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Strong T cell tolerance in parent----F1 bone marrow chimeras prepared with supralethal irradiation. Evidence for clonal deletion and anergy
Journal of Experimental Medicine (JEM)
Abstract
T cell tolerance induction was examined in long-term H-2-heterozygous parent----F1 chimeras prepared with supralethal irradiation (1,300 rad). Although these chimeras appeared to be devoid of host-type APC, the donor T cells developing in the chimeras showed marked tolerance to host-type H-2 determinants. Tolerance to the host appeared to be virtually complete in four assay systems: (a) primary mixed lymphocyte reactions (MLR) of purified lymph node (LN) CD8+ cells (+/- IL-2); (b) primary MLR of CD4+ (CD8-) thymocytes; (c) skin graft rejection; and (d) induction of lethal graft-vs.-host disease by CD4+ cells. Similar tolerance was observed in chimeras given double irradiation. The only assay in which the chimera T cells failed to show near-total tolerance to the host was the primary MLR of post-thymic CD4+ cells. In this assay, LN CD4+ cells regularly gave a significant antihost MLR. The magnitude of this response was two- to fourfold less than the response of normal parental strain CD4+ cells and, in I-E(-)----I-E+ chimeras, was paralleled by approximately 70% deletion of V beta 11+ cells. Since marked tolerance was evident at the level of mature thymocytes, tolerance induction in the chimeras presumably occurred in the thymus itself. The failure to detect host APC in the thymus implies that tolerance reflected contact with thymic epithelial cells (and/or other non-BM-derived cells in the thymus). To account for the residual host reactivity of LN CD4+ cells and the incomplete deletion of V beta 11+ cells, it is suggested that T cell contact with thymic epithelial cells induced clonal deletion of most of the host-reactive T cells but spared a proportion of these cells (possibly low affinity cells). Since these latter cells appeared to be functionally inert in the thymus (in contrast to LN), we suggest that the thymic epithelial cells induced a temporary form of anergy in the remaining host-reactive thymocytes. This anergic state disappeared when the T cells left the thymus and reached LN.
... Later studies suggested that incomplete deletional tolerance of these recipientreactive donor T cells was achieved, reflecting the absence of a self-renewing source of recipient APCs to ensure complete deletion of host-reactive donor T cells in the thymus. Nevertheless, functional tolerance to the recipient was achieved by a combination of mechanisms that involve thymic stromal cells, which are of recipient origin (12,13) (Figures 1A, B). ...
Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal.
... Besides their location in the periphery, cDCs and pDCs also reside in the steady-state thymus (Bendriss-Vermare et al., 2001;Vandenabeele et al., 2001;Okada et al., 2003;Wu and Shortman, 2005), where they play a key role in the establishment of central tolerance by inducing autoreactive T cell deletion and regulatory T cell generation (Gao et al., 1990;Brocker et al., 1997;Watanabe et al., 2005;Martín-Gayo et al., 2010), but the developmental origin of thymic DCs is far less well understood than the origin of peripheral DCs. The initial view that thymic DCs were derived in situ from intrathymic T/DC lymphoid progenitors (Ardavin et al., 1993;Shortman and Wu, 1996) was later challenged by cell-fate mapping ex-periments supporting separate lymphoid and myeloid origins for T cells and DCs, respectively (Schlenner and Rodewald, 2010). ...
A key unsolved question regarding the developmental origin of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) resident in the steady-state thymus is whether early thymic progenitors (ETPs) could escape T cell fate constraints imposed normally by a Notch-inductive microenvironment and undergo DC development. By modeling DC generation in bulk and clonal cultures, we show here that Jagged1 (JAG1)-mediated Notch signaling allows human ETPs to undertake a myeloid transcriptional program, resulting in GATA2-dependent generation of CD34+ CD123+ progenitors with restricted pDC, cDC, and monocyte potential, whereas Delta-like1 signaling down-regulates GATA2 and impairs myeloid development. Progressive commitment to the DC lineage also occurs intrathymically, as myeloid-primed CD123+ monocyte/DC and common DC progenitors, equivalent to those previously identified in the bone marrow, are resident in the normal human thymus. The identification of a discrete JAG1+ thymic medullary niche enriched for DC-lineage cells expressing Notch receptors further validates the human thymus as a DC-poietic organ, which provides selective microenvironments permissive for DC development.
... In hindsight, apparently conflicting results regarding the contribution of antigen presentation by mTECs to central tolerance might, at least in part, stem from different read-out systems that have been used to address this question (e.g. superantigen-reactivity, graft-versus-hostdisease or mixed lymphocyte reactions with hematopoietic APCs) [12][13][14][15]. Irrespective of these considerations, the topologically restricted expression of TRAs might provide reason enough to hypothesize that central tolerance to mTEC-derived self-determinants, rather than being autonomously promoted by mTECs, might involve antigen capture and presentation by thymic DCs. ...
Promiscuous expression of 'peripheral' tissue-restricted antigens (TRAs) by medullary thymic epithelial cells (mTECs) is essential for central tolerance. Remarkably, the expression of individual TRAs varies among mTECs and is confined to a perplexingly small number of cells. To reconcile this with the ensuing robust state of tolerance, one might envisage that mTECs serve primarily as an antigen reservoir, whereas tolerogenic recognition of TRAs would ultimately require antigen uptake and presentation by dendritic cells (DCs). Here, we survey the evidence for this 'antigen-spreading' scenario and relate it to findings that document autonomous antigen-presentation by mTECs. We suggest that DC-dependent and autonomous tolerogenic functions of mTECs operate in parallel, and the underlying mechanisms remain to be established.
T cells undergo rigorous selection in the thymus to ensure self-tolerance and prevent autoimmunity, with this process requiring innocuous self-antigens (Ags) to be presented to thymocytes. Self-Ags are either expressed by thymic stroma cells or transported to the thymus from the periphery by migratory dendritic cells (DCs); meanwhile, small blood-borne peptides can access the thymic parenchyma by diffusing across the vascular lining. Here we describe an additional pathway of thymic Ag acquisition that enables circulating antigenic macromolecules to access both murine and human thymi. This pathway depends on a subset of thymus-resident DCs, distinct from both parenchymal and circulating migratory DCs, that are positioned in immediate proximity to thymic microvessels where they extend cellular processes across the endothelial barrier into the blood stream. Transendothelial positioning of DCs depends on DC-expressed CX 3 CR1 and its endothelial ligand, CX 3 CL1, and disrupting this chemokine pathway prevents thymic acquisition of circulating proteins and compromises negative selection of Ag-reactive thymocytes. Thus, transendothelial DCs represent a mechanism by which the thymus can actively acquire blood-borne Ags to induce and maintain central tolerance.
Dendritic cells (DCs) are a unique class of immune cells that act as a bridge between innate and adaptive immunity. The discovery of DCs by Cohen and Steinman in 1973 laid the foundation for DC biology, and the advances in the field identified different versions of DCs with unique properties and functions. DCs originate from hematopoietic stem cells, and their differentiation is modulated by Flt3L. They are professional antigen-presenting cells that patrol the environmental interphase, sites of infection, or infiltrate pathological tissues looking for antigens that can be used to activate effector cells. DCs are critical for the initiation of the cellular and humoral immune response and protection from infectious diseases or tumors. DCs can take up antigens using specialized surface receptors such as endocytosis receptors, phagocytosis receptors, and C type lectin receptors. Moreover, DCs are equipped with an array of extracellular and intracellular pattern recognition receptors for sensing different danger signals. Upon sensing the danger signals, DCs get activated, upregulate costimulatory molecules, produce various cytokines and chemokines, take up antigen and process it and migrate to lymph nodes where they present antigens to both CD8 and CD4 T cells. DCs are classified into different subsets based on an integrated approach considering their surface phenotype, expression of unique and conserved molecules, ontogeny, and functions. They can be broadly classified as conventional DCs consisting of two subsets (DC1 and DC2), plasmacytoid DCs, inflammatory DCs, and Langerhans cells.
https://authors.elsevier.com/b/1aAOr_i3fF7PDJ
Thymic negative selection renders the developing T-cell repertoire tolerant to self-major histocompatability complex (MHC)/peptide ligands. The major mechanism of induction of self-tolerance is thought to be thymic clonal deletion, ie, the induction of apoptotic cell death in thymocytes expressing a self-reactive T-cell receptor. Consistent with this hypothesis, in mice deficient in thymic clonal deletion mediated by cells of hematopoietic origin, a twofold to threefold increased generation of mature thymocytes has been observed. Here we describe the analysis of the specificity of T lymphocytes developing in the absence of clonal deletion mediated by hematopoietic cells. In vitro, targets expressing syngeneic MHC were readily lysed by activated CD8+ T cells from deletion-deficient mice. However, proliferative responses of T cells from these mice on activation with syngeneic antigen presenting cells were rather poor. In vivo, deletion-deficient T cells were incapable of induction of lethal graft-versus-host disease in syngeneic hosts. These data indicate that in the absence of thymic deletion mediated by hematopoietic cells functional T-cell tolerance can be induced by nonhematopoietic cells in the thymus. Moreover, our results emphasize the redundancy in thymic negative selection mechanisms.
Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.
Self tolerance is under the strict control of T lymphocytes and is imposed during early T cell differentiation in the thymus [1–5]. Which particular cell types in the thymus induce self tolerance (negative selection) is controversial [5,6]. The prevailing view is that tolerance induction is largely a reflection of T cells encountering the contingent of bone-marrow-derived cells in the medulla [7–9]. These cells, especially dendritic cells [9], have a proven role in tolerance induction and are strategically positioned at the cortico-medul 1 ary junction. The role of thymic epithelial cells (TEC) in tolerance induction is less clear. In this paper we review our recent studies on the tolerogenicity of TEC in bone marrow (BM) and fetal liver (FL) chimeras. For allogeneic (mouse → mouse) chimeras, we present evidence that TEC are strongly tolerogenic for some T cells but only weakly tolerogenic for others. For xenogeneic (rat → mouse) chimeras, by contrast, TEC are almost nontolerogenic.
