Deletion of donor-reactive T cell clones after human liver transplant.

We recently developed a high throughput T cell receptor ß chain (TCRß) sequencing-based approach to identifying and tracking donor-reactive T cells. To address the role of clonal deletion in liver allograft tolerance, we applied this method in samples from a recent randomized study, ITN030ST, in which immunosuppression withdrawal was attempted within 2 years of liver transplantation. We identified donor-reactive T cell clones via TCRß sequencing following a pre-transplant mixed lymphocyte reaction and tracked these clones in the circulation following transplantation in 3 tolerant and 5 non-tolerant subjects. All subjects showed a downward trend and significant reductions in donor-reactive TCRß sequences were detected post-transplant in 6 of 8 subjects, including 2 tolerant and 4 non-tolerant recipients. Reductions in donor-reactive TCRß sequences were greater than those of all other TCRß sequences, including 3rd party-reactive sequences, in all 8 subjects, demonstrating an impact of the liver allograft after accounting for repertoire turnover. Although limited by patient number and heterogeneity, our results suggest that partial deletion of donor-reactive T cell clones may be a consequence of liver transplantation and does not correlate with success or failure of early immunosuppression withdrawal. These observations underscore the organ- and/or protocol-specific nature of tolerance mechanisms in humans.

Murine thymic selection quantified using a unique method to capture deleted T cells.

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77(GFP) transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim(-/-) mice had an increased number of GFP(hi) cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.

Regulatory role of leukocyte-common-antigen-related molecule (LAR) in thymocyte differentiation.

The strength of interaction between the antigenic peptide-loaded MHC (MHC/p) and the TCR determines T-cell fate in the thymus. A high avidity interaction between the TCR and the MHC/p induces apoptosis of self-reactive T cells (negative selection), whereas a moderate avidity interaction rescues thymocytes from apoptosis and permits further differentiation to mature T cells (positive selection). Leukocyte common antigen-related molecule (LAR), a receptor-like protein tyrosine phosphatase, is expressed on immature thymocytes, but its role in thymocyte differentiation has not yet been fully elucidated. We analyzed LAR-deficient mice and demonstrated that LAR deficiency affected the differentiation and expansion of immature thymocytes as well as positive and negative selection. Furthermore, LAR deficiency resulted in a lower Ca2+ response. The results indicate that LAR is an important modulator of TCR signaling that controls thymocyte differentiation.

T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus.

Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

Tolerance without clonal expansion: self-antigen-expressing B cells program self-reactive T cells for future deletion.

B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells.

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein.

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

Human skin cells support thymus-independent T cell development.

Thymic tissue has previously been considered a requirement for the generation of a functional and diverse population of human T cells. We report that fibroblasts and keratinocytes from human skin arrayed on a synthetic 3-dimensional matrix support the development of functional human T cells from hematopoietic precursor cells in the absence of thymic tissue. Newly generated T cells contained T cell receptor excision circles, possessed a diverse T cell repertoire, and were functionally mature and tolerant to self MHC, indicating successful completion of positive and negative selection. Skin cell cultures expressed the AIRE, Foxn1, and Hoxa3 transcription factors and a panel of autoantigens. Skin and bone marrow biopsies can thus be used to generate de novo functional and diverse T cell populations for potential therapeutic use in immunosuppressed patients.

Gammadelta T cell development--having the strength to get there.

Gammadelta T cells play critical roles in immune regulation, tumour surveillance and specific primary immune responses. Mature gammadelta cells derive from thymic precursors that also generate alphabeta T cells. Recent reports have highlighted the impact of the strength of signal received via the T cell receptor on T cell lineage commitment, and the importance of cross-talk between committed alphabeta thymocytes and bipotential progenitors for normal gammadelta T cell differentiation. Studies on T cell receptor-mediated selection of gammadelta cells have supported the view that these unconventional T cells are positively rather than negatively selected on cognate self antigen.

Age-associated remodeling of thymopoiesis: role for gonadal hormones and catecholamines.

