Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat

Bio-Breeding rat T lymphocytes proliferate poorly in response to alloantigen. Transplantation of Bio-Breeding rats with fetal thymus tissue from diabetes resistant rats leads to an improvement in the T cell proliferative response, but only if the thymus contains bone marrow-derived, radiation-resistant thymic antigen presenting cells of the diabetes-resistant phenotype. The current study provides evidence that thymus transplantation leading to the restoration of Bio-Breeding T cell proliferative function can also significantly reduce the incidence of insulitis and prevent the development of diabetes. It appears that a defect in the bone marrow-derived thymic APC population contributes to an abnormal maturation of Bio-Breeding T lymphocytes which in turn predisposes animals to insulitis and diabetic disease.     

The specificity of the syngeneic mixed leukocyte response, a primary anti-I region T cell proliferative response, is determined intrathymically

In previous studies, the syngeneic MLR of peripheral T cells was shown to be predominantly an I region-restricted function. In this report we show that adult thymocytes are also capable of responding to syngeneic irradiated stimulator cells in a syngeneic MLR, provided that TCGF is added to the culture system. Using this assay, it was possible for the first time to examine the pattern of I region restriction within the thymus itself. Analysis of the thymocyte syngeneic MLR in thymuses from radiation-induced bone marrow chimeras demonstrated that the MHC preference seen in the peripheral T cell population also existed in cells resident within the thymus. Experiments utilizing congenitally athymic mice transplanted with allogeneic thymic grafts demonstrated that both peripheral T cells and thymocytes from such animals displayed a strong preferential proliferation toward stimulator cells bearing thymic-type MHC determinants. The results in the nude model thus demonstrate that the thymus by itself is sufficient to impart such restriction specificity on a developing T cell repertoire. These results are consistent with the notion that the thymus exerts selective pressure on maturing T cell populations that results in a skewing of the T cell repertoire toward the recognition of thymic-type I region products, and that this MHC preference exists before expansion of T cells in the periphery.     

Characteristics of the immature cells involved in T cell-mediated enhancement of syngeneic tumor growth.

Enhancement of tumor growth was observed when non-sensitized thymocytes were injected together with tumor cells into syngeneic mice, although this tumor enhancement was less pronounced than that caused by tumor-sensitized T lymphocytes. The cells within the thymus which are responsible for this tumor enhancement were found to be rapidly dividing and to be absent from the thymus a day after cortisone administration. At a longer time interval the cortison-depleted thymus was repopulated by dividing cells which exhibited tumor-enhancing reactivity. The characteristics of these cells suggest that they are in the early stages of thymic processing. The enhancing thymocytes were sensitive to treatment with the thymic humoral factor which functions in T cell maturation, and their enhancing activity was cancelled by such treatment. These results are compatible with our hypothesis that exposure of immature T cells to a tumor stimulus may lead to tumor enhancement whereas interaction between mature T lymphocytes and tumor cells may be required for tumor inhibition.     

Ontogeny of T cells: development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

Synergy among rat T cells in the proliferative response to alloantigen.

A synergistic interaction in the proliferative response to alloantigen is described for mixtures of rat thymus and lymph node cells. The optimal conditions for synergy are quantitatively defined. Regression analysis of the slope of the dose-response curve has been utilized to estimate the degree of interaction in thymus-lymph node cell mixtures. The slope of the response of cell mixtures was noted to be significantly greater than the slope for the response of lymph node cells alone. Irradiation was shown to have a differential effect on the response of thymus and lymph node cells in mixtures. Irradiated thymus cells retained the capacity for synergy in mixtures, whereas irradiated lymph node cells did not. Additional studies have demonstrated that both de novo protein synthesis and specific antigen recognition by both responding cell populations in mixtures was required for maximal synergy. These studies demonstrate that synergy cannot be explained as an artifact of altered cell density in vitro. They establish that thymus cells and lymph node cells represent distinct subsets which manifest qualitatively different functions in the proliferative response to alloantigen. Thymus cells can respond directly to alloantigen by proliferation but also have the capacity to amplify the proliferative response of lymph node cells—a capacity which is resistant to X irradiation but requires recognition of alloantigen and de novo protein synthesis. Lymph node cells may similarly respond by proliferation to alloantigen but lack the amplifier activity of thymus cells. Synergy for rat lymphoid cells, like mouse lymphoid cells, has been shown to involve an interaction of thymus-derived lymphocytes.     