Dendritic cells (DCs) are uniquely well equipped antigen (Ag)-presenting cells. Their classic function was thought to be that of potent initiators of innate and adaptive immunity to infectious organisms and other Ags (including transplanted organs). Evidence has emerged, however, that DCs have a central and crucial role in determining the fate of immune responses toward either immunity or tolerance. This dichotomous function of DCs, coupled with their remarkable plasticity, renders them attractive therapeutic targets for immune modulation. In transplantation, much recent work has focused on the ability of DCs to silence immune reactivity in an Ag-specific manner in the hope of preventing rejection and diminishing reliance on potentially harmful immunosuppressive agents. Experimental strategies have included in vivo targeting of DCs, as well as ex vivo generation of regulatory (or tolerogenic) DCs with subsequent reinfusion (i.e. cell therapy). Different approaches to 'program' DC toward tolerogenic properties include genetic (transgene insertion), biologic (differential culture conditions, anti-inflammatory cytokine exposure) and pharmacologic manipulation. Recent data suggest a promising role for pharmacologic treatment as a means of generating potent regulatory DCs and have further stimulated speculation regarding their potential clinical application. Herein, we discuss evidence that the potential of regulatory DC therapy is considerable and that there are compelling reasons to evaluate it in the setting of organ transplantation in the near future.
Induction of donor-specific tolerance would be desirable and might even be essential to the success of clinical xenotransplantation for several reasons:
1.
It would eliminate the risk of acute or chronic rejection. Chronic rejection currently leads to eventual graft loss in a high percentage of allograft recipients, despite recent improvements in immunosuppressive therapy. In view of the frequently greater difficulty that is encountered in attenuating xenoresponses than alloresponses [1], it seems likely that both acute and chronic rejection might be a major limitation to xenogeneic organ transplantation, even if the initial natural antibody-induced hyperacute rejection problem could be overcome.
2.
A state of tolerance would obviate the need for chronic imunosuppressive therapy with its attendant risks of opportunistic infection, malignancy and organ toxicity.
Long-term H-2-heterozygous a----(a x b)F1 bone marrow (BM) chimeras prepared with supralethal irradiation (1,300 rad) are devoid of Ia+ host BM-derived antigen-presenting cells (APC), but show quite strong host Ia expression in germinal centers, probably on follicular dendritic cells (a class of nonhemopoietic stromal cells). To examine whether Ia expression on these non-BM-derived cells is capable of inducing post-thymic tolerance of T cells, thymectomized irradiated (a x b)F1 mice were reconstituted with parent alpha stem cells and then, 6 mo later, given parent alpha thymus grafts. As measured by primary mixed lymphocyte reactions and V beta expression, the CD4+ cells differentiating in the thymus-grafted mice showed no detectable tolerance to the H-2 (Ia) antigens of the host. To examine whether the thymus-grafted mice contained immunologically significant quantities of host Ia antigens, long-term alpha----(alpha x b)F1 chimeras were injected with normal strain alpha CD4+ cells; the donor cells were recovered from thoracic duct lymph of the chimeras and tested for host reactivity in vitro. The results showed that Ia expression in the chimeras was sufficient to cause selective trapping of a substantial proportion of host-Ia-reactive CD4+ cells soon after transfer and, at later stages, to induce strong priming. Tolerance was not seen. The data place constraints on the view that T cell recognition of antigen expressed on cells other than typical BM-derived APC leads to tolerance induction.
This review addressed basic and preclinical aspects of allograft tolerance from the perspective of the veto cell mechanism of immunoregulation. Our studies in rhesus monkeys focus on an encouraging strategy for inducing specific tolerance to kidney allografts with posttransplant RATG and donor BM in a preclinical model. In addition, they have led to seminal observations on the role of veto cells in allograft tolerance induced by donor hematopoietic cells. Evidence for the role of veto cells in allograft tolerance is now substantial and draws from multiple models and observations. The veto cell mechanism is an attractive explanation for many forms of tolerance, and may provide a conceptual guide for developing more effective approaches to tolerance in clinical transplantation.
Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells.
Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry.
The precursor frequency of dividing CD4+ T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency of alloantigen-specific CD4+ T cells.
A novel method for the direct calculation of the frequency of alloreactive CD4+ T cells is described. This technique allows the determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.
IntroductionMechanisms of T-cell toleranceMechanisms of B-cell toleranceApproaches to inducing HVG transplantation toleranceAchieving HVG tolerance with HCTSummary and future directions
To examine the development of T cells within an allogeneic or xenogeneic environment, we engrafted the fetal thymus from AKR mice or F344 rats under the kidney capsule of SCID mice (mTG and rTG mice). T lymphopoiesis developed in SCID mice 2 months after transplantation, although the ratio of CD4/CD8 in both experimental groups was different from that of normal control. T cells in mTG mice did not show in vitro proliferation or cytotoxicity against either host-type C.B-17 (H-2d) or donor-type AKR (H-2k) cells, while they exerted potent activities against third-party BIO (H-2b) cells. In contrast, T cells in rTG mice exhibited proliferation against both host-type C.B-17 and donor-type F344 rat cells. Consistently, graft-vs.-host disease symptoms developed in these mice and histological examination showed impressive infiltration of lymphocytes into the skin or into the mucosal layers of the stomach. Activated state of T cells in rTG mice was also evidenced by the positive expression of interleukin-2 receptor. Taken together, fetal thymus appears to contain progenitor cells which are sufficient for in vivo reconstitution of T lymphopoiesis, but species-specific environment is important for the induction of tolerance. In mTG mice, Vβ6+ T cells reactive to donor Mlsa determinants and Vβ3+ T cells reactive to host Mlsc determinants were deleted, suggesting that tolerance was regulated mainly by clonal deletion. By contrast, Vβ11+ T cells reactive to Mlsf determinants were not deleted possibly due to the lack of their ligands.
Athymic nude mice reconstituted at birth with allogeneic thymic epithelia (TE) from day 10 embryos (E10), show life‐long specific tolerance to skin and heart grafts, but eliminate B lymphocytes of the TE donor haplotype, nearly as well as those from a third strain. Previous immunizations with B cells do not alter the state of tolerance to skin grafts, but specifically accelerate elimination of lymphocytes. In contrast, transplantation of E15 allogeneic thymuses already seeded by hematopoietic cells resulted in chimeras tolerant to both skin and B lymphocytes. In vitro reactivities towards stimulator spleen cells of the haplotype of the thymus were observed in both E10 TE and E15 thymus chimeras.
We conclude that induction of full in vivo tolerance to B cells requires hematopoietic cells, while this is not the case for induction of tolerance to skin and heart tissues; furthermore, in vitro reactivity to stimulator spleen cells of the tolerized haplotype is independent of in vivo tolerance.
Dendritic cells (DC) are rare but extremely important bone marrow (BM)- derived antigen-presenting cells (APC) that initiate
and regulate immune responses [1-4]. Extensive recent investigations [reviewed in refs. 3,4] have improved understanding of DC lineage development, differentiation, activation and function. These studies have revealed
that, in addition to the well-recognized immunostimulatory properties of DC that offer potential for therapy of infectious
disease and cancer, DC also have potential tolerogenicity [reviewed in refs. 2,5,6]. This capacity of DC is manifested by suppression of T cell responses in experimental models of T cell ontogeny, tumor rejection,
autoimmunity, and allograft rejection. Moreover, exposure to a wide variety of agents, e.g. ultraviolet B (UVB) irradiation,
IL-10, transforming growth factor β (TGFβ), corticosteroids, prostaglandin E2 (PGE2), or the chimeric fusion protein cytotoxic T lymphocyte antigen (Ag) 4 immunoglobulin (CTLA4Ig) can confer tolerogenic properties
on DC. In addition, geneticallyengineered DC expressing immunosuppressive molecules, such as viral (v) IL-10, TGFβ, Fas Ligand
(L) (CD95L) or CTLA4Ig may offer potential for the suppression/deletion of auto- or alloAg-specific T cells, with implications
for tolerance induction.
The thymus serves as the central organ of immunologic self-nonself discrimination. Thymocytes undergo both positive and negative selection, resulting in T cells with a broad range of reactivity to foreign antigens but with a lack of reactivity to self-antigens. The thymus is also the source of a subset of regulatory T cells that inhibit autoreactivity of T-cell clones that may escape negative selection. As a result of these functions, the thymus has been shown to be essential for the induction of tolerance in many rodent and large animal models. Proper donor antigen presentation in the thymus after bone marrow, dendritic cell, or solid organ transplantation has been shown to induce tolerance to allografts. The molecular mechanisms of positive and negative selection and regulatory T-cell development must be understood if a tolerance-inducing therapeutic intervention is to be designed effectively. In this brief and selective review, we present some of the known information on T-cell development and on the role of the thymus in experimental models of transplant tolerance. We also cite some clinical attempts to induce tolerance to allografts using pharmacologic or biologic interventions.
Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.
Recent years have seen a major advance in our understanding of the organization of the dendritic cell (DC) compartment. Particularly rewarding in this respect have been studies investigating DC origins, based on the identification of transcription factor and growth factor requirements, as well as direct demonstrations of precursor/progeny relationships by adoptive cell transfers. However, to fully understand the organization of the DC compartment, functional definitions of DC subsets must be provided and potential task divisions revealed that distinguish DC from other immune cells, including the closely related mononuclear phagocytes, such as macrophages. In fact, functional definitions might eventually replace the current distinction between DC and macrophages, which is in part based on arbitrary historic considerations, i.e. mononuclear phagocytes identified before the advent of DC in the mid 1970s generally termed macrophages. In this article, we review recent insight in the functions of classical DC in the mouse, focusing on our own work involving conditional and constitutive cell ablation.
Dendritic cells (DCs) are key coordinators of the immune response, governing the choice between tolerance and immunity. DCs are professional antigen-presenting cells capable of presenting antigen on MHC molecules and priming CD4 and CD8 T-cell responses. They form a heterogeneous group of cells based on phenotype, location, and function. In this review, murine DCs will be discussed regarding their function with special emphasis on their tissue distribution. Recent findings on DC homeostasis during cancer progression will be presented. Finally, the developmental pathways leading to DC differentiation from their precursors will be summarized.
Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (TRs). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of TRs. TDCs include two conventional dendritic cell (DC) subtypes, CD8loSirpαhi/+ (CD8loSirpα⁺) and CD8hiSirpαlo/− (CD8loSirpα⁻), which have different origins. We found that the CD8hiSirpα⁺ DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8loSirpα⁺ DCs demonstrate a superior capacity to induce TRs in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce TR generation and negative selection.
• thymic selection
• migratory dendritic cells
• tolerance
Dendritic cells are critically involved in the promotion and regulation of T cell responses. Here, we report a mouse strain that lacks conventional CD11c(hi) dendritic cells (cDCs) because of constitutive cell-type specific expression of a suicide gene. As expected, cDC-less mice failed to mount effective T cell responses resulting in impaired viral clearance. In contrast, neither thymic negative selection nor T regulatory cell generation or T cell homeostasis were markedly affected. Unexpectedly, cDC-less mice developed a progressive myeloproliferative disorder characterized by prominent extramedullary hematopoiesis and increased serum amounts of the cytokine Flt3 ligand. Our data identify a critical role of cDCs in the control of steady-state hematopoiesis, revealing a feedback loop that links peripheral cDCs to myelogenesis through soluble growth factors, such as Flt3 ligand.