The present review summarizes recent data on age-related thymic changes termed thymic involution, and highlights the putative role of perturbances in extrathymical and, possibly, intrathymical production of gonadal steroids and catecholamines in this process. Thymic involution has been envisaged as an extremely complex process involving multifactorial mechanisms along the bone marrow-thymic axis that accounts for the major manifestations of immunosenescence. These mechanisms include basic cell aging processes (for example, cell replication and programmed cell death) and processes unique to the immune system (such as generation of the T cell receptor repertoire and control of potentially autoreactive cells). Given that the onset of age-associated thymic involution coincides with the rise in gonadal steroid levels at puberty, a causal link between these events has been suggested. It has been shown that: (1) peripubertal gonadectomy causes substantial decrease in the level of noradrenaline in adult male and female thymus and (2) catecholamines, acting via alpha- and beta-adrenoceptor, produce suppressive effects on the thymic cellularity and production of both effector and regulatory T cells. Furthermore, the possibility that gonadal steroids contribute to thymic involution is discussed in this paper. In light of recent data indicating that effects of gonadal hormone deprivation on the thymic cellularity and function are long lasting but transitory, a putative role for the intrathymic sex steroid/catecholamine production in assuring the organ involution, under conditions of their limited supply by extrathymic sources, is also considered.

Negative selection and autoimmunity.

The impaired elimination of self-reactive T cells is one factor that contributes to autoimmunity. Although the mechanism of thymic negative selection has been studied for decades, recent data demonstrate that the mechanisms underlying this fundamental process remain extremely controversial. Nonetheless, new models such as the Aire-deficient mice have demonstrated the importance of thymic negative selection in autoimmune disease progression in mice and humans.

Fractionated dosing of cyclophosphamide for establishing long-lasting skin allograft survival, stable mixed chimerism, and intrathymic clonal deletion in mice primed with allogeneic spleen cells.

BACKGROUND: Injection of allo-spleen cells (SC) followed by a single dose of cyclophosphamide (CP) can induce tolerance of tumor and/or skin allografts in mice. To minimize the damage caused by CP, fractionation of CP that can establish long-lasting skin graft survival, stable mixed chimerism, and intrathymic clonal deletion in the host was investigated in the present study. METHODS: Allo-SC (10(8)) were given intravenously on day 0. CP at 200 mg/kg was given intraperitoneally on day 2 in a single dose (CP 200x1 group). CP at 100, 66, 50, 40, and 33 mg/kg was given daily from day 1 through days 2, 3, 4, 5 and 6, respectively, in the fractionated doses (CP 100x2, 66x3, 50x4, 40x5, and 33x6 groups; total dose=200 mg/kg). Allografting was performed on day 14. RESULTS: In a fully allogeneic combination of C57BL/6 (H2b)-->AKR (H2k, Mls-1a), an EL-4 tumor (H2b) was specifically accepted to kill the AKR mice in all of the SC+CP 200x1, 100x2, 66x3, 50x4, 40x5, and 33x6 groups (n=6), but C57BL/6 skin graft survival was not prolonged in any of the tumor-tolerant groups. In an H2-identical combination of AKR-->C3H (H2k, Mls-1b), AKR skin graft survival was prolonged remarkably (80-90 days) in the SC+CP 200x1, 100x2, and 66x3 groups (n=5-11), but was prolonged moderately (20-60 days) in the SC+CP 50x4 and 40x5 groups. In both of the SC+CP 200x1 and 66x3 groups in the AKR-->C3H combination, mixed chimerism was maintained for as long as 100 days after tolerance induction in both the spleen and thymus, associated with intrathymic clonal deletion of Vbeta6+ T cells. The decreases in leukocyte count, hemoglobin level, spleen weight, SC count, and body weight were significantly smaller in the SC+CP 66x3 group than in the SC+CP 200x1 group. CONCLUSIONS: Fractionated CP is effective in ameliorating the compromised state induced by a single dose of CP. To induce a long-lasting skin allograft survival associated with stable mixed chimerism and intrathymic clonal deletion in an H2-identical combination, 200 mg/kg of CP can be divided into three or fewer fractions.

