Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage.

The process of thymocyte development culminates in the maturation of helper (CD4+) and cytotoxic (CD8+) T cells from their common precursors, the CD4+CD8+ double-positive cells. A crucial step during lineage specification is the termination of expression of either the CD4 or the CD8 coreceptor. A silencer element within the first intron of the CD4 gene is sufficient for CD4 transcriptional repression in cells of the cytotoxic lineage, as well as in thymocytes at earlier stages of differentiation. Here we show that the function of the CD4 silencer is required only at distinct stages of development. Its deletion before the initiation of lineage specification resulted in CD4 derepression throughout thymocyte differentiation. By contrast, once cells committed to the cytotoxic CD8+ lineage, the CD4 locus remained silent through subsequent mitoses, even when the silencer element was excised. The epigenetic inheritance of the silenced CD4 locus was not affected by the inhibition of DNA methylation or histone deacetylation, and may thus involve other mechanisms that ensure a stable state of gene expression.

Targeting macrophages in the tumour environment to enhance the efficacy of αOX40 therapy.

The treatment of high-grade tumours must consider a tumour environment dominated by cells that support cancer growth. In addition to directing angiogenesis and invasion, alternatively activated macrophages in the tumour provide protection from adaptive immunity and permit tumour growth. Agonist antibodies to the tumour necrosis factor receptor family member OX40 are an effective therapy for cancer in a range of murine models; however, as with many immune therapies, αOX40 therapy is less effective as the tumour grows and develops an immune suppressive environment. We demonstrate that αOX40 directly activates T cells and that this T-cell activation alters macrophage differentiation in the tumour environment. We demonstrate that macrophages in the tumour limit the efficacy of αOX40 therapy, and that combining αOX40 therapy with inhibitors of arginase significantly enhances survival of tumour-bearing mice. These data demonstrate that macrophages in the tumour environment limit the effectiveness of OX40-based immunotherapy, and combination therapies that target both the cell-mediated immune response and the suppressive tumour environment will be required for translation of effective immunotherapies to patients with established tumours.

Response of tertiary addictions services to opioid dependence during the COVID-19 pandemic.

The emergence of the COVID-19 pandemic has presented the addiction services with an unprecedented set of challenges. Opioid users are particularly vulnerable because of their high level of pre-existing health problems and lifestyle factors. In order to minimise their risks to self and to others in the current Covid-19 crisis, addiction services sought to urgently identify vulnerable individuals, and induct them into opioid substitution treatment (OST) promptly. Additionally, several guidelines were created and regularly updated by the health and safety executive (HSE) for any healthcare staff working with opioid users. These include guidance documents, to facilitate prompt induction of patients onto the OST programme, the prescribing of naloxone to all patients at risk of overdose, eConsultation, medication management for those in self-isolation, and the delivery of injecting equipment. The guidance documents and resources will provide a template for a new way of working for the sector during these challenging times and into the future.

Homeostatic competition among T cells revealed by conditional inactivation of the mouse Cd4 gene.

Absence of CD4 impairs the efficiency of T cell receptor (TCR) signaling in response to major histocompatibility complex (MHC) class II-presented peptides. Here we use mice carrying a conditional Cd4 allele to study the consequences of impaired TCR signaling after the completion of thymocyte development. We show that loss of CD4 decreases the steady-state proliferation of T cells as monitored by in vivo labeling with bromo-deoxyuridine. Moreover, T cells lacking CD4 compete poorly with CD4-expressing T cells during proliferative expansion after transfer into lymphopenic recipients. The data suggest that T cells compete with one another during homeostatic proliferation, and indicate that the basis of this competition is TCR signaling.

Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes.

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR-zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.

Requirement for CD8-major histocompatibility complex class I interaction in positive and negative selection of developing T cells.

The interaction of the T cell surface glycoprotein CD8 with major histocompatibility complex (MHC) class I molecules on target cells is required for effective T cell activation. Mutations in the alpha 3 domain of the MHC class I molecule can disrupt binding to CD8, yet leave antigen presentation unaffected. Here we show that such a mutation can interfere with positive and negative selection of T cells bearing T cell receptors (TCRs) that interact specifically with the mutant class I molecule. Autoreactive T cells in male mice expressing a transgenic TCR specific for the male antigen H-Y and H-2Db were not deleted in the context of a transgenic Db molecule bearing a mutation at residue 227. Similarly, CD8+ cells were not positively selected in female mice expressing both the TCR and mutant class I transgenes. Endogenous MHC class I molecules were competent to bind CD8, but were unable to rescue the defect, indicating a requirement for coordinate recognition of antigen/MHC by a complex of the TCR and CD8 coreceptor for both positive and negative selection of thymocytes.