Central tolerance is established through the negative selection of self-reactive thymocytes and the induction of T-regulatory cells (T-regs). A role for thymic epithelial cells in mediating both negative selection and T-reg induction has been clearly shown. The role of thymic dendritic cells (DCs) in these processes has not been clearly determined but has been the focus of recent studies. Thymic DCs include two conventional DC (cDC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+) herein) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-) herein). It has been shown that these DC subsets have distinct developmental origins, the CD8(hi)Sirpalpha(-) cDCs developing intra-thymically and the CD8(lo)Sirpalpha(+) migrating into the thymus from the periphery. Furthermore, an important role for thymic DCs in the induction of T-regs has been shown. In this review, the role of DCs in the development and education of T cells in the thymus will be reviewed, with emphasis on the role of circulatory DCs in mediating these processes.
The expression of MHC class I molecules protects cells against lysis by natural killer (NK) cells. It is possible that NK cells are 'educated' to recognize self MHC class I molecules and that the combination of self peptide and MHC class I molecule blocks NK-mediated lysis. Here, Rogier Versteeg compares and contrasts models of education and self-nonself discrimination by T cells and NK cells, and presents a hypothesis for the evolution of T cells from NK cells.
Grafting of thymic anlagen from day-10 DBA/2 (H-2d; Mls-1a) embryos to newborn athymic BALB/c (H-2d; Mls-1b) mice leads to reconstitution of T cell populations in the recipients. Analysis of adult chimeras shows that their V beta T cell receptor (TcR) repertoires, particularly V beta 6 and V beta 8.1, do not significantly differ in most animals (10 out of 13) from those scored in control chimeras that received syngeneic thymic anlagen. In all cases analyzed, such Mls-1a-reactive T cells could be stimulated at levels comparable to control responses, both in vitro and in vivo. The few cases in which Mls-1a reactive V beta TcR were reduced seem to reflect the variability in TcR V beta repertoires found in this experimental system. In contrast, BALB/c mice, injected at birth with DBA/2 spleen cells show a marked, albeit variable, reduction in the frequencies of V beta 6- and V beta 8.1-bearing CD4+ T cells, and lower frequencies of Mls-1a-reactive T cells in limiting dilution analyses. It appears, however, that V beta 6- and V beta 8.1-bearing T cells remaining in these mice are functionally competent. We conclude that Mls-1 antigens are not expressed by thymic epithelium.
To study the role of thymic medullary epithelium in tolerance induction, the third and fourth branchial clefts of embryos from E mu-Kb transgenic mice, which express the major histocompatibility complex class I antigen H-2Kb exclusively on medullary thymic epithelium, were grafted to athymic nude mice. The grafts differentiated into tissue that morphologically resembled normal thymus. These grafts expressed the H-2Kb antigen appropriately and gave rise to a functional T cell repertoire. In vivo tolerance to H-2Kb disparate skin grafts was invariably found in mice expressing H-2Kb in the medulla or in both medulla and cortex of C57BL/6 branchial cleft-grafted controls. In marked contrast, in vitro cytotoxicity assays demonstrated reactivity toward H-2Kb in the presence of interleukin 2, and limiting-dilution analyses showed similar frequencies of cytolytic T cell precursors reactive to H-2Kb and to third-party stimulators. Medullary epithelium can, therefore, induce split tolerance, in which in vivo tolerance is accompanied by strong in vitro responses in the presence of interleukin 2.
During thymic development, thymocytes that can recognize major histocompatability complex (MHC) molecules on thymic epithelial cells are selected to survive and mature (positive selection), whereas thymocytes that recognize MHC on hematopoietic cells are destroyed (negative selection). It is not known how MHC recognition can mediate both death and survival. One model to explain this paradox proposes that thymocytes whose T cell antigen receptors (TCRs) recognize MHC with high affinity are eliminated by negative selection, whereas low affinity TCR-MHC interactions are sufficient to mediate positive selection. Here we report that, while the expression of a 2C TCR transgene leads to positive selection of thymocytes in H-2b mice, expression of both a CD8 transgene and a 2C TCR transgene causes negative selection. This observation indicates that quantitative differences in TCR-MHC recognition are a critical determinant of T cell fate, a finding predicted by the affinity model for thymic selection.
The specificity of mature CD8+ and CD4+ T lymphocytes is controlled by major histocompatibility complex (MHC) class I and
class II molecules, respectively. The MHC class specificity of T cells is stringent in many assays, but is less evident when
cells are supplemented with exogenous lymphokines. The repertoire of T cells is shaped through contact with MHC molecules
in the thymus and involves a complex process of positive selection and negative selection (tolerance). Tolerance of immature
T cells to MHC molecules can reflect either clonal deletion or anergy and results from intrathymic contact with several cell
types, including epithelial cells and cells with antigen-presenting function. Unlike immature T cells, mature T cells are
relatively resistant to tolerance induction. In certain situations partial unresponsiveness of mature T cells can be achieved
by exposing T cells to foreign MHC molecules expressed on atypical antigen-presenting cells. Tolerance is rarely complete,
however, and the precise requirements for tolerizing mature T cells are still unclear.
New experimental protocols for the induction of transplantation tolerance continue to be developed. In the past year, encouraging data have been reported from a clinical trial using a protocol specifically designed to induce tolerance to the histocompatibility antigens of the kidney donor. Progress has also been made in our understanding of the mechanisms responsible for the induction and maintenance of tolerance to alloantigens in vivo; it is becoming increasingly clear that more than one mechanism can be involved, particularly at different phases in the response.
Mice given short courses of anti-CD4 and anti-CD8 monoclonal antibodies became tolerant of allogeneic skin grafted at the same time. Tolerance could be obtained without T cell depletion across multiple minor antigen mismatches, both in naive and primed animals, demonstrating that peripheral T cells could be tolerized, even if they had been previously activated. Where donor and recipient were incompatible across the whole major histocompatibility complex, specific tolerance could be achieved by using a combination of depleting followed by non-depleting antibodies, where each alone was unsuccessful. Although mice clearly tolerated their original skin grafts, we observed in some strain combinations that a second fresh, but genotypically identical graft, was slowly rejected. Such mice also possessed T cells which could proliferate to donor-type stimulator cells in vitro. Whatever the mechanisms, we have demonstrated that operational transplantation tolerance can be achieved with simple, non-toxic antibody therapy. The introduction of comparable tolerance-inducing regimens in clinical organ transplantation could obviate the need for long-term immunosuppression and its unfortunate side effects.
One prediction from the complex series of steps in intrathymic T-cell differentiation is that to regulate it the stroma controlling the process must be equally complex: the attraction of precursors, commitment to the T-cell lineage, induction of T-cell receptor (TCR) gene rearrangement, accessory molecule expression, repertoire expansion, major histocompatibility complex (MHC) molecule-based selection (positive and negative), acquisition of functional maturity and migratory capacity must all be controlled. In this review, Richard Boyd and Patrice Hugo combine knowledge of T-cell differentiation with thymic stromal cell heterogeneity to offer an integrated view of thymopoiesis within the thymic microenvironment.
To attempt to resolve the controversy on the role of thymic epithelial cells (TEC) in tolerance induction, athymic mice were grafted with allogeneic day-14 fetal thymuses treated with deoxyguanosine in vitro. The data indicate that the tolerogenicity of TEC varies considerably according to the antigen and the subpopulation of T cells studied. For cytotoxic CD8+ cells responding to H-2 class I antigens, TEC induce minimal tolerance. For proliferative responses of CD4+ cells, by contrast, TEC induce significant tolerance to H-2 class II antigens but no detectable tolerance to Mlsa antigens.
Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.
Athymic mice grafted at birth with allogeneic thymic epithelium (TE) display life-long tolerance to tissue grafts of the TE donor strain, in spite of harboring peripheral T cells capable of rejecting those grafts. Tolerance is maintained in these chimeras by TE-specific regulatory CD4 T cells. We presently address the quantification and the mechanisms of this dominant tolerance process. C57BL/6 mice containing variable but defined numbers of peripheral, resident T cells received cell transfers of graded numbers of peripheral T cells from B6(BALB E10) chimeras (C57BL/6 nude mice grafted with TE from 10-day-old BALB/c embryos), resulting in a series of animals containing a wide range of donor (tolerant) versus host (non-tolerant) T cell chimerism. Increasing the relative representation of donor T cells results in a progressive delay in the rejection of BALB/c skin grafts, life-long tolerance being achieved at a ratio of tolerant and non-tolerant T cell populations of 1. In recipients displaying full tolerance, graft-reactive non-tolerant T cells were not deleted, anergized or committed to noninflammatory functions. Thus, sorted host T cells from tolerant recipients readily rejected BALB/c skin grafts upon transfer to immunodeficient animals. Finally, measurements of "helper" and inflammatory activities, as well as interleukin-4 and interferon-gamma production, failed to discriminate between T cell populations from tolerant and non-tolerant animals after specific in vitro stimulation. We conclude that: (a) TE-selected regulatory T cells can suppress, in a quantitative manner, in vivo T cell responses against major and minor histocompatibility antigens expressed by the TE and, (b) this suppressive activity neither inactivates mature non-tolerant T cells, nor does it seem to drive their differentiation along noninflammatory pathways.
It is not surprising that the recent explosion of interest in physiological cell death has been centered particularly on lymphocytes. Physiological cell death responses are singularly important in the biology of T lymphocytes, especially in the establishment and maintenance of a diverse, non-autoreactive, and self-limiting repertoire. Cell death responses can be triggered in T cells by a variety of stimuli; sensitivity to these inducers is altered as a function of differentiation, activation, aging, and transformation. The elimination of autoreactive T cells occurs by a process that involves comitogenic stimulation at high dose with antigenic and/or mitogenic agents. The control of susceptibility to this activation-driven cell death with differentiation and with prior activation provides a mechanistic explanation for the development of central and peripheral tolerance. Enhanced lymphocyte activation with aging also leads to an augmented activation-driven cell death response. However, aging does not alter cell death responses generally, and aging-associated changes in cell death responses cannot account for aging-associated immunopathology. Oncogenic transformation also alters the activation-driven cell death response by supplanting one of the required signals for activation-driven cell death. This difference provides a rationale for selective anti-tumor therapy. A single mechanism underlies all cases of physiological cell death and involves out-of-phase mitotic activity. We now know that of the two hallmarks of cell death, genome digestion is dispensable and mitotic-like events associated with cell cycle arrest are critical. T cells triggered to undergo physiological cell death arrest in a post-mitotic compartment of the cell cycle and die when they attempt a precocious and abortive mitosis.
