Thymocyte Compartment Research Articles

Reduced Regulatory T Cell Diversity in NOD Mice Is Linked to Early Events in the Thymus

The thymic natural regulatory T cell (Treg) compartment of NOD mice is unusual in having reduced TCR diversity despite normal cellularity. In this study, we show that this phenotype is attributable to perturbations in early and late stages of thymocyte development and is controlled, at least in part, by the NOD Idd9 region on chromosome 4. Progression from double negative 1 to double negative 2 stage thymocytes in NOD mice is inefficient; however, this defect is compensated by increased proliferation of natural Tregs (nTregs) within the single positive CD4 thymocyte compartment, accounting for recovery of cellularity accompanied by loss of TCR diversity. This region also underlies the known attenuation of ERK-MAPK signaling, which may preferentially disadvantage nTreg selection. Interestingly, the same genetic region also regulates the rate of thymic involution that is accelerated in NOD mice. These findings highlight further complexity in the control of nTreg repertoire diversity.

Defective positive selection results in T cell lymphopenia and increased autoimmune diabetes in ADAP‐deficient BDC2.5‐C57BL/6 mice

Adhesion and degranulation promoting adapter protein (ADAP), a positive regulator of T cell receptor (TCR) signaling, is required for thymocyte development and T cell homeostasis. To investigate the role of ADAP in a T cell-driven autoimmune response, we generated ADAP-deficient, BDC2.5 TCR transgenic, diabetes-prone (C57BL/6) mice (BDC/B6). We observed a striking enhancement of diabetes incidence in ADAP-deficient mice, both in animals homozygous for I-Ag7, and in mice carrying one I-Ab allele (BDC/B6g7/b). Increased disease correlates with significantly reduced numbers of pathological CD4(+) T cells in the mice. Consistent with a state of functional lymphopenia in ADAP-deficient BDC/B6g7/b mice, T cells display increased homeostatic proliferation. Transfer of syngeneic lymphocytes or T cells both blocks ADAP-dependent diabetes and relieves exaggerated homeostatic T cell proliferation observed in ADAP-deficient mice. Marked attenuation in cellularity of the CD4+ single-positive thymocyte compartment in ADAP-deficient BDC/B6g7/b animals suggests a mechanism for induction of the lymphopenia. We conclude that inefficient positive selection in ADAP deficiency results in lymphopenia that leads to enhanced autoimmune diabetes in the BDC/B6g7/b model. Our findings support the notion that ineffective thymic T cell output can be a powerful causative factor in lymphopenia-driven autoimmune diabetes.

Germline transcription from T-cell receptor Vβ gene is uncoupled from allelic exclusion

Allelic exclusion operates in B and T lymphocytes to ensure clonal expression of antigen receptors after V(D)J recombination. Germline transcription, which proceeds V(D)J recombination, has been postulated to provide an instructive signal for allelic exclusion. Here, we use a genetic marker to track germline transcription from a Vbeta gene within the TCRbeta locus. We find that developing thymocytes exhibit uniformed, bi-allelic activation of the Vbeta gene before V-DJ recombination, a process subject to allelic exclusion. We further show that V-DJ rearrangement promotes activation rather than silencing of germline transcription from the remaining Vbeta genes on either the functionally or non-functionally rearranged chromosome. Results presented here suggest that germline transcription, although necessary for V(D)J recombination, is not sufficient to instruct allelic exclusion.

Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7.

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.

Cross-linking of T-cell receptors on double-positive thymocytes induces a cytokine-mediated stromal activation process linked to cell death.

To investigate molecular events associated with the intrathymic process of negative selection, we established an in vivo system using an anti-CD3 epsilon monoclonal antibody to induce synchronous apoptosis in the thymus of AND T-cell receptor (TCR) transgenic RAG-2-/- mice in a non-selecting haplotype. This model eliminates endogenous negative selection as well as gene activation in the mature thymocyte compartment, offering an ideal source of tester (anti-CD3 epsilon-treated) and driver (untreated) thymus RNA for representational difference analysis (RDA). Fourteen mRNA sequences that are up-regulated in the thymuses of such mice 2-6 h after anti-CD3 epsilon treatment were identified. Surprisingly, the majority of these transcripts were derived from stromal cells rather than the TCR-cross-linked CD4+CD8+TCRlow thymocytes including the macrophage products IL-1, the chemokine Mig and the transcription factor LRG-21. IFN-gamma secretion from the CD4+CD8+TCRlow thymocytes regulates macrophage Mig production. Three other cytokines (IL-4, GM-CSF and TNF-alpha), known to activate a variety of stromal cells, are also induced in the same thymocyte population undergoing apoptosis. Expression of a TNF-alpha-inducible gene, B94, in stromal cells after TCR ligation further supports the notion of cross-talk between thymocytes and stroma. Thus, TCR-triggered immature thymocytes elaborate cytokines which may regulate the delivery of further signals from stromal cells required for apoptosis.