Characterization of the proliferative response of a CD4-8- thymic T lymphoma cell line to stimulation by thymic cellular elements

E710.2 is a cloned T cell line that was isolated from an AKR/J thymic tumor. This clone expresses Thy-1, heat-stable Ag, and the CD3/TCR complex but does not express CD4 or CD8. When the E710.2 cell line is injected into syngeneic mice, it grows as a malignant tumor in lymphoid organs and the thymus. In contrast, this cell line does not grow in vitro under standard culture conditions. This latter property allowed us to analyze the in vitro responsiveness of this CD4-CD8- cell line to stimulation by pharmacologic agents and cellular elements from the spleen and thymus. E710.2 cells proliferate when stimulated with phorbol esters or when cocultured with thymocytes or splenocytes. We could not detect soluble stimulatory factors in cultures of E710.2 and/or lymphoid cells, suggesting that cell contact might be required for this response. The stimulatory activity in thymus and spleen appears to be broadly expressed, because all cell subsets that were examined from these tissues stimulate this cell line. The stimulation of E710.2 cells is not MHC-restricted and is not inhibited by anti-MHC mAb. Furthermore, the responsiveness of these cells is not decreased when the TCR/CD3 complex is modulated from the cell surface. Similarly, TCR/CD3-deficient E710.2 variant clones retain their responsiveness to thymic and splenic cell stimulation. These findings suggest that there is a TCR-independent pathway of activation in E710.2 that is stimulated by a broadly expressed, non-MHC-encoded molecules(s).     

Reciprocal T cell responses in the liver and thymus of mice injected with syngeneic tumor cells.

We investigated the T cell responses in various tissues, especially in the liver and thymus, of mice injected with syngeneic tumors. This study was undertaken since recent evidence indicated that the liver is one of the important immune organs for T cell proliferation. When C3H/He mice were intraperitoneally injected with mitomycin-treated syngeneic MH134 tumors (1 x 10(7)/mouse), a transient increase of liver mononuclear cells (MNC) was induced, showing a peak at Day 4 after injection. Histological study of such liver showed a sinusoidal dilatation and an accumulation of MNC in the sinusoids. The most predominant MNC induced were double negative (CD4-8-) alpha beta T cells and gamma delta T cells. These gamma delta T cells varied, showing unique time-kinetics. Despite a continuous increase of whole liver MNC and alpha beta T cells, the proportion of gamma delta T cells in the liver decreased beginning 4 days after injection. In contrast with the response in the liver, a striking decrease in the cell number of thymocytes was induced after tumor injection, showing a basal level at Day 6. This hypocellularity in the thymus appears to be an inverted response of the lymphocytosis in the liver. At this time, a corresponding decrease in the proportion of double positive (CD4+8+) T cells was always seen in the thymus. Analysis of cell proliferative response showed that the increase of liver MNC after tumor injection was accompanied by augmented proliferation, whereas the decrease of thymocytes was accompanied by depressed proliferation. The present results indicate that there exists a unique, reciprocal response of T lymphocytes between the liver and thymus, and that the presence of tumor appears to stimulate T cell response in the liver but alternatively inactivates such response in the thymus.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

A CXCR4-dependent chemorepellent signal contributes to the emigration of mature single-positive CD4 cells from the fetal thymus

Developing thymocytes undergo maturation while migrating through the thymus and ultimately emigrate from the organ to populate peripheral lymphoid tissues. The process of thymic emigration is controlled in part via receptor-ligand interactions between the chemokine stromal-derived factor (SDF)-1, and its cognate receptor CXCR4, and sphingosine 1-phosphate (S1P) and its receptor S1PR. The precise mechanism by which S1P/S1PR and CXCR4/SDF-1 contribute to thymic emigration remains unclear. We proposed that S1P-dependent and -independent mechanisms might coexist and involve both S1P-induced chemoattraction and SDF-1-mediated chemorepulsion or fugetaxis of mature thymocytes. We examined thymocyte emigration in thymi from CXCR4-deficient C57BL/6 embryos in a modified assay, which allows the collection of CD62L(high) and CD69(low) recent thymic emigrants. We demonstrated that single-positive (SP) CD4 thymocytes, with the characteristics of recent thymic emigrants, failed to move away from CXCR4-deficient fetal thymus in vitro. We found that the defect in SP CD4 cell emigration that occurred in the absence of CXCR4 signaling was only partially overcome by the addition of the extrathymic chemoattractant S1P and was not associated with abnormalities in thymocyte maturation and proliferative capacity or integrin expression. Blockade of the CXCR4 receptor in normal thymocytes by AMD3100 led to the retention of mature T cells in the thymus in vitro and in vivo. The addition of extrathymic SDF-1 inhibited emigration of wild-type SP cells out of the thymus by nullifying the chemokine gradient. SDF-1 was also shown to elicit a CXCR4-dependent chemorepellent response from fetal SP thymocytes. These novel findings support the thesis that the CXCR4-mediated chemorepellent activity of intrathymic SDF-1 contributes to SP thymocyte egress from the fetal thymus.     