Two cloned thymic epithelial cell (TEC) lines, D2.TEC-A3 and AKR TEC-K1, were established from minor lymphocyte-stimulating (Mls)-1a-positive normal, 4-week-old DBA/2 (H-2d, Mls-1a2a) mice and AKR (H-2k, Mls-1a2b) mice, respectively. Both cell lines were MHC class I and class II (both I-A and I-E) positive without stimulation by interferon-gamma. They were capable of infolding immature thymocytes to form thymic nurse cells (TNC; we call this type of TEC "nursing TEC") and induced apoptosis with DNA fragmentation in immature thymocytes. Using a primary Mls mixed lymphocyte reaction (MLR) we demonstrated that self-superantigen Mls-1a was expressed on these cloned nursing TEC lines. D2.TEC-A3 cells stimulated nylon-wool-purified splenic T cells obtained from H-2d-compatible BALB/c (Mls-1b2a) and B10.D2 (Mls-1b2b) mice with a maximal response at a stimulator:responder ratio of 1:40 after 4 days of the coculture. AKR TEC-K1 cells also stimulated purified T cells from H-2k-compatible C3H/He mice (Mls-1b2a) in a similar manner. The Mls MLR induced by the nursing TEC lines was completely inhibited in the presence of anti-mouse I-A and anti-mouse I-E monoclonal antibodies. These results suggest that nursing TEC/TNC could be involved in negative selection due to apoptosis.
CBA/Ca mice may be made tolerant to minor histoincompatible B10.BR skin grafts by treatment with a short course of non-depleting anti-mouse CD4 and CD8 monoclonal antibodies (mAb), during the transplantation period. We wished to determine when, in relation to antibody therapy, the T cells became tolerant. This was investigated by a series of adoptive transfer experiments in which mAb-treated cells were removed from therapeutic antibody at defined times after skin grafting, and exposed to fresh antigen in the absence of further mAb treatment. We show here that T cells do not become fully tolerant until 5 weeks after skin grafting. If antibody therapy is continued for the full 5 weeks, T cell tolerance can still be established, suggesting that antibody therapy does not prevent lymphocytes from registering the presence of antigen. Once the tolerant state is established, it is difficult to break that tolerance by lymphocyte infusions from normal donors. This "resistance" is mediated by T cells of the tolerant host. We show that the maintenance of both tolerance and "resistance" requires a continuous supply of antigen. When tolerant cells were "parked" in T cell-depleted mice, tolerance and "resistance" were eventually lost by 6 months. In contrast, "parked" cells exposed to fresh antigen at any time up to 4 months remained tolerant and "resistant" indefinitely. Finally, we wished to establish whether "resistance" was peculiar to this form of peripheral tolerance, or whether it might also be present in tolerance considered to be classically central. We observed resistance to be greater in the mAb-treated peripherally tolerant group, but noted that some of the centrally tolerant animals also exhibited a level of resistance above that of T cell-ablated controls. This suggests that a tolerance mechanism whose role is only minor in central tolerance may have a major role in antibody-mediated peripheral tolerance.
The cellular requirements of T cell tolerance induction in the thymus by clonal deletion was investigated by using an in vitro assay: thymocytes from mice expressing a transgenic TcR specific for lymphocytic choriomeningitis virus (LCMV) and H-2Db were co-cultured with various H-2b cell types as antigen-presenting cells in the presence of the antigenic LCMV peptide. The results revealed that all cell lines examined including embryonic and transformed fibroblasts, melanoma cells, cortical thymic epithelial cells, lymphomas and neuronal cells induced an antigen dose-dependent deletion of CD4+8+ thymocytes. Similarly, highly enriched accessory cell populations from thymus and spleen (macrophages, dendritic and cortical epithelial cells, i.e. thymic nurse cells) could induce antigen-specific depletion of immature CD4+8+ thymocytes. Depending on the cell type examined micromolar to picomolar concentration of LCMV peptide were required to induce deletion. The effectiveness of deletion by the different cell types did not correlate with their major histocompatibility class I expression level; it was, however, influenced by the presence of ICAM-1 adhesion molecules.
We have established a set of transgenic mouse lines in which the HLA-DRA gene was expressed in different cell types. In one line (DR alpha-24), DR alpha E beta b molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow-derived antigen-presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DR alpha-30) was found on thymic medullary and cortical epithelial cells but not on bone marrow-derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DR alpha-24 and DR alpha-30 was performed. In DR alpha-30, T cells expressing TcR V beta 5 and V beta 11 were eliminated to comparable levels to those in DR alpha-24, suggesting that expression of the DR alpha E beta b molecules on thymic epithelial cells are sufficient for clonal deletion of the self-superantigen-reactive T cells. In addition, CD4+ T cells from DR alpha-30 as well as those from DR alpha-24 were tolerant to DR alpha-derived peptide/I-Ab complex expressed on spleen cells from DR alpha-24 even in the presence of exogenous interleukin-2. These observations suggest that expression of the DR alpha chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DR alpha-derived peptide bound to I-Ab molecules, probably via clonal deletion of the self-reactive T cells.
T cell tolerance to self antigens is at least partly a reflection of clonal deletion of immature T cells in the thymus. Although it is well accepted that intrathymic tolerance is primarily a reflection of T cell contact with bone-marrow (BM)-derived antigen-presented cells (APC), evidence is presented that thymic epithelial cells (TEC) can contribute to tolerance induction. Studies with thymocytes from BM chimeras suggest that selective contact with antigen on TEC induces clonal deletion of a subset of high-affinity T cells; these cells are primarily responsible for in vivo effector functions such as allograft rejection and induction of lethal graft-versus-host disease. Intrathymic contact with TEC fails to delete the typical low-affinity T cells which mediate cytotoxic responses in vitro when cultured with lymphokines. Deletion of these low-affinity T cells appears to require contact with BM-derived APC. Despite the evidence that self tolerance involves clonal deletion in the thymus, it is often stated that backup mechanisms for tolerance induction must exist in the post-thymic environment, but this has yet to be proved. The competing argument is that normal self/nonself discrimination is solely a reflection of intrathymic tolerance: the failure of T cells to react against tissue-specific antigens is not a reflection of post-thymic tolerance but simply that T cells and tissue-specific antigens are kept segregated.
Parent-->F1 bone marrow (BM) chimeras provide a useful model for studying self tolerance induction. When prepared with supralethal irradiation (1300 cGy) and conditioned with anti-T cell antibodies, parent-->F1 BM chimeras are devoid of host BM-derived cells; host H-2 expression is apparent in both the intrathymic and extrathymic environments but is limited to non BM-derived cells. When parent-->F1 chimeras are injected with T cells from normal parental strain mice, the expression of host H-2 antigens on nonprofessional APC might be expected to induce tolerance through induction of clonal anergy. In practice, this does not occur. Instead, a small proportion of the injected T cells is induced to proliferate and differentiate into effector cells. Tolerance is not seen. Similarly, tolerance is not apparent when thymectomized parent-->F1 chimeras are given parental strain thymus grafts. These findings suggest that the expression of host H-2 antigens in the post-thymic environment of chimeras is not intrinsically tolerogenic for mature T cells or recent thymic emigrants. Interestingly, post-thymic tolerance does occur when parental strain T cells differentiate in the endogenous thymus of chimeras. Thus, when mature CD8+ cells are prepared from thymus vs lymph nodes (LN) of parent-->F1 chimeras, tolerance to host class I antigens is more marked in LN than thymus; this applies to cytotoxic T lymphocyte (CTL) precursors, generated by limiting dilution analysis. It would appear therefore that many of the host-reactive CTL precursors generated in the thymus of chimeras undergo tolerance induction (deletion or irreversible inactivation) in the post-thymic environment. We suggest that such tolerance is a reflection of a covert form of tolerance induced in the thymus: intrathymic contact with host antigens on thymic epithelial cells (TEC) in chimeras does not delete typical CTL precursors, but these cells are rendered "semi-tolerant". When cultured in vitro in the presence of lymphokines, the cells are able to recover and differentiate into CTL. In vivo, however, the cells recognize antigen in the periphery in the relative absence of lymphokines and the cells die. Although host class I expression on TEC in chimeras deletes only a small proportion of CTL precursors, contact with TEC induces strong tolerance of CD8+ cells in terms of helper-independent proliferative responses in vitro and induction of lethal graft-versus-host disease in vivo. We postulate that these latter responses are controlled by high-affinity T cells, whereas typical CTL generated in LDA are predominantly low-affinity cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Extrathymic T cell differentiation pathways have been reported, although the thymus is the main site of T cell differentiation. The thymus is also known to produce several cytokines that induce proliferation of thymocytes. In the present study, we investigated the influence of thymus-derived cytokines on extrathymic T cell differentiation by intraperitoneal implantation with a diffusion chamber which encloses fetal thymus (we named it fetal thymus-enclosed diffusion chamber, FTEDC) in athymic BALB/c nu/nu mice. Increase in number of T cells bearing T cell receptor (TcR) alpha/beta was detected in lymph nodes and spleens of FTEDC-implanted nude mice 1 week after implantation, whereas no such increase was detected in control nude mice implanted with a diffusion chamber without thymus. The FTEDC-induced increase of T cells was suppressed by intraperitoneal injection of anti-interleukin-7 monoclonal antibody (mAb). The TcR alpha/beta T cells in FTEDC-implanted BALB/c nu/nu mice preferentially expressed V beta 11, although V beta 11-positive T cells are deleted in the thymus of euthymic BALB/c mice by clonal elimination of self-super-antigen Dvb11-specific T cells. TcR alpha/beta T cells in FTEDC-implanted nude mice were of CD4-CD8- phenotype and showed no proliferative response against anti-TcR monoclonal antibody stimulation. These results suggest that the thymus can induce extrathymic T cell differentiation through the influence of thymus-derived cytokine(s) including interleukin-7, and that such extrathymically differentiated T cells have acquired only a little or no ability for proliferation when they recognize antigen by their TcR.
After immunization, normal H-2 heterozygous mice (for example H-2(b) × H-2(d)) generate two populations of cytotoxic effector
T cells, one specific for target cells expressing H-2(b)-plus-antigen and the other specific for H- 2(d)-plus-antigen. With
a multideterminant antigen, these two populations have about the same activity. We show here that the H-2 type of resident
cells in the thymus determines the H-2 preference of cytotoxic T lymphocytes. F(1)(B 10 × B 10.D2) (H-2(b) × H-2 (d)) mice
were thymectomized, lethally irradiated, and reconstituted with T-cell-depleted syngeneic hematopoietic cells. Groups of such
ATXBM mice were grafted subcutaneously with neonatal thymus lobes from parental mice, either B10 (H-2 (b)) or B10.D2 (H-2(d)).