Role of intrathymic rat class II+ cells in maintaining deletional tolerance in xenogeneic rat-->mouse bone marrow chimeras.

BACKGROUND: Mixed xenogeneic bone marrow chimerism and tolerance can be induced in mice conditioned with a nonmyeloablative regimen followed by injection of T cell-depleted rat bone marrow cells. We hypothesized that, despite a gradual decline in rat hematopoiesis observed in these chimeras, as long as rat class II+ antigen-presenting cells remain in their thymi, tolerance will persist as a result of deletion of donor-reactive thymocytes. METHODS: The level of chimerism and of mouse Vbeta5 and Vbeta11 T-cell deletion was followed over time. These results were correlated with the presence of rat class II+ cells in the thymus by immunohistochemistry and the presence of tolerance in long-term chimeras by in vivo and in vitro assays. RESULTS: (1) Proliferation and cytotoxicity assays, as well as skin graft survival, demonstrated the presence of specific tolerance to host and to donor rat, with normal reactivity to third-party rat and mouse stimulators, even as late as 85 weeks after bone marrow transplantation. (2) The absence of mature Vbeta5+ and Vbeta11+ host T cells in the thymus and periphery was always associated with the presence of rat class II+ cells in the thymus, and incomplete deletion of T cells expressing these Vbeta families was observed in thymi in which rat class II+ cells were not detectable. CONCLUSIONS: Donor-specific T-cell tolerance is maintained during the period when donor-type reconstitution declines, and is most likely mediated by intrathymic clonal deletion of T cells that recognize antigens expressed on class II+ rat cells.

Procaspase-3 and its active large subunit localized in both cytoplasm and nucleus are activated following application of apoptotic stimulus in Ramos-Burkitt lymphoma B cells.

Ramos-Burkitt lymphoma (Ramos-BL) B cell line is a neoplastic model of normal B cell selection by apoptosis at the germinal center site during maturation of the humoral immune response and can be triggered into apoptosis by cross-linking their surface antigen receptor with antibodies directed against immunoglobulin (Ig)M (anti-IgM) or by treating with the calcium ionophore ionomycin. We have recently demonstrated that anti-IgM and ionomycin trigger significant activation of caspase-3, -7 and -8 and for cleavage of the resident nuclear proteins poly(ADP-ribose) polymerase (PARP) and lamin B1 in Ramos-BL B cells, suggesting that these caspases may be localized to the nucleus as well as to the cytoplasm of Ramos-BL B cells. In order to examine this hypothesis further, we fractionated Ramos-BL B cells into their cytosolic and nuclear components and examined for expression of the endogenous proform and active large subunit of caspase-3; procaspase-3 and its active p17 large subunit were identified in both the cytosolic and nuclear fractions of Ramos-BL B cells. Immunofluorescence staining together with ordinary and confocal microscopy confirmed the observations that procaspase-3 immunoreactivity was clearly identified in the cytoplasm and nucleus while Fas ligand staining was localized to the cell surface and PARP immunoactivity to the nucleus, which were used as controls; procaspase-3 exhibited granular nuclear immunoreactivity whereas PARP displayed diffuse nuclear immunoreactivity; both of which was more intense in the internucleolar regions. Taken together, we now present evidence that procaspases and their active large subunits are found in both the cytoplasm and the nucleus and that procaspases localized not only in the cytoplasm but also in the nucleus are activated following application of apoptotic stimulus in Ramos-BL B cells.

Genetic elimination of α3(IV) collagen fails to rescue anti-collagen B cells.

Organ deposition of autoantibodies against the noncollagenous-1 domain of the α3 chain of type IV collagen leads to severe kidney and lung injury in anti-glomerular basement membrane disease. The origin and regulation of these highly pathogenic autoantibodies remains unknown. Anti-α3(IV) collagen B lymphocytes are predicted to mature in vivo ignorant of target antigen because α3(IV) collagen expression is highly tissue restricted and pathogenic epitopes are cryptic. However, a recent analysis of an anti-α3(IV)NC1 collagen autoantibody transgenic mouse model revealed that developing B cells are rapidly silenced by deletion and editing in the bone marrow. To dissect the role of collagen as central tolerogen in this model, we determined B cell fate in autoantibody transgenic mice genetically lacking α3(IV) collagen. We found that absence of the tissue target autoantigen has little impact on the fate of anti-α3(IV)NC1 B cells. This implies a more complex regulatory mechanism for preventing anti-glomerular basement membrane disease than has been previously considered, including the possibility that a second antigen present in bone marrow engages and tolerizes anti-α3(IV)NC1 collagen B cells.