Feedback Regulation of T Cell Development in the Thymus

Recent findings suggest that mature T cells in the thymus may regulate the growth and differentiation of immature thymocytes. Here we use mathematical modeling and computer simulations to identify the thymocyte subsets that might serve as targets for regulation, and the processes that might be affected by regulation. our results suggest that thymocyte development is subject to regulation through two feedback loops: mature CD4 +T cells exert a positive feedback on the single positive CD4 +8 −thymocyte compartment, by reducing CD4 +8 −cell death and possibly accelerating the differentiation of CD4 +8 +thymocytes into CD4 +8 −thymocytes; they may also exert a negative feedback on the double-positive CD4 +8 +thymocyte compartment, by reducing the proliferation or accelerating the maturation of these cells.

Insertional mutagenesis affecting programmed cell death leads to thymic hyperplasia and altered thymopoiesis.

The mouse mutant Ft displays thymic hyperplasia and fused toes (Ft) of the forelimbs. Both phenotypic abnormalities are caused by transgene insertion in the D region of chromosome 8. While the forelimb defect is probably caused by developmentally dysregulated programmed cell death, the mechanism underlying thymic hyperplasia has not been characterized. In this work, we show that expansion of the thymocyte compartment progresses with time, is polyclonal, and affects all major thymocyte subsets, including the earliest CD4-8- subset, i.e., CD44+ CD25- cells; hyperplasia is not an autonomous property of mutant T cells, but is caused indirectly by a primary defect in thymic stromal. The rate of cell division and the cell turnover of immature CD4-8- and CD4+8+ thymocytes under steady state conditions are not altered in hyperplastic Ft thymi. Immature CD4+8+ thymocytes of mutant mice, however, are less susceptible to induction in vitro of programmed cell death by different modes (TCR cross-linking, cortisone, or radiation). Increased production of thymocytes results in increased export of T cells, yet the size and composition of the peripheral T cell pool are normal. Overproduction of immature CD4+8+ thymocytes is offset partly by a reduced conversion rate of CD4+8+ double positive to single positive thymocyte growth control by epithelial cells, and may serve as a model to study the regulation of early thymopoiesis.

Dose-dependent systemic human immunodeficiency virus infection of SCID-hu mice after intraperitoneal virus injection

SCID mice were engrafted with human foetal liver, thymus and lung. Human cells were subsequently detected among peripheral blood leukocytes for 81% of tested animals and in tissue implants for 100% of tested animals. SCID-hu mice received intraperitoneal injections of human immunodeficiency virus type 1 (HIV1) at from 20 up to 20,000 median tissue culture infectious doses (TCID5). HIV1 infection was detected by means of cell culture and polymerase chain reaction both in blood and implants, up to 58 days after infection. The rate of infection was dependent upon the inoculated dose: the frequency of thymus infection ranged from 14% with 20-500 TCID50 up to 100% with 20,000 TCID50. HIV1 infection was detected less frequently in blood leukocytes than in thymus. Thymus virus load ranged from 40 to 50,000 HIV1 provirus copies per million cells and was not correlated with either infectious dose or viraemia. Thymus T-cell depletion was observed mainly in the CD1+4+8+ immature thymocyte compartment. The same rate of SCID-hu mouse infection was obtained using three different primary HIV1 isolates, suggesting that infection was not restricted to a few particular virus strains. The systemic infection of SCID-hu mice following intraperitoneal virus injection mimics some traits of human HIV infection and provides a promising, novel approach for future investigations in this field.

Intrathymically expressed c- kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo

To investigate the role of the receptor-type tyrosine kinase, c- kit and its ligand, stem cell factor (SCF) in T cell development, we analyzed c- kit (W/W) and SCF (SI/SI) deficient mice. We also engrafted wild-type or SCF-deficient fetal thymi onto wild-type recipient mice and analyzed the rate of proliferation by in vivo bromodeoxyuridine labeling. The results show that the most immature thymocyte compartment defined as CD3 −CD4 −CD8 − is significantly reduced in SI/SI grafts and W/W thymi compared with wild-type counterparts. Also, the expansion rate of these immature thymocytes in SI/SI graft is reduced by ∼50%. These experiments provide direct evidence for an important role for c- kit-SCF interactions in expansion of very early thymocytes.