2-3 mo later, the mice were immunized against the minor histocompatibility antigens on F(1)(BALB/c × BALB.B) cells and assayed
for cytotoxic T-cell activity. H-2(b) × H-2(d) ATXBM mice with H-2(b) thymus grafts responded to antigen-plus-H-2(b) much
better than to antigen-plus-H-2(d), and vice versa for the mice with H-2(d) thymus grafts. As judged by antiserum treatment,
the effector cells were of F(1) origin.
To explore the possibility that the "thymus preference" may have been due to suppression of T-cell activity, nonimmune spleen
and lymph node cells from normal H-2(b) × H-2(d) mice and cells from H-2(b) × H-2(d) mice bearing a homozygous thymus were
mixed 1:1 and immunized in adoptive transfer. The mixture responded to antigen-plus-H-2(b) and antigen-plus-H-2(d) equally
well, demonstrating that the cells that showed a "thymus preference" could not suppress a response to antigen in association
with the nonthymic H-2 type. We conclude from these and other experiments that H-2 antigens present on resident cells of the
thymus determine the spectrum of specificity of T cells which mature in that thymus and eventually make up the peripheral
T- cell pool.
Tetraparental bone marrow chimeras were produced by injecting lethally X-irradiated F1 hybrids with relatively high numbers of T-cell-depleted bone marrow cells from both allogeneic parental strains. The mice survival in excellent health and showed a stable, approximately 50:50 (parent:parent), lymphoid cell chimerism lasting for at least 7 mo after irradiation; regeneration of host-type hemopoietic cells was very limited. Thymus, lymph node, and thoracic duct lymphocytes showed specific unresponsiveness to host mixed leukocyte reaction (MLR) determinants. Similarly specific tolerance to H-2 antigens of host type was demonstrated in spleen and lymph node. No suppressor cells could be demonstrated in either system and blocking serum factors could not be found. The results suggest specific deletion of functional T cells reactive to host-type MLR and cell-mediated lympholysis determinants.
Semiallogenetic radiation chimeras were prepared by injecting heavily irradiated F1 hybrid mice with bone marrow cells from one parental strain; the bone marrow cells were treated with anti-theta serum and complement to remove T cells and injected in large numbers (2 times 10-7 cells). The mice survived in excellent health until sacrifice 6 mo later. Thoracic duct cannulation at this stage showed that the mice possessed normal numbers of recirculating lymphocytes. Close to 100% of thoracic duct lymphocytes and lymph node cells were shown to be of donor strain origin. The capacity of lymphocytes from the chimeras to respond to host-type determinants was tested in mixed leukocyte culture and in an assay for cell-mediated lympholysis (CML). Mixed leukocyte reactions (MLR) were measured both in vitro and in vivo; tumor cells and phytohemmaglutinin-stimulated blast cells were used as target cells for measuring CML. While responding normally to third party determinants, cells from the chimeras gave a definite, though reduced MLR when exposed to host-type determinants. However, this proliferative response to host-type determinants, unlike that to third party determinants, was not associated with differentiation into cytotoxic lymphocytes. No evidence could be found that unresponsiveness in this situation was due to blocking serum factors or suppressor T cells. It is argued that the results support the concept that lymphocytes responsive in mixed leukocyte culture have a different specificity to those exerting cell-mediated lympholysis.
Self tolerance induction in the thymus is known to delete T cells expressing certain V beta TCR molecules. In particular, V beta 17a+ and V beta 11+ T cells are selectively deleted in mice expressing H-2 I-E molecules. Although this finding implies that V beta 17a+ and V beta 11+ T cells have specificity for self I-E molecules, studies with V beta 11+ hybridomas prepared from mature lymphocytes taken from I-E- mice have shown that the vast majority of these hybridomas do not display I-E alloreactivity, at least in vitro. To examine whether V beta 11+ T cells are capable of reacting to I-E antigens in vivo, normal unprimed T cells from I-E- B10.A(4R) mice were transferred to irradiated I-E+ B10.A(2R) hosts and harvested from thoracic duct lymph of the recipients at various intervals. The donor T cells recovered in early lymph collections showed no reactivity to the I-E antigens of the host in vitro, presumably as a reflection of selective sequestration of the host-reactive cells in the lymphoid organs. Significantly, the disappearance of functional host-reactive cells from TDL was paralleled by a 90-95% reduction of V beta 11+ CD4+ cells. Blast cells were rare in early lymph collections but accounted for nearly all of the lymph-borne cells by day 3 after transfer. These blast cell populations contained a surprisingly high proportion of V beta 11+ cells, i.e., up to 25% in some experiments. Interestingly, the enrichment for V beta 11+ cells in the blast populations applied to CD8+ cells as well as to CD4+ cells. Collectively, the data suggest that in marked contrast to the failure of V beta 11+ cells to respond to I-E antigens in vitro, a high proportion of normal resting V beta 11+ cells are capable of reacting to I-E alloantigens in vivo.
We have generated an mAb, RR3-15, that recognizes murine TCRs containing the V beta 11 domain. Using this antibody to stain peripheral T cells, we have demonstrated that V beta 11-bearing T cells are largely absent from strains of mice that express the class II MHC molecule, I-E. Studies with F1 mice demonstrate that this effect is dominant, consistent with tolerance. The clonal deletion of V beta 11-bearing T cells appears to occur intrathymically, as immature but not mature V beta 11+ T cells are present in the thymus of I-E-bearing mice. Examination of B6 x DBA/2 recombinant inbred strains demonstrates that the expression of I-E molecules is necessary for the clonal deletion of V beta 11-bearing T cells, but that other non-MHC genes control the clonal deletion process, as well. Paradoxically, only a small fraction of V beta 11+ T cell hybridomas are I-E reactive.
In light of the widely accepted view that Ia-restricted L3T4+ T helper cells play a decisive role in controlling the differentiation of Lyt-2+ cells, experiments were designed to examine whether Lyt-2+ cells can respond to antigen in the absence of L3T4+ cells. The results showed that highly purified Lyt-2+ cells gave high primary mixed lymphocyte reactions (MLR) to various class I differences, including both mutant and allelic differences; responses to class II (Ia) differences were generally undetectable with Lyt-2+ cells. The intensity of MLR to class I differences was not affected by addition of anti-L3T4 monoclonal antibodies (mAb) to the cultures or by removing T cells from the stimulator populations. Negative selection experiments showed that Lyt-2+ cells could respond to class I differences across Ia barriers. MLR of purified Lyt-2+ cells peaked on days 3-4 and then fell sharply; background responses with syngeneic stimulators (auto-MLR) were virtually absent. Parallel experiments with purified L3T4+ cells showed that this subset responded in MLR only to class II (Ia) and not class I differences, reached peak responses only on day 6 rather than days 3-4, and often gave high auto-MLR. Within the first 3-4 d of culture, MLR were generally higher with Lyt-2+ cells than L3T4+ cells. Although no evidence could be found that Ia-restricted L3T4+ cells were required for the response of Lyt-2+ cells, presentation of antigen by Ia+ cells appeared to be essential. Thus, responses were ablated by pretreating stimulator cells with anti-Ia mAb plus C'. Significantly the failure of Lyt-2+ cells to respond to anti-Ia plus C'-treated stimulators could not be restored by adding syngeneic spleen cells; addition of IL-2 led to only a minor (15%) restoration of the response. It is suggested that Ia+ cells provide an obligatory second signal required by Lyt-2+ cells.
Highly purified populations of C57BL/6 (B6) L3T4+ and Lyt-2+ T cell subsets were compared for their capacity to exert alloreactivity to class I vs. class II H-2 differences in vivo. B6 Lyt-2+ cells responded strongly to the class I different mutant, bm1, as manifested by DNA synthesis in the spleen of irradiated mice followed by entry of blast cells into thoracic duct lymph, induction of splenomegaly in newborn mice, production of lethal GVHD in irradiated mice, and skin allograft rejection. By all of these parameters, B6 Lyt-2+ cells showed almost total unresponsiveness to the class II-different mutant, bm12. Reciprocal results were observed with B6 L3T4+ cells, these cells responding strongly against bm12 but not against bm1. In the case of purified T cell subsets from other strains, CBA/Ca and B10.BR L3T4+ cells both responded well to a full H-2 difference. Responses by Lyt-2+ cells from these strains were weaker, especially for CBA/Ca cells. The implications of these findings are discussed.
We studied the development of thymus-dependent immunity in congenitally athymic nude rats after implantation of cultured thymic fragments (CTF), particularly the development of in vitro alloreactivity in allogeneic combinations. CTF of DA (RT1a), PVG (RT1c), and RP (RT1p(u,1] origin were implanted in nude rats of WAG (RT1u) origin. In analysis 14 to 18 wk later, all recipients exhibited thymus-dependent immunocompetence assessed by (immuno)-histology of lymphoid organs and responsiveness to in vitro concanavalin A stimulation and in vivo ovalbumin immunization. Control nude animals were unresponsive. Also, in vitro alloreactivity was observed, measured by mixed lymphocyte reaction and cell-mediated lympholysis. The alloresponse to the allogeneic CTF donor haplotype was as to a third party, but that to the recipient was negative. The CTF before implantation were devoid of lymphoid elements and revealed epithelial-like cells as the major component. Cells in CTF showed expression of RT1 class I and class II antigens. CTF at autopsy had the architecture of a normal thymus. In immunohistochemistry using haplotype-specific antibodies, lymphocytes showed RT1u class I expression as in the normal WAG thymus. In the cortex-like area of CTF, stromal cells revealed class I and class II haplotype expression of the donor thymus, but in the medulla-like area, class II haplotype expression was that of the recipient WAG rat. These data indicate that after implantation in nude rats, CTF become populated not only with lymphoid elements, but also with stromal components from the recipient. In induction of thymus-dependent immunity, these acceptor-derived stromal (dendritic) cells may be involved in generation of allospecificity; class I and class II haplotype expression by the donor cortex (epithelial) compartment is ignored in this process.
