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.     

NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells

NK1.1+ T cells represent a specialized T cell subset specific for CD1d, a nonclassical MHC class I-restricting element. They are believed to function as regulatory T cells. NK1.1+ T cell development depends on interactions with CD1d molecules presented by hematopoietic cells rather than thymic epithelial cells. NK1.1+ T cells are found in the thymus as well as in peripheral organs such as the liver, spleen, and bone marrow. The site of development of peripheral NK1.1+ T cells is controversial, as is the nature of the CD1d-expressing cell that selects them. With the use of nude mice, thymectomized mice reconstituted with fetal liver cells, and thymus-grafted mice, we provide direct evidence that NK1.1+ T cells in the liver are thymus dependent and can arise in the thymus from fetal liver precursor cells. We show that the class I+ (CD1d+) cell type necessary to select NK1.1+ T cells can originate from TCRalpha-/- precursors but not from TCRbeta-/- precursors, indicating that the selecting cell is a CD4+CD8+ thymocyte. 5-Bromo-2'-deoxyuridine-labeling experiments suggest that the thymic NK1.1+ T cell population arises from proliferating precursor cells, but is a mostly sessile population that turns over very slowly. Since liver NK1.1+ T cells incorporate 5-bromo-2'-deoxyuridine more rapidly than thymic NK1.1+ T cells, it appears that liver NK1.1+ T cells either represent a subset of thymic NK1.1+ T cells or are induced to proliferate after having left the thymus. The results indicate that NK1.1+ T cells, like conventional T cells, arise in the thymus where they are selected by interactions with restricting molecules.     

Characterization of an endogenous Lyt2+ T-suppressor-cell population regulating autoreactive T cells in vitro and in vivo

Autoreactive T cells have been defined by their capacity to respond to self-Ia antigens expressed on non-T cells. Several recent studies have suggested that these cells may play important immunoregulatory functions. However, it is not clear what regulates the responsiveness of autoreactive T cells and why such cells are not demonstrably stimulated in vivo, where they are in the constant presence of self-Ia antigens. In the present study we examined the role of T suppressor (Ts) cells in regulating autoreactive T cells. We observed that enhanced autoreactivity occurred in vitro when Lyt2+ T cells were depleted from the responding and/or stimulating spleen cells in a syngeneic mixed-lymphocyte reaction. Similarly, addition of irradiated Lyt2+ T cells but not L3T4+ T cells inhibited the response of L3T4+ T cells to self-Ia antigens. The activity of the suppressor cells was specific to the autoreactive T cells since antigen-specific and alloreactive T-cell proliferation were not inhibited. Furthermore, depletion of Lyt2+ T cells by in vivo treatment of mice with anti-Lyt2 monoclonal antibodies caused enhanced endogenous proliferation of lymph node and splenic T cells and increased the T-cell response to self-Ia antigens in vitro. These studies, therefore, suggest that T-cell tolerance to self-Ia antigens in vivo may be maintained by naturally occurring Lyt2+ Ts. Mice having enhanced autoreactivity may provide a useful tool to address the role of autoreactive T cells in the immune response to foreign antigens and in the pathogenesis of autoimmune diseases.     

Isolation of a thymus-homing Lyt-2-, L3T4- T-cell line from mouse spleen

We have selectively isolated and transformed a population of T-cell-receptor+, Lyt-2-, L3T4- cytotoxic T cells from mouse spleen following stimulation in vivo with a radiation leukemia virus-induced thymoma, C6VL/1. The two sublines analyzed here were found to induce tumors with primarily thymic involvement and one of these has been shown to have specific homing capacity for the thymus. Properties displayed by this cell line are evidence that T cells do exist in peripheral lymphoid tissue which can traffic back to the thymus and that Lyt-2-, L3T4- immature T cells can enter peripheral lymphoid organs.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

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.     

