Search

A factor secreted by thymocytes of immunized rabbits totally suppressed both the initiation of, and ongoing synthesis and secretion of, lectin (PWM)-induced synthesis of IgM and IgG immunoglobulins by the circulating B lymphocytes of normal humans, and of twenty consecutive patients with rheumatoid arthritis and twelve consecutive patients with systemic lupus erythematosus. The suppressor factor, referred to as human Ig synthesis/secretion suppressor factor or HISSF, is not HLA restricted in its activity and is not cytotoxic to the circulating human mononuclear cells (B cells, T cells, Null cells and monocytes). It was demonstrated that T cells precultured with HISSF were transformed into suppressor cells which, when added to fresh cultures of autologous B cells, suppressed the synthesis and secretion of IgM and IgG. On the basis of its suppressive and non-cytotoxic properties in vitro, HISSF may be an effective immunosuppressant in the treatment of patients with autoimmune diseases.

We analyzed the genetic restriction of direct cell-cell interactions between thymocytes and a) cortical epithelial cells, b) macrophages, and c) medullary dendritic cells in the mouse thymus. Thymectomized (C3H X C57BL/Ka)F1 hybrid mice were doubly grafted with P1 and P2 neonatal thymus grafts, were lethally irradiated, and were reconstituted with a mixture of P1 and P2 bone marrow cells which differed in the Thy-1 locus. The contributions of both parental inocula to the composition of the free and stromal cell-associated T cell compartments were analyzed separately in thymic grafts of each parental strain. The lymphoid composition in both compartments essentially reflected the peripheral T cell-chimerism in the host. The development of lymphostromal complexes was not restricted by the genotype of the partner cells. Statistical analysis of the distributions of P1 and P2 T cells among free thymocytes and within individual lymphostromal complexes, however, suggests that the T cells of an individual complex are the progeny of oligoclonal proliferation. Thus, both epithelial cells and bone marrow-derived stromal cells seem to be involved in different stages of intrathymic lymphopoiesis.

Using the technique of competitive labelling (Kaplan, H., Stevenson, K.J. and Hartley, B.S. (1971) Biochem. J. 124, 289-299) with 1-fluoro-2,4-dinitro-[3,5-3H]benzene ([3H]Dnp-F), the chemical reactivity of the alanyl amino-terminus of H3, the prolyl amino-terminus of H2B and the sulphydryl groups of H3 within calf thymus chromatin were determined. Over pH values from 6 to 10, none of the groups had pH-reactivity profiles which fit titration curves, indicating that they are involved in specific interactions with other structural elements of chromatin. The alanyl amino-terminus is completely nonreactive over the entire pH range studied. The prolyl amino-terminus is reactive but its reactivity is considerably lower than expected for a secondary amino group. An unusual feature of this group is that its pH-reactivity profile has sharp discontinuities at pH values of 7.8 and 9. The average reactivity of the two sulphydryl groups of H3, unlike the two amino-termini, appears to approximate that expected of a free sulphydryl group.

BALB/c mice infused with 30 x 10(6) C57BL/Ka bone marrow (BM) cells 1 day after treatment with fractionated total lymphoid irradiation (TLI) (17 fractions of 200 rads each) became stable mixed chimeras without clinical graft-vs-host disease (GVHD). Mice given 18 fractions of 100, 50, or 25 rads each followed 1 day later by C57BL/Ka BM did not become chimeric, indicating that a critical cumulative radiation dose is required for this effect. Animals given TLI with lead shielding placed over the thymus also developed stable chimerism without GVHD. Thus susceptibility to tolerance induction and protection from GVHD after TLI and allogeneic BM transplantation is not due to alteration of the thymic microenvironment by fractionated irradiation. A delay of 7 or 21 days between completion of TLI and BM administration resulted in a high incidence of graft rejection. Sensitization to minor histocompatibility antigens of the BM donor strain by blood transfusion either before or during TLI resulted in marrow graft rejection in a high percentage of animals.

Transplantation of thymus and bone marrow cells from irradiated C57BL/Ka mice demonstrated the presence of potentially neoplastic cells in the thymus at 30 to 60 days postirradiation. During the same interval, no such cells could be detected in the bone marrow; moreover, the capacity of bone marrow cells to repopulate the thymus was impaired severely. These observations suggest that the primary site of neoplastic transformation in irradiated C57BL/Ka mice is the thymus rather than the bone marrow and that impaired thymic regeneration is a critical step in radiation leukemogenesis in mice.