We have prepared a monoclonal antibody, KJ16-133, from the cells of a rat immunized with the purified receptor for antigen plus I-A of a BALB/c T cell hybridoma, DO-11.10. Unlike most other monoclonal anti-receptor antibodies that have been described before, KJ16-133 is not clone specific. It reacts with approximately 20% of the receptors on T cells of normal BALB/c mice. It also reacts with about the same percentage of antigen-specific, major histocompatibility complex (MHC)-restricted or allogeneic I-region specific T cell hybridomas. Reaction of KJ16-133 with a given T cell hybridoma does not seem to depend on the antigen specificity or MHC-restricting element of the T cell in question. The determinant recognized by KJ16-133 has some unexpected properties. It is absent in several strains of mice including SJL/J and SJA/20, but present on the T cells of most other commonly used strains. The determinant recognized therefore does not map to Igh. Our experiments suggest that a clone-specific "antiidiotypic" antibody and KJ16-133 recognize determinants on different parts of the receptor. For example, the binding of a clone-specific antibody to target T cells is relatively temperature insensitive, whereas KJ16-133 binds well to cells at 37 degrees C but poorly to cells at 4 degrees C. The determinant recognized by a clone-specific antibody is sensitive to reduction and alkylation of the receptor, whereas KJ16-133 reactivity is not. Finally, binding of KJ16-133 at saturating concentrations to target T cells does not block the binding of a clone-specific antibody. Similarly, binding of a clone-specific antibody only marginally inhibits binding of KJ16-133. Taken together, these results suggest that KJ16-133 is directed against an allelic determinant on T cells that may be close to the membrane, and not in the receptor binding site for antigen plus MHC. The antibody may recognize an allele of a constant region isotype, or an allele of a J region.
Chimeric thymus, formed by fusing the prelymphoid third pharyngeal pouches of fetal mice with fetal liver, have been allowed to develop entirely in vitro. Syngeneic and allogeneic chimeras were prepared and both types of thymus were shown to contain substantial numbers of functional cytotoxic T lymphocyte precursors reactive against "third party" alloantigens. However, alloreactivity specific for H-2 antigens present on either the third pharyngeal pouch or the fetal liver was minimal. In three different allogeneic chimeric thymuses, the frequencies of cytotoxic T lymphocyte precursors reactive to H-2 antigens present on the third pharyngeal pouches were reduced to 1%, 4%, and 0% of control values, whereas, in the one allogeneic chimera tested for alloreactivity to H-2 antigens present on the fetal liver, the cytotoxic T lymphocyte precursor frequency was reduced to less than 1% of control values. The phenotype of the H-2 tolerance is shown to be one of functional clonal deletion of the cytotoxic T lymphocyte precursor.
Potent cytotoxic T lymphocyte (CTL) activity can be derived from cultures of thymocyte responders and minor H different spleen cell stimulators. As is the case of the spleen cell response previously reported, this cytotoxic activity requires in vivo priming. We performed several experiments designed to determine whether the in vivo priming effect is due to the in situ priming of the thymocyte CTL precursors, to contamination of thymus cell preparations with cells of neighboring lymph nodes, or to the appearance in the thymus of antigen-reactive peripheral T cells. We show by depletion of peripheral cells with antilymphocyte serum and part body irradiation that recent thymic immigrants derived from the bone marrow contribute to the primed thymic response. Thymic CTL were primed in animals in which peripheral T cell responses were completely eliminated by repeated treatment in vivo with monoclonal anti-Thy-1 reagents. Primed, antigen-activated lymph node cells were also demonstrated to contribute to the thymus-derived CTL response. Thus, the minor H-specific thymic CTL response is due both to in situ priming and the immigration of activated peripheral T cells. We discuss the possible significance for models of T cell differentiation of the presence within the thymus of antigen and antigen-reactive mature T cells.
The expression and distribution of antigens coded by the K and I regions of the major histocompatibility complex in the developing thymus of normal and nude mice has been investigated using monoclonal antibodies. Both immunohistological studies of intact rudiments and in vitro labeling of cultures derived from microdissected rudiments indicate that, while K region antigens are present on epithelial and mesenchymal elements, I region antigens are only detectable on the epithelium. This view is also substantiated by the selective absence of I region antigens in the abnormal nude thymic rudiment where the defect is considered to be epithelial in nature. The findings are considered in relation to the role of the thymus in providing an environment for the differentiation and selection of developing T cells, and it is proposed that the Ia-expressing epithelial elements play a central role in these functions.
The authors reinvestigated the basis of tolerance to histocompatibility antigens in X irradiation chimeras. No evidence could be found that tolerance in bone marrow chimeras was due to active suppression, leading to the conclusion that immunologic unresponsiveness reflects clonal deletion of histocompatibility reactive T cells. This decision probably occurs within the environment of the thymus at an early stage of T cell differentiation. T cells primed in axchimeric environment will cooperate with primed allogenic B cells but allogenic TB collaboration does not take place when the priming occurs in the separate strains. (Blum - Terre Haute, Ind.)
CELL mediated immune reactions in mice involving cytotoxic thymus derived lymphocytes have two important features; they are antigen-specific1 and the structures coded for by the major murine histocompatibility gene complex (H-2) play an important part in killer cell-target cell interaction1. This finding is not unique to mice since it has also been reported for rats, humans and chickens2-4. Two hypotheses have been suggested to explain this dual specificity of T killer cells; one proposes that T killer cells possess two separate structures, one of which recognises self H-2 structures and the other a non-H-2 antigen (dual recognition). The other hypothesis proposes that T killer cells possess one receptor which recognises an H-2 antigen modified by a non-H-2 antigen (modified self)1,5. Evidence to test these hypotheses was sought by preparing chimaeras made by reconstituting lethally irradiated F1 mice with bone marrow stem cells of one parent (Parent-->F) refs 6-8) or neonatally tolerant mice9 (mice of a parental H-2 type injected with F1 cells as neonates). As a result, Parent --> F1 chimaeric lymphocytes lysed infected and trinitrophenyl (TNP)-modified targets of both parental H-2 types, suggesting that H-2 antigens of the killer cells were not involved in the lytic interaction. In contrast, lymphocytes from neonatally tolerant mice did not lyse infected targets from the incompatible parent9. Although these results can be explained by postulating a single receptor, they also fit the dual recognition hypothesis6-10. Bevan has shown that irradiated bone marrow chimaeras of the type (A × B)F1 --> Parental A recipient generated preferentially cytotoxic T cells against minor histocompatibility antigens in association with the H-2 haplotype of the recipient A; he concluded that the chimaeric host determines the specificity of T cell restriction either through a thymic selection process as proposed by Jerne or alternatively through an antigen presentation mechanism.
After immunization, normal H-2 heterozygous mice (for example H-2(b) x H-2(d)) generate two populations of cytotoxic effector T cells, one specific for target cells expressing H-2(b)-plus-antigen and the other specific for H- 2(d)-plus-antigen. With a multideterminant antigen, these two populations have about the same activity. We show here that the H-2 type of resident cells in the thymus determines the H-2 preference of cytotoxic T lymphocytes. F(1)(B 10 x B 10.D2) (H-2(b) x H-2 (d)) mice were thymectomized, lethally irradiated, and reconstituted with T-cell-depleted syngeneic hematopoietic cells. Groups of such ATXBM mice were grafted subcutaneously with neonatal thymus lobes from parental mice, either B10 (H-2 (b)) or B10.D2 (H-2(d)). 2-3 mo later, the mice were immunized against the minor histocompatibility antigens on F(1)(BALB/c x BALB.B) cells and assayed for cytotoxic T-cell activity. H-2(b) x H-2(d) ATXBM mice with H-2(b) thymus grafts responded to antigen-plus-H-2(b) much better than to antigen-plus-H-2(d), and vice versa for the mice with H-2(d) thymus grafts. As judged by antiserum treatment, the effector cells were of F(1) origin. To explore the possibility that the "thymus preference" may have been due to suppression of T-cell activity, nonimmune spleen and lymph node cells from normal H-2(b) x H-2(d) mice and cells from H-2(b) x H-2(d) mice bearing a homozygous thymus were mixed 1:1 and immunized in adoptive transfer. The mixture responded to antigen-plus-H-2(b) and antigen-plus-H-2(d) equally well, demonstrating that the cells that showed a "thymus preference" could not suppress a response to antigen in association with the nonthymic H-2 type. We conclude from these and other experiments that H-2 antigens present on resident cells of the thymus determine the spectrum of specificity of T cells which mature in that thymus and eventually make up the peripheral T- cell pool.
Transgenic mice expressing a T cell receptor heterodimer specific for a fragment of pigeon cytochrome c plus an MHC class II molecule (I-Ek) have been made. We find that H-2k alpha beta transgenic mice have an overall increase in the number of T cells and express a 10-fold higher fraction of cytochrome c-reactive cells than H-2b mice. Surface staining of thymocytes indicates that in H-2b mice, T cell development is arrested at an intermediate stage of differentiation (CD4+8+, CD310). Analyses of mice carrying these T cell receptor genes and MHC class II I-E alpha constructs indicate that his developmental block can be reversed in H-2b mice by I-E expression on cortical epithelial cells of the thymus. These data suggest that a direct T cell receptor-MHC interaction occurs in the thymus in the absence of nominal antigen and results in the enhanced export of T cells, consistent with the concept of "positive selection".
The thymus has two important roles in controlling the specificity of T lymphocytes. First, T cells differentiating in the thymus are rendered tolerant of 'self' antigens, particularly antigens encoded by the major histocompatibility complex, the H-2 complex in mice. Second, the thymus imbues T cells with the property of H-2-restricted recognition of antigen, that is, the capacity of T cells to react with foreign antigens presented in association with self H-2 gene products. Until recently it has generally been assumed that self-tolerance and H-2-restricted specificity both reflect early T-cell contact with self H-2 determinants expressed on thymic epithelial cells. Recent evidence suggests, however, that intrathymic cells of the macrophage/dendritic cell (Mphi/DC) lineage also have a role in shaping T-cell specificity. In particular, it has been found that the tolerance to graft-type H-2 determinants which normally ensues when T cells differentiate in an H-2-different thymus fails to occur when the thymus is pretreated with deoxyguanosine (dGuo), a procedure that selectively destroys Mphi/DC but spares epithelial cells. In contrast to these findings on tolerance induction, evidence is presented here that dGuo-treated thymus grafts do imprint T cells with H--2-restricted specificity for antigen. It appears, therefore, that induction of tolerance and H--2 restriction are controlled by different cells in the thymus.