Peripheral T cell lymphopenia and concomitant enrichment in naturally arising regulatory T cells: the case of the pre-Talpha gene-deleted mouse

In pre-Talpha (pTalpha) gene-deleted mice, the positively selectable CD4+ CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+ FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTalpha(-/-) peripheral T cell phenotype could be reproduced with normal cells. In the pTalpha(-/-) thymus and peripheral lymphoid organs, FoxP3+ CD4+ cells were enriched. Parabiosis experiments showed that many pTalpha(-/-) CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+ CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTalpha(-/-) mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.     

Developmental regulation of the intrathymic T cell precursor population

The maturation potential of CD4-8- thymocytes purified from mice of different developmental ages was examined in vivo after intrathymic injection. As previously reported, 14-day fetal CD4-8- thymocytes produced fewer CD4+ than CD8+ progeny in peripheral lymphoid tissues, resulting in a CD4+:CD8+ ratio of less than or equal to 1.0. In contrast, adult CD4-8- thymocytes generated CD4+ or CD8+ peripheral progeny in the proportions found in the normal adult animal (CD4+:CD8+ = 2 to 3). Here we have shown that CD4-8- precursor cells from the 17-day fetal thymus also produced peripheral lymphocytes with low CD4+:CD8+ ratios. Precursors from full term fetuses produced slightly higher CD4+:CD8+ ratios (1.1-1.6) and precursors from animals three to 4 days post-birth achieved CD4+:CD8+ ratios intermediate between those produced by fetal and adult CD4-8- thymocytes. Parallel changes in the production of alpha beta TCR+ peripheral progeny were observed. Fetal CD4-8- thymocytes generated fewer alpha beta TCR+ progeny than did adult CD4-8- thymocytes. However, peripheral lymphocytes arising from either fetal or adult thymic precursors showed similar proportions of gamma delta TCR+ cells. The same pattern of progeny was observed when fetal CD4-8- thymocytes matured in an adult or in a fetal thymic stromal environment. In contrast to fetal thymic precursors, fetal liver T cell precursors resembled adult CD4-8- thymocytes by all parameters measured. These results suggest that fetal thymic precursors are intrinsically different from both adult CD4-8- thymocytes and fetal liver T cell precursors. Moreover, they lead to the hypothesis that the composition of the peripheral T cell compartment is developmentally regulated by the types of precursors found in the thymus. A model is proposed in which migration of adult-like precursors from the fetal liver to the thymus approximately at birth triggers a transition from the fetal to the adult stages of intrathymic T cell differentiation.     

The induction of peripheral T cell unresponsiveness in adult mice by monomeric human gamma-globulin

Monomeric human gamma-globulin (HGG), when injected into adult mice, induces a state of specific immunologic unresponsiveness to further challenge with immunogenic forms of HGG. In this report we have directly determined the role of the thymus in the induction of HGG tolerance and the proliferative responsiveness of T cells from normal and HGG-tolerant mice. Draining lymph node T cells were isolated from HGG-tolerized and -challenged mice, and tested for their proliferative response to HGG in vitro. T cells from untreated but challenged adult CBA/CaJ and A/J mice proliferate in response to HGG, whereas such mice given monomeric HGG before challenge fail to show an HGG-specific proliferative response. APC from tolerant or nontolerant mice were equally effective in the support of Ag-specific proliferation of primed T cells. The influence of the thymus gland on HGG-induced T cell unresponsiveness was assessed by determining whether thymectomized mice could be tolerized to HGG. The results suggest that the generation of T cell tolerance to HGG is independent of thymic function as assayed by both antibody production in vivo and T cell proliferation in vitro. Unresponsiveness of T cells from tolerant mice was not a result of the presence of CD8+ cells since removal of CD8+ cells from lymph node T cells did not alter unresponsiveness to HGG in vitro. Further, mixing tolerant T cells with normal HGG-primed T lymphocytes did not inhibit proliferation of the HGG-primed cells. The results of this investigation suggest that this mouse model of tolerance to HGG represents a thymus-independent unresponsiveness of mature peripheral T cells to a nonself-Ag. Understanding the regulation of tolerance to HGG may give additional insight into the mechanisms required for the maintenance and possibly the induction of tolerance to certain self-Ag in peripheral lymphoid organs.     