A role for CD4 in peripheral T cell differentiation.

Naive CD4+ T helper cells (Th) differentiate into one of two well-defined cell types during immune responses. Mature Th1 and Th2 cells regulate the type of response as a consequence of the unique cytokines that they secrete. CD4 serves a prominent role in potentiating antigen recognition by helper T cells. We have examined the role of CD4 in peripheral T cell differentiation by studying helper T cells from mice with a congenital defect in CD4 expression. After protein immunization or infection with Leishmania major, CD4-deficient mice were incapable of mounting antigen-specific Th2 responses, but retained their Th1 potency. CD4-deficient, T cell receptor transgenic T cells were also incapable of Th2 differentiation after in vitro activation. Expression of a wild-type CD4 transgene corrected the Th2 defect of CD4-deficient mice in all immune responses tested. To investigate the role of the cytoplasmic domain, mice reconstituted with a truncated CD4 molecule were also studied. Expression of the tailless CD4 transgene could not rescue the Th2 defect of CD4-deficient mice immunized with protein or CD4-deficient transgenic T cells activated in vitro, raising the possibility that the cytoplasmic domain of CD4 may influence Th2 generation. Expression of the tailless transgene was, however, capable of restoring Th2 development in CD4-deficient mice infected with L. major or CD4-deficient transgenic T cells activated in the presence of recombinant IL-4, demonstrating that the cytoplasmic domain is not absolutely required for Th2 development. Together, these results demonstrate a previously undescribed role of the CD4 molecule. The requirement for CD4 in Th2 maturation reflects the importance of molecules other than cytokines in the control of helper T cell differentiation.

Itk negatively regulates induction of T cell proliferation by CD28 costimulation.

CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses.

Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains.

Immature thymocytes express a pre-T cell receptor (pre-TCR) composed of the TCRbeta chain paired with pre-Talpha. Signals from this receptor are essential for passage of thymocytes through a key developmental checkpoint in the thymus. These signals were efficiently delivered in vivo by a truncated form of the murine pre-TCR that lacked all of its extracellular immunoglobulin domains. De novo expression of the truncated pre-TCR or an intact alphabetaTCR was sufficient to activate characteristic TCR signaling pathways in a T cell line. These findings support the view that recognition of an extracellular ligand is not required for pre-TCR function.

A subset of CD4+ thymocytes selected by MHC class I molecules.

To complete their maturation, most immature thymocytes depend on the simultaneous engagement of their antigen receptor [alpha beta T cell receptor (TCR)] and their CD4 or CD8 coreceptors with major histocompatibility complex class II or I ligands, respectively. However, a normal subset of mature alpha beta TCR+ thymocytes did not follow these rules. These thymocytes expressed NK1.1 and a restricted set of alpha beta TCRs that are intrinsically class I-reactive because their positive selection was class I-dependent but CD8-independent. These cells were CD4+ and CD4-8- but never CD8+, because the presence of CD8 caused negative selection. Thus, neither CD4 nor CD8 contributes signals that direct their maturation into the CD4+ and CD4-8- lineages.

Helper T cells without CD4: control of leishmaniasis in CD4-deficient mice.

Expression of either the CD4 or CD8 glycoproteins discriminates two functionally distinct lineages of T lymphocytes. A null mutation in the gene encoding CD4 impairs the development of the helper cell lineage that is normally defined by CD4 expression. Infection of CD4-null mice with Leishmania has revealed a population of functional helper T cells that develops despite the absence of CD4. These CD8- alpha beta T cell receptor+ T cells are major histocompatibility complex class II-restricted and produce interferon-gamma when challenged with parasite antigens. These results indicate that T lymphocyte lineage commitment and peripheral function need not depend on the function of CD4.

A role for CD5 in TCR-mediated signal transduction and thymocyte selection.

CD5 is a transmembrane protein that is expressed on the surface of T cells and a subset of B cells. The absence of CD5 rendered thymocytes hyperresponsive to stimulation through the T cell antigen receptor (TCR) in vitro. Selection of T cells expressing three distinct transgenic TCRs was also abnormal in CD5-deficient mice. These observations indicate that CD5 can influence the fate of developing thymocytes by acting as a negative regulator of TCR-mediated signal transduction.

Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action.

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2-/- mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2-/- animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.

T-cell regulation: Thy-1 - hiding in full view.

Thy-1 is a highly abundant glycoprotein on the surface of thymocytes and neurons. A null mutation in the gene encoding Thy-1 deregulates T-cell receptor signaling and causes abnormal thymocyte development; does this mean that Thy-1 has a signaling function?