In an attempt to resolve the issue of whether H-2-restricted T cell specificity is controlled by thymic epithelial cells or by cells of the macrophage/dendritic cell (M phi/DC) lineages, long-term F1----parent chimeras were subjected to secondary irradiation and reconstitution with F1 marrow cells. The rationale was that if F1 M phi/DC enter the thymus only quite slowly after irradiation, as claimed by other investigators, leaving F1----parent chimeras for a period of several months before re-irradiation would ensure that the new wave of T cells generated in the thymus of the chimeras would have no difficulty in making contact with donor-derived F1 M phi/DC. According to the view that M phi/DC rather than epithelial cells control H-2 restriction, the T cells differentiating in these chimeras would be expected to show H-2 restriction to both parental strains. In practice, T cells from twice-irradiated (1000 + 800 rad) chimeras showed strong restriction to host (thymic) H-2 determinants, the degree of restriction to host determinants being as marked as with T cells from once-irradiated chimeras. This finding applied both to T proliferative responses to KLH assayed in vitro and to T helper function for sheep erythrocytes measured in vivo. Preliminary experiments established that the initial dose of irradiation used for preparing the chimeras (1000 rad) resulted in almost total replacement of intrathymic M phi/DC by donor-derived cells within 4 wk of irradiation; M phi/DC were typed by determining their capacity to stimulate mixed-lymphocyte reactions. Collectively, the data imply that, at least under the conditions used, H-2-restricted T cell specificity is controlled by epithelial cells rather than by M phi/DC.
Irradiation bone marrow chimeras were established by reconstitution of lethally irradiated AKR mice with C57BL/10 marrow cells to permit serial analysis of the developing reactivities of lymphocytes from such chimeras, [B10----AKR], against donor, host, or third party antigens. We found that substantial proliferative responses to Ia antigens of the recipient strain and also to third party antigens were generated by the thymocytes obtained from the irradiation chimeras at an early stage after bone marrow reconstitution. The majority of the responding thymocytes had surfaces lacking demonstrable peanut agglutinin receptors and were donor type Thy-1+, Ly-2-, and L3T4+ in both anti-recipient and anti-third party MLR. In anti-host responses, however, Ly-2+ thymocytes seemed to be at least partially involved. This capacity of thymus cells to mount a response to antigens of the recipient strain declined shortly thereafter, whereas the capacity to mount MLR against third party antigens persisted. The spleen cells of [B10----AKR] chimeras at the same time developed a more durable capability to exhibit anti-host reactivities and a permanent capability of reacting to third party allo-antigens. The stimulator antigens were Ia molecules on the stimulator cells in both anti-recipient and anti-third party MLR. The responding splenocytes were of donor origin and most of them had Thy-1+, Ly-1+2-, and L3T4+ phenotype.
One of the risks in attempting to give an overview of a subject as mired in contrversy as the immunobiology of T cells is that the reader fails to appreciate the distinction between established 'facts' and the idiosyncratic views of the authors. With this risk in mind, a short 'unbiased' summary of what is known and not about T cell specificity and function is given below. A. T cell specificity. Perhaps the most striking difference between T and B lymphocytes is that B cells have specificity for free antigen, whereas T cells react with antigens displayed in close association with H-2 molecules on the surface of living cells. The bulk of evidence suggests that the response of T cells to foreign non-H-2 antigens (antigen X) requires that the antigen be 'processed' (reduced to peptides or unfolded) by the APC. Processing allows small particles of antigen X to enter into an immunogenic alignment with surface class I or class II H-2 molecules; T cells recognize the association of self H-2 + X and are triggered. The evidence for antigen processing is quite strong for antigens seen in association with H-2 class II molecules, although recent evidence suggests that antigen processing might also be important for certain class I-restricted responses. Although there is continuing debate on whether antigen and H-2 molecules form a true self + X complex, two groups have now provided impressive evidence that certain peptide antigens do enter into firm physical association with high responder class II molecules; it remains to be seen whether this is a general phenomenon.
The thymus is the major site for T-cell receptor (TCR) gene rearrangement and T-cell maturation. The specific antigen recognition structure (TCR) on murine T cells has been shown to be dependent on a polymorphic set of disulphide-linked heterodimers, containing two integral membrane glycoprotein chains, TCR alpha and TCR beta, expressed in non-covalent association with an invariant complex of proteins, CD3 (T3). Recently, a novel TCR/CD3 complex, that includes the product of the TCR gamma gene, has been identified on a subset of both peripheral cells and thymocytes. Here we examine the expression of TCR/CD3 complexes in fetal ontogeny and in the adult thymus. The results demonstrate that CD3+4-8-(T3+,L3T4-,Lyt2-)cells are detected in day-15 fetal thymi, throughout fetal development and in adult thymus. In situ hybridization studies indicate that these early CD3+ cells express high levels of TCR gamma-specific RNA, low levels of TCR beta-specific RNA and no detectable TCR alpha-specific RNA. Day-16 CD3+,4-,8- fetal thymocytes can be activated to proliferate and demonstrate cytolytic activity when cultured in the presence of anti-CD3 monoclonal antibodies and interleukin-2 (IL-2). These results suggest that CD3-bearing cells, present early in thymic ontogeny, express a functional TCR and may, therefore, be important in repertoire development.
Immunological tolerance of self has been studied experimentally by the induction of unresponsiveness to antigens of the major histocompatibility complex (MHC) in neonatal mice. The specific unresponsiveness resulting from such neonatal tolerance induction is first demonstrable in the thymus, suggesting that neonatal tolerance is induced by some cellular component of the thymus, or at some prethymic stage. Recent transplantation studies suggest that thymic epithelium, derived by organ culturing fetal mouse thymus in the presence of deoxyguanosine, survives in an allogeneic host environment despite the continued expression of MHC donor antigens, but fails to induce allotolerance. We demonstrate here that embryonic thymus lobes organ cultured at 24 degrees C, a treatment that deletes the lymphohaematopoietic component of thymus leaving the epithelial matrix intact, when transplanted to intact histoincompatible recipients, similarly survive for a prolonged period and do not induce tolerance to donor MHC antigens. However, when such culture-derived thymic epithelium is allografted to athymic nude mice, host-derived lymphocytes from both the epithelial graft and recipient spleen are unresponsive to the MHC antigens of the epithelial donor. The results suggest that, when investigated in a system which precludes the possible involvement of extrinsic mature T cells, processed by the syngeneic host thymus, thymic epithelium may induce transplantation tolerance.
Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.
The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.
Differentiation of early thymocytes into mature T cells depends upon intrathymic T cell contact with major histocompatibility complex (MHC) molecules, i.e., H-2 molecules in mice. T cell recognition of H-2 molecules in the thymus has two consequences. First, some T cells undergo a process of positive selection which leads specifically-reactive immature thymocytes to survive and differentiate into mature functional T cells. Second, T cells with high affinity for H-2 molecules undergo negative selection (tolerance). We and others have argued that positive selection is controlled by thymic epithelial cells, especially cortical epithelium, whereas negative selection reflects contact with bone-marrow (BM) derived cells. This scheme appears to be an oversimplication because we have recently found evidence that a non-BM-derived component of the thymus, presumably epithelial cells, is highly tolerogenic for CD4+ cells. Whether tolerance of CD4+ cells is controlled by cortical epithelium or medullary epithelium is unclear. In this respect it is of interest that chronic injection of mice with cyclosporine A results in selective destruction of medullary epithelial cells and impaired induction of self tolerance.
Treatment of fetal thymuses with 2-deoxyguanosine depletes these organs of many haematopoietic cells, and if such thymuses are transplanted into allogeneic athymic nude mice, intrathymic development of cytolytic T-lymphocyte precursors (CTL-P) occurs, including those which are specific for class I major histocompatibility complex (MHC) antigens expressed by the thymus epithelium. Thus, T cells from BALB/c (H-2d) nude mice transplanted with allogeneic C57BL/6 (H-2b) thymic epithelium can be stimulated in vitro to produce CTL specific for H-2b class I MHC antigens. We report here that thymocytes and lymph node T cells from such mice are responsive in mixed leukocyte reaction in the absence of exogenous growth factors, indicating that lack of tolerance is manifest at the level of CTL-P and proliferating T cells. We also show that T cells from such mice are tolerant to minor histocompatibility antigens of the thymus donor in the context of MHC antigens of the recipient. The results indicate that haematopoietic rather than epithelial cells tolerize CTL-P and that donor-type minor but not major histocompatability antigens can be presented in tolerogenic form by haematopoietic cells expressing recipient-type MHC antigens.
The monoclonal antibody KJ23a reacts with T cell receptors utilizing the V beta segment V beta 17a. T cells bearing V beta 17a+ receptors react with very high frequency with the MHC class II protein, IE. In this paper we show that T cells expressing V beta 17a are selectively eliminated from the peripheral T cell and mature thymocyte pool of mice expressing IE, but are present in expected numbers in the immature thymocyte population of such animals. These results show that in normal animals tolerance to self-MHC is due to clonal elimination rather than suppression. In addition, they indicate that tolerance induction may occur in the thymus at the time immature thymocytes are selected to move into the mature thymocyte pool.
The E alpha MHC class II gene with 1.4 kb of 5'-flanking and 0.5 kb of 3'-flanking sequences was introduced into (H-2b X s)F2 mice, which do not express their endogenous E alpha gene. The transgene was expressed in thymic tissue and in adherent spleen cells and was induced in peritoneal exudate cells by gamma-interferon. In contrast to the normal E alpha gene, there was no expression in B lymphocytes. Since transgenic animals made with constructs containing 3.2 kb and 2 kb of 5'-flanking sequences show normal expression pattern of the E alpha gene, it appears that deletion of 5'-flanking sequences between -1.4 kb and -2 kb inactivated or eliminated regulatory sequences required for expression of E alpha specifically in B cells. The presence of pBR327 DNA linked to the -1.4 kb E alpha transgene suppresses expression in peripheral adherent cells, yielding mice expressing E alpha only in the thymus. These mice appear to be tolerant to I-E, as measured in mixed leukocyte response experiments.
Grafts of the anterior limb bud introduced at embryonic day 4 between histoincompatible chick embryos were subject to chronic,
mild rejection beginning from several weeks to several months after birth. In contrast, quail wing buds similarly grafted
into chickens started to be rejected at the first or second week after birth and finally autoamputated. Embryonic thymus epithelium
from donor quail (before it had been colonized by hemopoietic cells) was grafted into chicks. A chimeric thymic epithelial
stroma was generated in which the lymphocytes of the chick acquired the capacity to recognize the grafted limb as self either
permanently or for a protracted period of time. In such thymic chimeras the grafted wings were not rejected.
