Susan O. Sharrow

Characterization of murine thymocytes with CDS-associated T-cell receptor structures

The thymus is the major site for T-cell receptor (TCR) gene rearrangement and T-cell maturation1,2. 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, TCRα and TCRβ, expressed in non-covalent association with an invariant complex of proteins, CD3 (T3)3–6. Recently, a novel TCR/CD3 complex, that includes the product of the TCRγ gene, has been identified on a subset of both peripheral cells and thymocytes7–9. 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 TCRγ-specific RNA, low levels of TCRβ-specific RNA and no detectable TCRα-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.

Functional and phenotypic comparison of human T cell leukemia/lymphoma virus positive adult T cell leukemia with human T cell leukemia/lymphoma virus negative Sézary leukemia, and their distinction using anti-Tac. Monoclonal antibody identifying the human receptor for T cell growth factor.

Adult T cell leukemia (ATL) and Sézary leukemia are malignant proliferations of T lymphocytes that share similar cell morphology and clinical features. ATL is associated with HTLV (human T cell leukemia/lymphoma virus), a unique human type C retrovirus, whereas most patients with the Sézary syndrome do not have antibodies to this virus. Leukemic cells of both groups were of the T3, T4-positive, T8-negative phenotype. Despite the similar phenotype, HTLV-negative Sézary leukemic cells frequently functioned as helper cells, whereas some HTLV-positive ATL and HTLV-positive Sézary cells appeared to function as suppressors of immunoglobulin synthesis. One can distinguish the HTLV-positive from the HTLV-negative leukemias using a monoclonal antibody (anti-Tac) that appears to identify the human receptor for T cell growth factor (TCGF). Resting normal T cells and most HTLV-negative Sézary cells were Tac-negative, whereas all ATL cell populations were Tac-positive. The observation that ATL cells manifest TCGF receptors suggests the possibility that an abnormality of the TCGF-TCGF receptor system may partially explain the uncontrolled growth of these cells.

Basis of CTLA-4 function in regulatory and conventional CD4+ T cells

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4(+) T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4(+) T cells.

In vitro evidence that cytokine receptor signals are required for differentiation of double positive thymocytes into functionally mature CD8+ T cells

CD4+8+ double positive (DP) thymocytes differentiate into CD4+ and CD8+ mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4−8+ thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8+ thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells.

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL–AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4−/− MLL–AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL–AF9 blasts, which requires cyclin-dependent kinase inhibitor p21Cip1 (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

Cultures of purified human natural killer cells: Growth in the presence of interleukin 2

Abstract We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and NK K activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7–10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.

Strength of Signaling by CD4 and CD8 Coreceptor Tails Determines the Number but Not the Lineage Direction of Positively Selected Thymocytes

The present study has assessed the impact of the intracellular domains of CD4 and CD8 on positive selection and lineage direction of MHC class I-restricted thymocytes. Contrary to current presumption, we found that the CD4 tail promotes the generation of both CD4+ and CD8+ T cells without preference for the CD4+ T cell lineage. We also found that the identity of the coreceptor tail and hence the strength of coreceptor signaling determine the number of thymocytes undergoing positive selection but not their ultimate CD4/CD8 phenotype. These findings demonstrate that the strength of coreceptor signaling has a significant quantitative but not qualitative impact on positive selection and provide a simple explanation for the greater numbers of CD4+ than CD8+ T cells selected in the normal thymus.

Lck Availability during Thymic Selection Determines the Recognition Specificity of the T Cell Repertoire

Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire.

Unraveling a Revealing Paradox: Why Major Histocompatibility Complex I–signaled Thymocytes “Paradoxically” Appear as CD4+8lo Transitional Cells During Positive Selection of CD8+ T Cells

The mechanism by which T cell receptor specificity determines the outcome of the CD4/CD8 lineage decision in the thymus is not known. An important clue is the fact that major histocompatibility complex (MHC)-I–signaled thymocytes paradoxically appear as CD4+8lo transitional cells during their differentiation into CD8+ T cells. Lineage commitment is generally thought to occur at the CD4+8+ (double positive) stage of differentiation and to result in silencing of the opposite coreceptor gene. From this perspective, the appearance of MHC-I–signaled thymocytes as CD4+8lo cells would be due to effects on CD8 surface protein expression, not CD8 gene expression. But contrary to this perspective, this study demonstrates that MHC-I–signaled thymocytes appear as CD4+8lo cells because of transient down-regulation of CD8 gene expression, not because of changes in CD8 surface protein expression or distribution. This study also demonstrates that initial cessation of CD8 gene expression in MHC-I–signaled thymocytes is not necessarily indicative of commitment to the CD4+ T cell lineage, as such thymocytes retain the potential to differentiate into CD8+ T cells. These results challenge classical concepts of lineage commitment but fulfill predictions of the kinetic signaling model.