Cutting edge: thymic selection and autoreactivity are regulated by multiple coreceptors involved in T cell activation.

Immune responses are shaped by several processes that promote responses to pathogens and hinder responses to self. One mechanism that contributes to this polarization in response is negative selection, in which thymocytes that can respond to self-peptide/MHC complexes are deleted from the T cell repertoire. I found here that several coreceptors known to contribute to mature T cell activation also participate in negative selection. Interestingly, these molecules appeared to act in a cooperative fashion. Blocking the contribution of these molecules in fetal thymus organ culture not only prevented negative selection in the CD4+ lineage, but also induced the appearance of autoreactive thymocytes. This is the first demonstration that blocking coreceptor interactions during thymic development can produce autoreactive T cells. The contribution of negative selection to the mature T cell repertoire and to autoimmunity is discussed in light of these results.     

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analysis revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1-, Lyt 2- and Thy 1+, Lyt 1-, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1-, Lyt 2- cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2-responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1-, Lyt 1-, and Lyt 2-. On the other hand, a predominant type of the IL 2-responsive cells in BALB/c nu/nu mice were Thy 1-, Lyt 1-, and Lyt 2-, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.     

Tolerance induction in T helper (Th1) cells by thymic macrophages.

Most macrophages in the peripheral tissues present Ag optimally to a variety of functionally distinct Th cells. Although thymic macrophages have been implicated in deleting autoreactive thymocytes, their role in influencing the functional capacities of mature T cells is not clear. We have established a normal untransformed macrophage cell line, named TMC, from the mouse thymus. The TMC line presents protein Ag to an IL-4-producing Th2 type Th clone after IFN-gamma treatment as evidence by T cell proliferation and the release of IL-3 and IL-4. However, these thymic macrophages are inefficient at stimulating a well characterized cytochrome C-specific IL-2-producing Th1 clone, A.E7. Ag presentation by TMC results in the production of IL-3 but not IL-2 production or proliferation of A.E7 cells. This selective Ag presentation defect to Th1 cells is corrected by the addition of live but not fixed allogeneic irradiated spleen cells, suggesting that the thymic macrophages lack the expression of costimulatory activity required for Th1 activation. This is further demonstrated by the failure of live thymic macrophages to provide costimulatory activity to A.E7 cells stimulated with fixed spleen cells plus the antigenic peptide 81-104. Exposure of A.E7 cells to paraformaldehyde-treated TMC in the presence of 81-104 peptide induces specific hyporesponsiveness, anergy. These data demonstrate that thymic macrophages can have a profound influence on the response of selected T cells to Ag. Furthermore, the nature of the T cell stimulus is also critical because Th1 and Th2 cells responded equally well to the T cell mitogen, Con A, and a bacterial superantigen presented by the thymic macrophages.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.     

Migration of putative progenitor T cells in response to thymus-derived chemotactic factors

Progenitor T cells reach the thymus through the circulation from hematopoietic organs and then migrate toward the site of differentiation in the thymus. The mechanism that regulates such intrathymic migration is not well understood. In order to clarify this mechanism, in vitro chemotactic activity for murine thymocytes was assayed in the extracts and culture supernatants of thymic tissue elements. A potent thymocyte chemotactic activity was found in the extract and culture supernatant from Ig-, Ia- thymic stromal cells. Peanut agglutinin-positive (PNA+1), Thy 1+, TL-, Lyt 1+2-, L3T4- thymocytes, Ig-, Thy 1- bone marrow cells, and mononuclear cells of spleen and peripheral blood, but neither B cells nor lymph node cells, were chemotactically attracted by the factor(s). The chemotactic activity was found in none of the following materials tested: the extract and culture supernatant of thymocytes, culture supernatant of lymph node stromal cells, normal mouse serum, and zymosan-activated serum. The chemotactic activity was found in three molecular fractions by gel chromatography. The activity in all three fractions was destroyed by trypsin digestion or by heating at 56 degrees C for 30 min. These results suggest that Ig-, Ia- thymic stromal cells but not thymocytes secrete a chemotactic factor(s) for progenitor T cells with three molecular species. The factor is considered to play an important role in the migration of intrathymic progenitor T cells into the site of differentiation.     

Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4(+)CD25(+) regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4(+)CD25(+) regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4(+)CD25(+) thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.     

Characterization of T lymphocyte colony-forming cells in the mouse. The colony-forming cell is confined to Lyt-1,2,3+ cell subsets in the thymus and the lymph nodes

When cell populations from the thymus were studied with FACS, it was found consistently that the brightly labeled Thy-1.2+ populations contained very few T colony-forming cells (CFC), while these latter cells were numerous in the cell populations showing lower Thy-1.2 antigen density. This was paralleled by findings after peanut agglutinin (PNA) separation that showed enrichment of CFC in the PNA-negative medullary population, and by sorting based on TL, T-200, and H-2 determinants or light scatter properties of the cells. By FACS sorting of Lyt-labeled thymic cells, it was also shown that CFC were predominantly present in cell populations that were brightly Lyt-1+, and exclusively in populations that were Lyt-2+ and Lyt-3+. After FACS sorting of lymph node cells, no major differences in colony formation were found between dully- and brightly-labeled Thy-1.2+ or Lyt-1+ populations, or between lymphoid cells showing different light scatter characteristics. In addition, it was shown that CFC--like thymic CFC--were of the Lyt-1,2,3+ phenotype. It is concluded that the CFC may be present in several differentiation steps of Lyt-1,2,3+ cell lines, and that the frequency of these cells increases from the thymic cortex via the medulla and to peripheral lymphoid tissues.     

The role of LFA-1/ICAM-1 interactions during murine T lymphocyte development.

We have examined the expression and function of the cell adhesion molecules LFA-1 (CD11a/CD18), ICAM-1 (CD54), and ICAM-2 in murine fetal thymic ontogeny and in the adult thymus. On fetal days 14 and 15, 40 to 50% of thymocytes coexpress high levels of LFA-1 and ICAM-1, as determined by flow cytometry. By day 16, more than 90% of fetal thymocytes are LFA-1+ ICAM-1hi, and all IL-2R+ cells are located in this population. Although LFA-1 expression remains unchanged thereafter, ICAM-1 expression appears to be differentially regulated in different thymocyte subpopulations, with CD4+8+ cells being ICAM-1lo and CD4-8- thymocytes remaining ICAM-1hi. ICAM-2 surface expression is dull on both fetal and adult thymocytes. Surprisingly, the expression of ICAM-1 is differentially up-regulated on T cells having a mature phenotype in thymus and in peripheral lymphoid organs, with CD8+ T cells bearing the highest amount of surface ICAM-1. Addition of anti-ICAM-1 or anti-LFA-1 antibodies to fetal thymic organ cultures results in the impaired generation of CD4+8+ cells. These results indicate that LFA-1/ICAM-1 interactions facilitate murine thymic development and suggest that cell adhesion molecules mediate important events in T cell differentiation.     

Augmentation of T cell levels and responses induced by androgen deprivation

Androgen has been implicated as a negative regulator of host immune function and a factor contributing to the gender dimorphism of autoimmunity. Conversely, androgen deprivation has been suggested to potentiate male host immunity. Studies have shown that removal of androgen in postpubertal male mice produces an increase in size and cellularity of primary and peripheral lymphoid organs, and enhances a variety of immune responses. Yet, few details are known about the effect of androgen removal on T cell-mediated immunity. In this study, we demonstrate two pronounced and independent alterations in T cell immunity that occur in response to androgen deprivation, provided by castration, in postpubertal male mice. First, we show that levels of T cells in peripheral lymphoid tissues of mice are increased by androgen deprivation. Second, T cells from these mice transiently proliferate more vigorously to TCR- and CD28-mediated costimulation as well as to Ag-specific activation. In addition, androgen deprivation accelerates normalization of host T and B cell levels following chemotherapy-induced lymphocyte depletion. Such alterations induced by androgen deprivation may have implications for enhancing immune responses to immunotherapy and for accelerating the recovery of the immune system following chemotherapy.