Differential expression of Mtv loci in MHC class II-positive thymic stromal cells.

Despite the important role played by endogenous superantigen in shaping the T cell repertoire, little is known concerning the expression of the different Mtv loci in cells of the thymic microenvironment involved in repertoire selection. Here we have examined the expression of a panel of Mtv Ags by different MHC class II+ stromal cel types using reverse transcriptase-PCR and monitored the effects of these stromal cells on the development of cells expressing Mtv-reactive TCR V beta elements in closed thymic organ culture systems. Although Mtv-6 and Mtv-8/9 mRNAs are expressed in normal thymus lobe organ cultures, no Mtv expression was detected in MHC class II+ thymic epithelial cells. In contrast a striking pattern of differential expression was observed in dendritic cells of thymic origin that were devoid of Mtv-8/9 but expressed readily detectable levels of Mtv-6. This pattern of Mtv gene expression correlated well with TCR V beta repertoire development. TCRV beta 3+ T cells, normally deleted in response to Mtv-6, were virtually absent from the single positive thymocyte compartment in thymic organ cultures where dendritic cells are present but were present in reaggregate cultures where the only MHC class II-positive cells were thymic epithelial cells. On the other hand, V beta 11+ T-cells were not deleted in organ cultures, possibly reflecting the absence of Mtv-8/9 expression in dendritic cells. Our studies suggest that the influence Mtvs have on shaping the T cell repertoire not only depends on their expression within a particular strain but also on their tissue specific expression in relation to MHC class II, which is necessary for their presentation.

Apoptosis and T-cell repertoire selection in the thymus.

Thymic tolerance depends on induction of apoptosis (programmed cell death) in immature thymocytes by antigen/MHC complexes on dendritic cells (and possibly other bone marrow-derived APCs). Interactions with antigen/MHC complexes on thymic epithelial cells promote maturation of double-positive thymocytes to single-positive cells. However, the nature of the antigen/MHC complexes on thymic epithelial cells is unknown, and if the stromal cell interaction model as just outlined is correct, then presumably these complexes must be different from those presented on dendritic cells, otherwise all cells signaled for positive selection would be subject to negative selection by similar complexes on APCs. The nature of the signals provided by thymic epithelial cells versus dendritic cells is unknown and may provide a key to understanding the processes of positive and negative selection within the thymus. Clearly the intense selection of the T-cell repertoire within the thymus explains the high level of cell death observed within the immature thymocyte compartment. Such intensive selection shapes the T-cell repertoire in a way that provides an explanation for the genetic basis of immune responsiveness and for the susceptibility of certain individuals to autoimmunity.

Acute lymphoblastic leukemia. An unusual case of primary relapse in the uterine cervix

Female genital tract involvement by hematologic neoplasms is uncommon and is usually associated with disseminated disease. Lymphoid neoplasms, which involve the cervix and uterus primarily, are usually of either diffuse large cell or small cleaved cell type, and rarely Burkitt's (small non-cleaved) lymphoma or granulocytic sarcoma. In this report the authors describe young woman with acute stem cell leukemia which was terminal deoxyribonucleotidyl transferase positive, and involved the bone marrow and peripheral blood. After induction of remission and a 2-year disease-free interval, she relapsed with involvement localized to the uterus and cervix. At relapse the leukemic cells expressed CD1, CD2, CD5, CD7, and CD4 or CD8, antigens of the common thymocyte compartment. The authors postulate that the T-cell antigens, known extracellular matrix receptors, may have determined the tissue infiltration: an unusual pattern of relapse.

Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte binding protein, T11, represents an essential cell surface component of a human T-cell-lineage activation pathway. Furthermore, it is known that the human T-cell antigen-major histocompatibility complex (MHC) receptor complex T3-Ti is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2 receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and may possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.

Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte-binding protein, T11, represents an essential cell surface component of a human T-cell lineage activation pathway. Furthermore, it is known that the human T3-Ti T-cell antigen/major histocompatibility complex receptor complex is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2-receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and must possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.

The expression, function, and ontogeny of a novel T cell-activating protein, TAP, in the thymus.

