Linda K. Clayton

Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1.

Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context–specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

The Structural Biology of CD2

The CD2 molecule is a 50-55KD transmembrane glycoprotein expressed on the vast majority of thymocytes and virtually all peripheral T lymphocytes. Its functions are two-fold: adhesion and activation. CD2 serves to facilitate conjugate formation between the T-lineage cell and its cognate partner via intermolecular interaction of CD2 and LFA-3 on the former and latter cells, respectively. Perturbation of the CD2 extracellular segment by certain combinations of anti-CD2 MAbs or LFA-3 and a single anti-CD2 MAb activate T-lineage function. These CD2-mediated activation events also synergize with signals mediated through the TCR to augment T-cell response. Based on microchemical analysis of immunoaffinity-purified human CD2 and cDNA and genomic cloning of mouse and human molecules, considerable structural information is now available. The mature surface human CD2 molecule consists of 327 amino acids: a 185 aa extracellular segment; a 25 aa hydrophobic transmembrane segment; and a 117 aa cytoplasmic domain rich in prolines and basic residues. The CD2 gene is comprised of five exons which span approximately 12 Kb on chromosome 1. A similar protein structure and gene exon organization is found for the mouse CD2 homologue. The CD2 adhesion domain is approximately 103 aa in length and is encoded by a single exon (exon 2). This domain is resistant to proteolysis, even though it lacks any intrachain disulfides and, like the entire extracellular segment protein expressed in a baculovirus system, binds to its cellular ligand, LFA-3. The latter occurs with a micromolar Kd. This relatively low affinity suggests that multivalent interactions among CD2 monomers on the T cells and individual LFA-3 structures on the cognate partner are important in enhancing the avidity of the T-cell interaction with its target or stimulator cell. The affinity of the CD2 extracellular segment for LFA-3 is not affected by truncations in the CD2 cytoplasmic domain, implying that ligand binding is not regulated by intracellular mechanisms. Given that CD2 mRNA expression and surface CD2 copy number are increased by more than one order of magnitude post-TCR stimulation, it is more likely that adhesion via CD2 is modulated by alteration in surface copy number. Analysis of early transduction events occurring via CD3-Ti (TCR) and CD2 including single channel Ca2+ patch-clamp recordings on living human T lymphocytes indicate a virtual identity of signals.(ABSTRACT TRUNCATED AT 400 WORDS)

Peptide-Induced Negative Selection of Thymocytes Activates Transcription of an NF-ΚB Inhibitor

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: The molecular basis of negative selection

T‐cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte‐specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double‐positive (DP) CD4+CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen‐induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti‐CD3ϵ monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

Interdependence of CD3-Ti and CD2 activation pathways in human T lymphocytes

Human T lymphocytes can be activated through either the antigen/MHC receptor complex T3‐Ti (CD3‐Ti) or the T11 (CD2) molecule to proliferate via an IL‐2 dependent mechanism. To investigate the relationship of these pathways to one another, we generated and characterized Jurkat mutants which selectively express either surface CD3‐Ti or CD2. Here we show that CD3‐Ti‐ mutants fail to be stimulated by either pathway to increase phosphoinositide turnover, mobilize calcium or induce the IL‐2 gene. The activation capacity of these mutants via CD2 as well as CD3‐Ti can be restored following reconstitution of surface CD3‐Ti expression upon appropriate DNA transfer (e.g. Ti beta subunit cDNA into Ti beta‐ Jurkat variants). Collectively, these results demonstrate that CD3‐Ti and CD2 pathways are interdependent and that phosphoinositide turnover is linked to the CD3‐Ti complex.