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.

The tissue distribution and extent of virus-specific antigen expression were studied by immunofluorescence as a function of time and of lymphoma development in adult C57BL/Ka (Fv-1(b)) mice after intravenous injection of radiation leukemia virus, a B-tropic murine leukemia virus. Viral antigens were detected earlier in the thymus (1 week) than in the bone marrow, spleen, or lymph nodes (2-3 weeks). Despite an initial virus-induced thymic involution, the percentage of immunofluorescence-positive cells in the thymus rapidly increased thereafter to 65-80%, at which level it remained until 9 weeks, at which time increases in size and weight, histological changes, and an increased number of blastoid cells indicated the onset of lymphoma development in the thymus. In contrast, the percentage of immunofluorescence-positive cells in the bone marrow, spleen, and nodes remained low, and gradually decreased to zero within 8 weeks after thymectomy. The selective thymic localization of antigens induced by radiation leukemia virus in C57BL/Ka mice is in striking contrast to the previously reported ubiquitous tissue distribution of the Gross-AKR virus, an N-tropic virus, in its natural host, the Fv-1(n), AKR strain with a high incidence of leukemia.

Thymic nurse cells (TNC), complexes between epithelial cells and thymocytes, are further characterized. Our results show that TNC are predominantly located in the subcapsular and outer cortical region of the mouse thymus. The enveloped thymocytes display the characteristics of cortical thymocytes. They are PNA+, Thy-1+, Lyt-123+, TL+, ThB+, H-2K+, IA+, and cortisone and radiation sensitive. In vitro cultivation of TNC in the presence of interleukin 2 induces a phenotype change to Lyt-1-, ThB-, Lyt-23+. The yield of TNC is highly dependent on the isolation conditions. Moreover, the thymocytes enveloped by an individual TNC do not constitute a single clone. The latter findings strongly suggest that complete envelopment is probably an isolation artifact. However, the cellular composition of the complexes is not random but rather the result of a preexisting in vivo association between an immature thymocyte subpopulation and outer cortical epithelial cells. TNC represent the in vitro correlate of this in vivo interaction. The implications of these results for intrathymic T cell differentiation are discussed.

Studies on the maturational lineages of thymic lymphocytes have revealed several subclasses which are distinguishable on the basis of cell size, topographic distribution within the thymus, DNA synthetic and mitotic activity, migratory behavior, and other properties. Strain C57BL/Ka mice were inoculated with radiation leukemia virus at different concentrations, and tissues were removed at defined intervals. Sequential sections were analyzed for virus-specific cytoplasmic antigen expression, for morphological evidence of neoplastic transformation, and for alkaline phosphatase activity. The first detectable sign of MuLV infection was the focal appearance of cytoplasmic viral antigens in cells of the outer thymic cortex, followed by coalescence of such foci and, several weeks later, by the appearance of morphologically transformed and alkaline phosphatase-positive cells, again often focally distributed in the outer thymic cortex. These observations strongly suggest that the large, mitotically active cells of the outer thymic cortex are the principal source of target cells for both productive infection and subsequent lymphoma induction by the virus.

A method of cultivation has been developed that yields thymus reticuloepithelial (TRE) cell monolayers composed almost entirely of epithelial-like cells, with no detectable macrophages or lymphocytes. These cultures show the capacity to induce responsiveness to lectin mitogens in immature thymocytes. Such monolayers induce Thy-1 expression in nude mouse spleen and bone marrow cells. The functional macrophage-lymphocyte depleted TRE monolayers can be cryptically infected by radiation leukemia virus (RadLV). However, RadLV-infected TRE cells lack the capacity to induce neoplastic transformation in normal weanling and newborn thymocytes. Cultures prepared from C57BL/Ka mice treated with a leukemogenic course of irradiation produce a B-fibrotropic nonleukemogenic virus, and normal thymocytes cocultivated with these monolayers do not undergo in vitro transformation. It is concluded that TRE monolayers depleted of macrophages and lymphocytes: (1) exhibit the functional capacity to induce some of the properties of mature T cells; (2) are infectable by RadLV; (3) are one of the sites for expression of ecotropic virus after leukemogenic fractionated irradiation; and (4) are not capable of transforming normal thymocytes in vitro.