The TAP molecule is an allelic 12,000 m.w. membrane protein that participates in T cell activation. This report analyzes the expression, function, and ontogeny of this molecule in the thymus. TAP is expressed on a small subset (10 to 20%) of thymocytes which is distinct from its expression on a majority (70%) of peripheral T lymphocytes. In the adult thymus, the majority of the TAP-bearing thymocytes are cortisone-resistant, Thy-1+, TL-, J11D-, and PNA-, which localizes TAP expression to medullary thymocytes. Cortical thymocytes do not bear this determinant. Parallel functional studies demonstrated that TAP+ thymocytes are required for Con A and MLR responsiveness. Anti-TAP MAb plus PMA specifically induces proliferation of mature thymocytes comparable in magnitude to the Con A response. These results demonstrate that TAP expression defines the immunocompetent thymocyte compartment and, further, that this molecule is functional on these cells. The ontogeny of TAP expression was also analyzed. TAP is expressed early in fetal thymic development at a time when most T cell markers (except Thy-1 and the iL2-R) are absent. The small sub-population of adult L3T4- and Lyt-2- thymocytes, which resemble early fetal thymocytes, also express TAP. These early thymocytes are capable of being activated through the TAP molecule. The implications of these findings for T cell development and, in particular, the relationship of TAP to T cell receptor expression and acquisition of immunocompetence are discussed.

Differentiation of human mature thymocytes: existence of a T3+4-8- intermediate stage.

A T3 complex-bearing subpopulation was characterized within an in vivo cycling T4-8- early thymocyte compartment which contains cells constitutively expressing interleukin 2 and transferrin receptors. We show differentiation in vitro of both mature subsets of thymocytes (T3+4+8- and T3+4-8+) from the above T4-8- compartment, their appearance being preceded by cells in a T3+4-8- intermediate stage. Furthermore, those mature thymocytes generated in vitro contain functionally competent cells which use T3, T4 and T8 structures for their cytolytic activity. The finding of T3+4-8- thymocytes in vivo, together with the observation that T3 antigen expression precedes that of T4 or T8 molecules in vitro, shows that T3 (and presumably Ti) is present early in ontogeny, and suggests that T3+4-8- cells constitute an "intermediate" stage relevant to the connection between early precursors and mature thymocytes during T lymphocyte ontogeny.

The human T cell receptor: Appearance in ontogeny and biochemical relationship of α and β subunits on IL-2 dependent clones and T cell tumors

The human T cell receptor for antigen (Ti) has recently been identified on IL-2 dependent T cell clones as a 90 kd disulfide-linked heterodimer comprised of one 49–51 kd alpha (α) and one 43 kd beta (β) chain. These subunits are noncovalently associated with a monomorphic 20–25 kd T3 molecule. Here, we produce monoclonal antibodies to a human tumor (REX) derived from an earlier stage of thymic differentiation in order to determine whether clonotypic structures are expressed and to define the ontogeny of Ti. The results of SDS-PAGE and peptide map analyses indicate that an homologous T3-associated heterodimer is synthesized and expressed by REX. This glycoprotein shares several peptides in common with clonotypic structures on an IL-2 dependent T cell clone. In addition, similar Ti related molecules appear during intrathymic ontogeny in parallel with surface T3 expression. The latter findings provide the structural basis for the immunological competence observed exclusively within the T3+ thymocyte compartment.

The Replacement of Monocytes and Interleukin-1 by Phorbol Ester in Lectin-Induced Proliferation of Human Thymocytes and T Cells

Small human thymocytes isolated by counterflow centrifugal elutriation (CCE) failed to respond to lectins, and therefore they were considered to represent the immature thymocyte (IT) population. In addition, these IT also failed to respond to the tumor promotor 12-0-tetradecanoyl-13 acetate (TPA). Proliferation of the IT was induced if in addition to lectins irradiated allogeneic monocytes, crude interleukin-1 (IL-1) preparations or TPA at concentrations of 1–10 −3 µg/ml were added. Allogeneic monocytes, IL-1 or TPA also acted similarly in their synergistic effects with lectins (at concentrations that induced optimal proliferation) in the proliferative responses of unfractionated thymocytes (UT) and the medium sized to large thymocytes (MLT) which represent the mature thymocyte compartment. Irradiated autologous monocytes or TPA were also found to act in a similar synergistic way with lectins at suboptimal concentrations in the proliferation of mature T cells. These results indicate that TPA mimics the signal provided by monocytes or IL-1 in the mitogenesis of human thymocytes and mature T cells. However, at optimal lectin concentrations autologous monocytes were found to restore the reduced mature T-cell responses, whereas in contrast TPA caused strong reductions in the 3H-thymidine ( 3H-TdR) uptake of these cells. The mechanism of this depressed 3H-TdR uptake remains unclear, but our data so far indicate that metabolites of TPA are toxic for those T cells that have relatively large quantities of lectins bound to their membranes, since the same metabolites had no toxic effects on mature T cells activated with suboptimal lectin concentrations.