PlexinD1 Glycoprotein Controls Migration of Positively Selected Thymocytes into the Medulla

Precise intrathymic cell migration is important for thymocyte maturation and organ architecture. The orchestration of thymocyte trafficking, however, is not well understood at a molecular level. Here, we described highly regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes. PlexinD1 expression was further affected by the engagement of T cell receptor complex. Activation of plexinD1 via the ligand, semaphorin 3E, repressed CCL25 chemokine signaling via its receptor CCR9 in CD69+ thymocytes. In the absence of plexinD1, CD69+ thymocytes remained in the cortex, maturing to form ectopic single positive (SP) thymocyte clusters in Plxnd1-deficient fetal liver cell-transplanted mice. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions was disrupted and medullary structures formed under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients.

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.

Functional role for IκBNS in T cell cytokine regulation as revealed by targeted gene disruption

Triggering of the TCR by cognate peptide/MHC ligands induces expression of IκBNS, a member of the IκB family of NF-κB inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of IκBNS in TCR triggering, we created a targeted disruption of the IκBNS gene. Surprisingly, mice lacking IκBNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, IκBNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-γ than wild-type cells. Transfection analysis demonstrates that IκBNS and c-Rel individually increase IL-2 promoter activity. The effect of IκBNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-κB rather than the CD28RE site; mutation of the NF-κB site extinguishes the induction of transcription by IκBNS in transfectants and prevents association of IκBNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-γ-linked transcripts in IκBNS knockout T cells. Collectively, our findings demonstrate that IκBNS regulates production of IL-2 and other cytokines induced via “strong” TCR ligation.

Targeted disruption within the CD3 zeta/eta/phi/Oct-1 locus in mouse.

To elucidate the role of the CD3 eta subunit of the T cell receptor (TCR) in thymic development, a CD3 eta ‐/‐ mouse was generated by gene targeting. Insertion of a neomycin resistance gene into exon 9 of the CD3 zeta/eta/phi locus disrupted expression of CD3 eta and CD3 phi without affecting the expression of CD3 zeta. Little difference was observed between wild type and CD3 eta ‐/‐ mice with regard to cellularity or subset composition in thymus and peripheral lymphoid organs. Furthermore, neither alloproliferative responses nor cytotoxic T lymphocyte generation and effector function was affected by the mutation. The effect of the CD3 eta ‐/‐ mutation on thymic selection was examined by crossing the CD3 eta knockout animals with anti‐HY TCR transgenic animals: the absence of the CD3 eta subunit altered neither positive nor negative selection. Thus, CD3 eta is not required for thymic selection. Of note, the birth rate of the CD3 eta ‐/‐ animals was significantly lower than that of wild type or heterozygous animals (P = 0.041‐0.002). This unexpected result is probably the consequence of an alteration in mRNA expression of the Oct‐1 nuclear transcription factor in CD3 eta ‐/‐ animals. The CD3 zeta/eta/phi locus partially overlaps the gene encoding Oct‐1 whose transcription is dysregulated by the CD3 eta ‐/‐ mutation. Our results clearly underscore the value of characterizing all products of a genetic locus disrupted by gene targeting.

Involvement of the TCR Cβ FG Loop in Thymic Selection and T Cell Function

The asymmetric disposition of T cell receptor (TCR) Cβ and Cα ectodomains creates a cavity with a side-wall formed by the rigid Cβ FG loop. To investigate the significance of this conserved structure, we generated loop deletion (βΔFG) and βwt transgenic (tg) mice using the TCR β subunit of the N15 CTL. N15βwt and N15βΔFG H-2b animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4−CD8− double-negative (DN) compartment. N15βΔFG facilitates transition from DN to CD4+8+ double-positive (DP) thymocytes in recombinase activating gene (RAG)-2−/− mice, showing that pre-TCR function remains. N15βΔFG animals possess ∼twofold more CD8+ single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Vα repertoire observed in N15βΔFG mice may confound the deletions effect, we crossed N15αβ TCR tg RAG-2−/− with N15βΔFG tg RAG-2−/− H-2b mice to generate N15αβ RAG-2−/− and N15αβ.βΔFG RAG-2−/− littermates. N15αβ.βΔFG RAG-2−/− mice show an 8–10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15αβ, N15αβ.βΔFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cβ FG loop facilitates negative selection of thymocytes and activation of T cells.