Bone marrow (BM) and skin allografts from C57BL/Ka (H-2b/b) mice were transplanted to BALB/c (H-2d/d) recipients treated with total lymphoid irradiation (TLI), whole-body irradiation (WBI), or fractionated thymic irradiation TLI prolonged skin allograft survival about five times as long as that in untreated controls, and allowed for permanent engraftment of BM cells in approximately equal to 90% of recipients. None of the BM recipients showed clinical signs of graft-versus-host disease (GVHD) (diarrhea, weight loss, hunched back, etc.). On the other hand, recipients given WBI and allogeneic BM cells developed severe clinical GVHD. The majority of the latter recipients died within 12 days after BM transplantation, and 95% died within 61 days. Although TLI protected BALB/c mice against GVHD induced by BM cells, all recipients given TLI and allogeneic spleen cells developed lethal GVHD. Thymic irradiation alone marginally prolonged skin allograft survival, and did not allow for allogeneic BM engraftment. These results suggest that TLI may be a useful regimen in clinical BM transplantation, since this form of radiotherapy is used extensively in humans and has few severe side effects.

We describe the isolation and purification of multicellular complexes composed of lymphocytes and bone marrow-derived stromal cells ("thymocyte rosettes") from the mouse thymus. These rosettes are the structural in vitro correlate of in vivo associations between lymphoblasts and I-A/E negative macrophages or medullary I-A/E positive dendritic-like cells. Both types of rosettes are preformed in vivo. The rosette-associated thymocytes display a surface antigen phenotype typical of immature thymocytes. In radiation chimeras, replacement of host thymocytes by injected bone marrow cells follows a regular pattern: donor type T cells appear first at day 11 as clusters around I-A negative macrophages and approximately 2 days later as similar clusters associated with either I-A positive cortical epithelial cells or I-A positive medullary dendritic cells. These data suggest (a) a defined sequence of lymphostromal interactions during intrathymic maturation and (b) a rapid proliferation of thymocytes after interaction with stromal cells.

The spread of virus replication was studied by electron microscopy in the thymuses of inbred C57BL/Ka mice after intrathymic inoculation of the radiation leukemia virus (RadLV). The first type C-budding virus particles appeared in scarce blast cells of the subcapsular zone. Most of these blast cells were "X-cells," i.e., the thymus lymphoid cells most actively engaged in DNA synthesis. Virus replication spread to the entire cortical blast cell population and, from day 7 on, to the small cortical lymphocytes. The first virus-producing cells were derived from a very few target cells (approximately 0.001-0.003% of thymocytes) susceptible to RadLV infection. For determination of the phenotypes of these target cells, various thymocyte subpopulations obtained through a battery of cell separation methods were tested for their ability to support the replication of RadLV/VL3 virus in short-term culture. Most of these target cells were sensitive to the lytic effect of hydrocortisone and migrated in the fastest fraction of a 1Xg sedimentation gradient, together with the majority of [3H] thymidine-incorporating blast cells. They exhibited an intermediate density and expressed H-2 and Thy 1,2 cell surface antigens, although they were not found preferentially among the high Thy 1,2 population to which most of the cortical blast cells belonged. The spread of RadLV within the thymus and the surface phenotype characteristics of target cells indicate that these cells correspond to the thymocyte subset at the earliest stage of thymic lymphopoiesis and may be transitional between the prothymocytes and the subcapsular blast cell population.

Short-term lymphocyte cultures from mouse thymus, spleen, or lymph nodes were studied for the presence of murine leukemia virus group-specific antigens with an immunofluorescence test using rat immune sera against syngeneic cells infected with the radiation leukemia virus or its pseudotype of murine sarcoma virus and goat and rabbit antisera against purified murine leukemia virus group-specific antigen. Antigens reacting with these sera appeared in the cultured lymphocytes within 24 hr, and the proportion of immunofluorescent-positive cells increased to 25-80% by the second or third day of cultivation, concomitantly with a decrease in cell viability. The appearance of these antigens can be suppressed by inhibitors of DNA (mitomycin-C), RNA (actinomycin-D, cordycepin, and polyadenylic acid), and protein (cycloheximide) synthesis. No infectious virus could be detected by the immunofluorescence and XC-cell tests. The observed phenomenon appears to represent the spontaneous partial derepression of endogenous murine leukemia virus replication in lymphocytes during short-term in vitro cultivation.