The role of Ia molecules in the activation of T lymphocytes. IV. The basis of the thymocyte IL 1 response and its possible role in the generation of the T cell repertoire

The activation requirements for thymocyte proliferation were investigated. Thymocytes proliferate in the presence of exogenous interleukin 1, which has been used as the classic assay for this factor. This response, however, is greatly decreased in cultures of purified thymic T cells. Purified thymic T cells will proliferate in the presence of IL 1 if accessory cells are added to culture. The requisite accessory cell is a non-T, adherent, radioresistant cell found in macrophage/dendritic cell-enriched fractions of both thymus and spleen. This cell bears Ia molecules, which are critically involved in the activation of thymocytes. This thymocyte-accessory cell interaction is not dependent on exogenous nominal antigens. Therefore, it appears that IL 1 allows the expansion of thymocytes with specificity for self-class II MHC antigens. This response was found to be unique to this stage of T cell development and can be observed with both mature and immature thymic T cell subsets. The implications of these findings for the physiologic expansion of self-restricted T cells in the thymus are discussed.     

Human T cell antigen expression during the early stages of fetal thymic maturation

Human thymus tissue was examined from 7 wk of gestation through birth for the expression of antigens reacting with a panel of anti-T cell monoclonal antibodies. Additionally, the reactivities of reagents against the transferrin receptor, against leukocytes, against low m. w. keratins, and against major histocompatibility complex antigens were studied on human fetal thymic tissue. Frozen tissue sections were evaluated by using indirect immunofluorescence assays. At 7 wk of gestation, no lymphoid cells were identified within the epithelial thymic rudiment; however, lymphoid cells reacting with both antibody 3A1, a pan T cell marker, and antibody T200, a pan leukocyte reagent, were identified in perithymic mesenchyme. After lymphoid colonization of the thymic rudiment at 10 wk of fetal gestation, fetal thymic tissue reacted with antibodies T1, T4, and T8. At 12 wk of gestation, antibodies T3, T6, A1G3 (anti-p80, a marker of mature thymocytes), and 35.1 (anti-E rosette receptor) all reacted with thymic tissue. Our findings indicate that T cell antigens were acquired sequentially on thymocytes at discrete stages during the first trimester of human fetal development. The 3A1 antigen was present on fetal lymphocytes before lymphoid cell colonization of thymic epithelium, suggesting that passage through the thymus was not required for the expression of the 3A1 antigen by T cell precursors. The appearance of mature T cell antigens, T3 and p80, on thymocytes by 12 wk of gestation implies that the T cell antigen repertoire may be established in the thymus during the first trimester. Thus, a critical period of T cell maturation appears to occur between 7 and 12 wk of human fetal gestation.     

Differences in the pattern of proliferative response with age in thymocytes undergoing spontaneous and induced proliferation

The proliferative capacity of thymocytes from C3H/HeJ mice decrease as the animals attain maturity. The proliferative response of thymocytes from 24- to 28-week-old mice to stimulation with concanavalin A (Con A) is only 20% of that observed at 4 weeks of age. The decreased proliferative capacity of thymocytes in response to Con A stimulation observed between 4 and 24 weeks of age closely correlates to the drop in thymic weight and cellularity observed during this period. In contrast, the spontaneous proliferative capacity of thymocytes, as well as proliferation of thymocytes in response to stimulation with phorbol myristate acetate (PMA) and ionomycin, drops only slightly during this period, as proliferation under these condition in thymocytes from 24- to 28-week-old mice is approximately 65-70% of that observed in 4-week-old animals. We have previously shown that cytoplasmic extracts from proliferating lymphoid cells contain a factor, termed the activator of DNA replication (ADR), which is capable of inducing DNA synthesis in isolated, quiescent nuclei. We show in this study that the decreased proliferative capacity of thymocytes during whole organism maturation and thymic involution is associated with decreased endogenous levels of ADR, while nuclear sensitivity of thymocyte to ADR was retained during these process. The diminution of ADR activity during thymic involution was quantitatively greater than the loss in proliferative capacity.     

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics

Summary In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1–2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.     

IL-4 (B cell stimulatory factor 1) exhibits thymocyte growth factor activity in the presence of IL-2

The proliferative activity of thymocytes cultured with IL-2 and submitogenic concentrations of PHA is increased by 3- to 10-fold in the presence of IL-4. In contrast, IL-4 alone is unable to induce proliferative activity in thymocyte cultures and its synergistic activity is only apparent to concentrations of IL-2 above 1 U/ml. The costimulatory activity of IL-4 is abrogated by the monoclonal anti-IL-4 antibody 11B11. Furthermore, potentiation of the IL-2-mediated thymocyte proliferation is not seen with IL-1, IL-3, IFN-gamma, and granulocyte-macrophage CSF. Thymocytes are at least as responsive to IL-4 as B cells and the IL-4 costimulatory activity in fractionated thymocytes appears to be restricted mainly to the Lyt-2+/L3T4- population. In contrast, purified resting mature T cells do not respond to IL-4 plus IL-2, although they did proliferate in response to IL-4 in combination with PMA. These findings indicate that thymocytes and mature T cells are responsive to the costimulatory activity of IL-4 under quite different conditions, and that IL-4 may play an important role in thymocyte maturation in the thymus.     