A new model has been developed to address the question of whether T cells that traverse an allogeneic thymus during early and late life become restricted to interact, in vivo, with other leukocytes and target cells that display the major histocompatibility complex (MHC) antigens of the thymus haplotype. Chimeras were made microsurgically with pairs of 24-h-old Xenopus embryos such that the anterior region of an embryonic chimera contained the thymus anlagen and was of one MHC genotype, whereas the posterior region contained the anlagen of all hemopoietic cells and was of another genotype. Assays to determine the MHC haplotype restriction specificity of T cells in chimeras that had been reared through metamorphosis involved: specific antibody responses (IgM and IgG) to dinitrophenylated keyhole limpet hemocyanin; rejection of minor H locus disparate skin grafts that expressed the MHC antigens of either the thymus donor or the lymphocyte donor; and mixed leukocyte culture. MHC-mismatched chimeras displayed split tolerance since they accepted skin grafts of the thymus haplotype but had lymphocytes that proliferated in response to MHC antigens of the thymus donor strain as well as to MHC antigens of third-party donors. IgM responses of MHC-matched and MHC-mismatched chimeras and of nonchimeric controls did not differ. However, the IgG responses of MHC-mismatched thymus/lymphocyte chimeras peaked later than those of MHC-matched chimeras and normal controls. Data from skin grafting protocols were consistent with the proposition that there may be in vivo selection of T cells reactive to minor H antigens presented in association with the MHC antigens of the thymus rather than the MHC antigens of the lymphocytes themselves. These data suggest that although it is not absolute, there is thymic selection of the T cell repertoire in Xenopus.
Five rat monoclonal antibodies have been derived that express specificities for determinants present on the molecular complex bearing the Lyt 2 antigen. SDS-polyacrylamide gel electrophoresis of 125I-labeled polypeptides precipitated by each of these antibodies reveal 3 components (150,000, 75,000, and 33,000 daltons), and 2 components (44,000 and 33,000 daltons) when analyzed under nonreducing and reducing conditions, respectively. Two of these antibodies are IgG and are specific for the Lyt 2.2 determinant; the other 3 are IgM and react with determinants other than Lyt 2.2, which are nonpolymorphic. Each of the 5 antibodies can block the cytolytic activities of 5-day MLC cells or of cloned cytolytic T cells in the absence of C. Treatment of responding spleen cells with any of these antibodies and C inhibits the generation of cytolytic activity in MLC.
We describe here the properties of mAb GK1.5, which recognizes a cell surface molecule designated L3T4; the determinant on L3T4 recognized by mAb GK1.5 is designated L3T4a. We present evidence here that: i) the expression of L3T4a by murine T cell clones correlates primarily with class II MHC antigen-reactivity; ii) mAb GK1.5 blocks all class II MHC antigen-specific functions (cytolysis, proliferation, release of lymphokines) by murine class II MHC antigen-reactive T cell clones, although there appears to be clonal heterogeneity in the degree to which these functions are blocked by mAb GK1.5; iii) mAb GK1.5 blocks class II MHC antigen-specific release of IL-2 from cloned T cell hybridomas by blocking class II MHC antigen-specific binding; and iv) L3T4 is very similar to the human Leu3/T4 antigen. The properties of mAb GK1.5 (complement fixation, reactivity with all mouse strains tested, profound blocking of all class II MHC antigen-specific functions by murine T cells, usefulness for FACS analyses, and usefulness for immuno-precipitation/SDS-PAGE analyses) make it suitable for investigating both the role of class II MHC antigen-reactive T cells in various immunological phenomena and the mechanistic basis, at the molecular level, of class II MHC antigen-reactivity by murine T cells.
Intravenous injection of class I incompatible spleen cells into mice results in a drastic reduction of the recipient's cytotoxic T lymphocyte (CTL) response against the injected, but not against third party, class I antigens when measured in bulk cultures initiated 5 to 6 days after the injection. This specific suppressive effect is partly due to T cells but can also be seen when high numbers of anti-Thy-1 and complement-treated spleen cells of nude mice are injected. Such cells suppressing CTL responses against self histocompatibility antigens are called "veto cells." The precursor frequency of CTL specific for the injected class I antigen is found to be reduced greater than 200-fold at days 5 to 6 after the injection, whereas the frequencies of CTL specific for third party class I antigens are not significantly changed. These results indicate that there is a functional clonal deletion of the CTL recognizing class I incompatible veto cells in vivo. The role of such a veto phenomenon in the induction and maintenance of self tolerance and allograft tolerance is discussed.
BALB/c nu/nu mice were grafted with embryonic 14-day-old C57BL/6 thymi which were transplanted either nontreated or after elimination of hemopoietic cells with 2-deoxyguanosine. In both types of grafts host cells developed normally into functional thymocytes. Thymocytes from 2-deoxyguanosine-treated but not from untreated grafts contained as many cytolytic T lymphocyte precursors specific for class I MHC antigens on thymus epithelium as normal BALB/c thymocytes. As cytolytic T lymphocyte precursors were neither suppressed nor activated in these grafts it is concluded that thymocytes ignore class I MHC antigens expressed on thymus epithelium.
Intrathymic selection of T-cell specificity has been shown to be influenced by self-major histocompatibility complex (MHC) antigens encoded by radioresistant thymic stromal cells. The role of non-MHC antigens in intrathymic T-cell differentiation, in particular induction of antigen-specific tolerance, has been unclear and the access of non-MHC antigens to the thymus is controversial. Here we present evidence that circulating protein antigens enter the thymus and are presented by thymic stromal cells. At least three distinct types of stromal cells are thought to be associated with intrathymic lymphopoiesis; after intravenous (i.v.) injection of antigen only I-A/E-positive medullary dendritic cells, but not I-A/E-negative macrophages or I-A/E-positive cortical epithelial cells co-purified with antigen-specific stimulation of cloned T-helper cells in vitro. Antigen presentation by thymic stromal cells was dependent on the dose of antigen injected and the time interval after injection.
Eleven hybridoma antibodies directed against mouse major histocompatibility complex products of the H-2b haplotype have been produced and characterized. Of 7 antibodies reacting to H-2Kb and/or H-2Db antigens, all cross-reacted with other H-2 antigens, and 5 exhibited no correspondence with a known H-2 specificity established in the H-2 chart. Four anti-Iab antibodies all reacted with antigens encoded by the I-A subregion. Some of these antibodies showed no cross-reaction with other haplotypes, indicating reactions to private specificities of the I-Ab antigen. In addition, these anti-Ia antibodies appeared to be capable of distinguishing fine determinant differences, which corresponding alloantisera failed to reveal. A high frequency of hybridomas secreting IgM antibodies was found after fusions of spleen cells obtained from C3H anti-C3H.SW immunized mice, in contrast to the dominance of IgG hybridomas produced previously by fusions of spleen cells from mice immunized in the reverse direction. An isotype analysis of conventional cytotoxic alloantisera from the same strain combinations was therefore performed. The same correlation with respect to isotype expression was found, indicating that hybridoma antibodies reflect normal antibody responses and suggesting H-2-linked control of this expression.
A description is given of a rat anti-mouse hybridoma antibody, JIId, which reacts with erythrocytes, neutrophils, greater than 90% of thymus cells, and most B cells. JIId does not have detectable activity for mature T cells, pluripotential stem cells, platelets, or cells of the monocyte-macrophage lineage. Although the JIId antigen is present on 90 to 95% of typical small B lymphocytes, pretreatment of spleen cells with JIId plus complement has no effect on secondary IgG antibody responses; by contrast, primary IgM responses and proliferative responses to lipopolysaccharide are substantially reduced. Unlike the precursors of IgG antibody-producing cells (AFC), IgG AFC per se are strongly JIId-positive. Rapid acquisition of the JIId antigen also applied to the early progeny of pluripotential stem cells.
Maturation of polyspecific B cells to Ig secretion was induced in response to the interaction of helper T cells with soluble antigen. The in vitro propagated, keyhole limpet hemocyanin (KLH)‐primed C57BL16 T cells used in these experiments proliferated in response to antigen presented on I‐A‐compatible antigen‐presenting cells and were depleted of alloreactive cells. These T cells induced a strong polyspecific plaque‐forming cell response from normal C57BL/6 B cells over a wide range of KLH concentrations (100 μg/ml to 0.01 μg/ml). Culture conditions were established whereby H‐2‐restricted T cell‐macrophage interactions were nonlimiting, allowing a direct analysis of H‐2‐restricted T‐B cell interactions involved in polyspecific B cell induction.
The requirements for B cell induction/amplification were dependent on the size of the responding B cell population. Small B cells were activated only at high concentrations of KLH and required a direct H‐2‐restricted interaction with KLH‐primed helper T cells. In contrast, large B cells were induced/amplified over the full range of KLH concentrations tested in an H‐2‐unrestricted manner, limited only by the H‐2‐restricted, antigen‐dependent activation of T helper cells. The requirement for high antigen concentration in the activation of small B cells is proposed to reflect a requirement for antigen binding to the B cell surface via nonspecific interactions. This binding was not in itself stimulatory, but expression of antigen in conjunction with B cell surface Ia antigens provided a focus for a direct interaction with Ia‐restricted, antigen‐specific T helper cells (or factors). In contrast, the H‐2‐unrestricted induction/ amplification of large B cells was mediated solely as a consequence of the helper T cell activation which occurred at the lowest concentrations of KLH tested. T helper cell activation is proposed to lead to the production of non‐antigen‐specific, non‐Ia‐restricted factors supporting the continued growth of blasted B cells.
Thus, while an H‐2‐restricted T cell‐macrophage interaction is an obligate requirement for polyspecific B cell responses, the requirement for a direct H‐2‐restricted T‐B cell interaction varies with the pre‐existing state of activation of the responding B cell population.
Hybridoma cell lines secreting antibodies to mouse H-2 or Ia antigens have been generated by fusing mouse immune lymphocytes with appropriate myeloma lines. Among the 11 established clones reported here, nine produce anti-H-2 antibodies and two produce anti-Ia antibodies. The specificities and cross-reactions of these monoclonal antibodies have been studied in detail. One hybridoma antibody reacted only to Kk antigens without any detectable cross-reactions, thus suggesting reaction to a private specificity of the Kk molecule. All other anti-H-2 hybridoma antibodies appeared to detect public specificities as defined either by reactions with products of more than one H-2 locus or with different alleles at one or more loci. The two anti-Ia antibodies both reacted with I-E/C products, but exhibited different cross-reactivity patterns. Strain distribution analyses so far indicate that the public specificities detected by these monoclonal antibodies are considerably different from those that had been established by traditional serology. Since public specificities defined by the hybridoma antibodies must by definition represent cross-reactions, these findings may have important implications relating to the structure and evolution of MHC gene products.
Tolerance induced by thymic epithelial grafts in birds
- H Ohki
- C Martin
- C Corbel
- M Coley
- N M Le Douarin
Ohki, H., C. Martin, C. Corbel, M. Coley, and N. M. Le Douarin. 1987. Tolerance
induced by thymic epithelial grafts in birds. Science (Wash. DC). 237 :1032.