cyclosporine-treated hosts migrated similarly—into lymph nodes and away from spleen of test animals, in contrast with the pattern seen in the control groups. The-coated and monoclonal-antibody-coated SL were preferentially trapped in the liver. Thus these studies provide direct evidence that Tc/s lymphocytes transfer specific unresponsiveness from cyclosporine-treated and grafted hosts.

Anti-self receptors. III. Lack of allelic exclusion and thymic epithelium dependence of H-2L-region-restricted receptors on lymphocytes.

Murine thymocytes and peripheral lymphocytes bind autologous erythrocytes via H-2L-region-restricted receptors. After inhibiting autorosetting with different erythrocyte sonicates the specificity of these anti-self receptors was examined in F1 hybrid and chimaeric mice. Most F1 lymphocytes simultaneously expressed receptors against both parental haplotypes. Furthermore, analysis of lymphocytes from allogeneic and semi-allogeneic chimaeras clearly demonstrated that radioresistant elements in the recipient thymus did not modify the haplotype specificity of the receptors on donor-derived lymphocytes.

Induction of suppressor cells by donor-specific blood transfusions and heart transplantation in rats.

In the BN/Rij to WAG/Rij rat donor-host combination, a single injection of 1 ml of donor blood 7 days before transplantation leads to permanent acceptance of BN/Rij hearts. In this model of specific unresponsiveness, it was investigated whether suppressor cells were present in the steady-state phase at 5 to 6 weeks after transplantation. Thymocytes, spleen cells, peripheral blood lymphocytes, and lymph node cells from blood-conditioned recipients were adoptively transferred to WAG/Rij recipients irradiated with 450-rad X-rays. BN/Rij heart transplantation was performed after 14 days. It was found that suppressor cells were present in the spleen and thymus of unresponsive recipients but not in the peripheral blood or lymph nodes. Adoptive transfer of 25 x 10(6) spleen cells led to permanent survival of BN/Rij hearts in four of nine cases, whereas transfer of 25 x 10(6) thymocytes always resulted in permanent graft survival. Fractionation of suppressor spleen cells into T and B cell-enriched populations and macrophages revealed that the suppression was mediated by T cells.

Evidence for the presence of precursor B cells in normal and in hormonally bursectomized chick embryos

Embryonic bone marrow of normal and hormonally bursectomized chicks was examined for the presence of hematopoietic precursor cells capable of migrating to the thymus and bursa and of differentiating into functional T and B cells, respectively. Following transfer of chromosomally marked bone marrow of normal and in ovo bursectomized 14-day-old embryos to 14-day-old γ-irradiated embryonic recipients, donor cells proliferated in the marrow, thymus, and bursa of recipients, and differentiated to PHA- and Con A-responsive T cells as well as to dextran sulfate- and anti-immunoglobulin-responsive B cells. In contrast, when marrow of 2-day-old hatched normal and in ovo-bursectomized donors was transferred to 14-day-old embryonic recipients, donor cells repopulated only the marrow and thymus of recipients which was followed by differentiation to Con A- or PHA-responsive T cells, but the same donor cells failed to proliferate in the bursa and there was no differentiation to functional B cells of donor type. The data were fitted to a model of T- and B-cell differentiation from the stem cell level and they suggest the presence of separate populations of committed precursor T (PT) and precursor B (PB) cells in the marrow of normal and in ovo bursectomized embryos with a bursa-independent selective disappearance of PB cells from the marrow during the late embryonic period.

Influence of the recipient thymus on the maturation of T-lymphocytes in H-2 different radiation chimeras.

Graft-versus-host reactions in mice can be suppressed by incubation of the donor cells with T-cell-specific antibodies prior to transplanting them into lethally irradiated recipients. Since reconstitution of the immune functions in these recipients requires the formation of a new T-lymphocyte population, we investigated whether H-2 differences between recipient thymus and donor stem cells as well as an age dependent thymic involution impaired the maturation of immunocompetent T-lymphocytes. For this purpose the immune response of different chimeric mice against SRBC and third party skin grafts as well as the repopulation of the lymphatic organs with T-lymphocytes was evaluated. Chimeras of the type CBL leads to CBL/CBA and CBL/CBA leads to CBA were used for the experiments. In addition chimeras which had been thymectomized, transplanted with the thymus of one parent strain, lethally irradiated and transfused with bone marrow from other parent strain were analyzed. In all chimeric combinations, even in the case of complete H-2 difference between donor cells and recipient thymus, a normal or almost normal reconstitution of the immune response against SRBC and incompatible skin grafts were detected. Furthermore age-involuted thymi returned to normal morphologically and functionally after bone marrow transplantation. The repopulation of the lymphatic organs in the investigated chimeric mice was remarkably similar to normal controls. This indicates that hemopoietic stem cells and/or lymphopoietic progenitors may differentiate in a thymus in spite of H-2 differences to functional T-lymphocytes able to respond against SRBC and incompatible skin grafts.