The differentiation of suppressor cell populations as revealed by studies of the effects of mitogens on the mixed lymphocyte reaction and on the generation of cytotoxic lymphocytes.

The regulation by concanavalin A (Con A) and bacterial lipoloysaccharide (LPS) of the mixed lymphocyte reaction (MLR) and of the generation of cytotoxic lymphocytes (CL) was studied in congenic resistant mice using cortisone resistant thymocytes as the responding cells. LPS enhances the generation of CL selectively when suboptimal numbers of allogeneic cells are present in mixed lymphocyte cultures and also results in the augmentation of the MLR. Mitogenic concentrations of Con A on the other hand suppress the generation of CL regardless of alloantigen dose. The mechanism of suppression cannot be ascribed to the presence of suppressor T cells, since the addition to the cultures of syngeneic cortisone resistant thymocytes activated by Con A does not change the immune response. However, prospective suppressor cells that can be activated by Con A are located in secondary lymphoid organs such as spleen and lymph node. Suppressor activity by those cells is abolished by anti θ plus complement. Con A activated spleen cells suppress the MLR, whereas Con A activated thymocytes amplify the proliferation of responding cells.     

Opposing reactivities of subpopulations of T lymphocytes toward syngeneic tumor cells: separation of early thymocytes.

The present communication is a continuation of earlier studies which indicated that interaction between syngeneic tumors and those lymphocytes in the early stages of thymic processing can result in enhanced tumor growth in vivo. The thymocytes involved in this tumor enhancement were found previously in the rapidly dividing subpopulation of subcapsular cortical thymocytes, both in the untreated thymus and in the thymus undergoing repopulation after cortisone depletion. In the present experiments we have isolated this small subpopulation of early thymocytes. After cortisone injection such cells could be separated from the medullary cortisone-resistant thymocytes since the latter cells exhibit a high level of surface H-2 antigens and were thus lysed preferentially by anti-H-2 serum and complement. The repopulating subcapsular early thymocytes, which were resistant to this treatment, were incapable of responding to PHA while their basal proliferation rate was undiminished, and the majority of the cells were found to be dividing. When such low H-2 early thymocytes were injected together with three different tumors into syngeneic mice their tumor-enhancing activity was evident. It is clear that such early thymocytes are not devoid of biologic reactivity and their release from the thymus could have decisive results.     

Effect of syngeneic thymocytes on the proliferation of the small intestine epithelium in mice

The effect of syngeneic thymocytes on the proliferative compartment of the small intestinal epithelium was studied in CBA mice. In mice that intravenously received thymus cells, the proliferative enterocyte activity was depressed, the proliferative zone reduced and the maturation zone enlarged. The data obtained indicate that medullary thymocytes are responsible for this effect. The regulatory role of the immune system with regard to cell proliferation is discussed.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

Signal transduction in thymus development.

Reciprocal interaction between bone marrow derived lymphoid precursor cells and the thymic environment leads, through a series of developmental events, to the generation of a diverse repertoire of functional T-cells. During thymopoiesis fetal liver or bone marrow derived precursors enter the thymus and develop into mature T-cells in response to cues derived from the environment. The thymic micro-environment provides signals to the lymphoid cells as a result of cell-cell interactions, locally produced cytokines, chemokines and hormones. Developing thymocytes, in turn, influence the thymic stroma to form a supportive micro-environment. Stage-specific signals provide an exquisite balance between cellular proliferation, differentiation, cell survival and death. The result of this intricate signaling concert is the production of the requisite numbers of well educated self-restricted T-cells. Mature T-cells are exported to the peripheral lymphoid organs, where, upon encountering antigen, naive T-cells further mature into effector cells that provide cytolytic or T helper functions. While there are extra-thymic locations for T-cell development, majority of T-cells in peripheral lymphoid organs are thymus derived. In mice and humans, T-cells develop throughout life although the efficacy declines significantly with age. It is not clear if this is a direct consequence of deterioration of the thymic environment by involution, a paucity of bone marrow derived precursors, or both. However, new data clearly shows that the involuted adult thymus retains the ability to generate new T-cells. Recent advances have revealed many components of an exquisitely balanced signaling cascades that regulate cell fate, cellular proliferation and cell death in the thymus. This article describes fundamental features of developing thymocytes and the thymic micro-environment as they relate to the signaling pathways.