Signaling checkpoints during the development of T lymphocytes.

Two major lineage decisions face immature T cells as they develop in the thymus. At an early stage in their development, they must first commit to either the gamma delta or alpha beta lineages. If they opt for the alpha beta lineage, then at a later stage they must also choose between a CD4+ or CD8+ fate before they can pass through the thymic medulla and exit to the periphery. Thymocyte survival at key developmental checkpoints is determined by signaling from cytokine receptors and the T-cell receptor. Recent advances have been made in contemporary understanding of the signals that regulate thymocyte survival, proliferation and lineage decisions.

The regulation and function of the CD4 coreceptor during T lymphocyte development.

The data reviewed in this chapter suggest that the primary developmental function of the CD4 and CD8 coreceptors is to improve the efficacy by which a thymocyte recognizes peptide/MHC. During positive selection, DP thymocytes down-regulate expression of either CD4 or CD8 in response to signals that originate from the TCR/coreceptor complex. Experiments with transgenic and MHC-null mice have shown that coreceptor down-regulation and lineage commitment can occur stochastically in a manner that is independent of TCR specificity for MHC. Nevertheless, the positive selection of a given thymocyte is contingent on sustained expression of the coreceptor that is appropriate for the MHC specificity of its TCR. In most cases, loss of the required coreceptor blocks developmental progression and results in thymocyte apoptosis. CD4 expression is controlled by both positive and negative regulatory sequences embedded in the CD4 gene and it is likely that similar sequences regulate the CD8 gene. The down-regulation of coreceptor expression is coupled to a functional commitment which ensures that mature CD4+ T cells have a helper phenotype and CD8+ T cells have a cytotoxic phenotype. The molecular basis for this coupling and the identity of the switching mechanism which governs coreceptor regulation remain to be determined.

Studies of HIV-1 envelope glycoprotein-mediated fusion using a simple fluorescence assay.

OBJECTIVE: To study HIV envelope glycoprotein (Env)-mediated entry using a sensitive fusion assay. DESIGN AND METHODS: CD4+ lymphocytes or T-cell lines were labelled with fluorescent cytoplasm or membrane markers. Fusion with Env-expressing adherent cells was monitored by observing dye transfer from CD4+ cells to Env cells. RESULTS: Cell-cell fusion began 20-30 min after co-cultivation at 37 degrees C. Pre-binding at 4 degrees C was observed not to decrease the lag phase before fusion. Cells expressing envelope glycoproteins from non-syncytium-inducing (NSI) HIV strains showed dye transfer between two cells without progression to syncytia. A glycosylphosphatidylinositol anchored Env was found to be incapable of mediating membrane fusion, as measured either by lipid or cytoplasm contents mixing. Primary mouse cells expressing human CD4 and mouse 3T3 cells stably expressing both human CD4 and human CD26 did not support fusion with our Env-expressing cells. CONCLUSIONS: Env-mediated cell-cell fusion is a relatively slow process, probably reflecting a multi-step process occurring after CD4 binding and requiring the transmembrane domain of gp41. Env proteins are able to mediate cell-cell fusion at least under some experimental conditions, indicating that lack of a syncytia phenotype does not rule out the possibility of fusion occurring between only two or a few cells.

The function of the CD4 coreceptor in the development of T cells.

T cells with helper activity can be found in mice that lack expression of the CD4 glycoprotein. The CD4 promoter is active in these cells; they respond to antigens presented by MHC class II molecules; they do not express CD8 and they do not depend on MHC class I for their development. By such criteria, these CD8- T cells resemble normal CD4+ helper T cells. The development of the helper lineage in CD4-null mice can be potentiated by expression of transgenes that encode either wild type CD4, or a deletion mutant of CD4 that lacks the cytoplasmic tail and therefore cannot interact with the tyrosine kinase p56lck. These observations suggest that CD4 is not absolutely required for the specification of the helper cell lineage. The role of the CD4 molecule in the development of T cells and possible mechanisms by which it achieves its functions are discussed.

New haplotype for the Glu104Asp mutation in triose-phosphate isomerase deficiency and prenatal diagnosis in a Spanish family.

First-trimester prenatal diagnosis was undertaken by chorionic villus DNA analysis in a Spanish family with the inherited Glu104Asp triose-phosphate isomerase deficiency. The fetus was heterozygous for the mutation and therefore predicted to be clinically unaffected. To investigate the evolutionary origin of this mutation, studies were conducted on the intragenic 2262A/G polymorphism and the CD4 pentameric tandem repeat marker. A different haplotype was found to the one previously described, suggesting a different origin of the Spanish mutation.