The VDAC2-BAK rheostat controls thymocyte survival.

The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.

Thymic stromal lymphopoietin in normal and pathogenic T cell development and function.

Thymic stromal lymphopoietin, a four helix-bundle cytokine, is expressed mainly by barrier epithelial cells and is a potent activator of several cell types, particularly myeloid dendritic cells. TSLP influences the outcome of interactions between dendritic cells and CD4+ thymocytes and T cells in many situations, such as the regulation of the positive selection of regulatory T cells, maintenance of peripheral CD4+ T cell homeostasis and induction of CD4+ T cell-mediated allergic inflammation.

Diabetes is predicted by the beta cell level of autoantigen.

Two novel transgenic (Tg) strains were created expressing hen egg-white lysozyme (HEL) in a pancreas-specific fashion. RmHP.111 mice had levels of HEL per cell similar to that of the established ILK-3 strain, while RmHP.117 mice had 10-fold lower levels (50,000 molecules per cell). When bred to 3A9 TCR Tg mice, negative selection occurred equally in all three double-Tg combinations, yet only ILK-3 x 3A9 and RmHP.111 x 3A9 mice became diabetic. Additionally, activated 3A9 cells readily transferred diabetes into ILK-3 or RmHP.111 mice, but only marginally into the RmHP.117 strain. In the peripancreatic lymph node, division of naive 3A9 cells was similar between RmHP.111 and RmHP.117 strains, but pancreatic APCs from RmHP.111 x 3A9 mice stimulated HEL-reactive cells to a much greater degree than those from RmHP.117 x 3A9 mice. In this model, diabetes was dependent upon both initial priming in the peripancreatic lymph node and subsequent presentation in the pancreas, with disease incidence predicted by the beta cell level of autoantigen.

On the plausibility of the clonal expansion theory of the immune system in terms of the combinatorial possibilities of amino-acids in antigen and self-tolerance.

The clonal expansion theory of the immune system was first proposed over 30 years age (Jerne, 1955; Burnet, 1959). It still forms the basis of current thinking about the immune system. It was not obvious to the authors of this paper that the combinatorial possibilities of the 20 available amino acids in antigen peptides could result in a system that could distinguish successfully between self and foreign antigen, utilizing only the trial and error method proposed by the clonal expansion theory. In this paper it is demonstrated that the theory is credible on this issue, at least with respect to major histocompatibility complex (MHC) restricted antigen recognition, and that the very high proportion of immature lymphocytes that die in the thymus is consistent with the theory.

Thymic selection revisited: how essential is it?

Intrathymic T cell development represents one of the best studied paradigms of mammalian development. Lymphoid committed precursors enter the thymus and the Notch1 receptor plays an essential role in committing them to the T cell lineages. The pre-T cell receptor (TCR), as an autonomous cell signaling receptor, commits cells to the alphabeta lineage while its rival, the gammadeltaTCR, is involved in generating the gammadelta lineage of T cells. Positive and negative selection of immature alphabetaTCR-expressing cells are essential mechanisms for generating mature T cells, committing them to the CD4 and CD8 lineages and avoiding autoimmunity. Additional lineages of alphabetaT cells, such as the natural killer T cell lineage and the CD25+ regulatory T cell lineage, are formed when the alphabetaTCR encounters specific ligands in suitable microenvironments. Thus, positive selection and receptor-instructed lineage commitment represent a hallmark of the thymus. Ectopically expressed organ-specific antigens contribute to thymic self-nonself discrimination, which represents an essential feature for the evolutionary fitness of mammalian species.