Negative Regulation of Zap70 by Lck Forms the Mechanistic Basis of Differential Expression in CD4 and CD8 T Cells.

Lck and Zap70, two non-receptor tyrosine kinases, play a crucial role in the regulation of membrane proximal TCR signaling critical for thymic selection, CD4/CD8 lineage choice and mature T cell function. Signal initiation upon TCR/CD3 and peptide/MHC interaction induces Lck-mediated phosphorylation of CD3 ITAMs. This is necessary for Zap70 recruitment and its phosphorylation by Lck leading to full Zap70 activation. In its native state Zap70 maintains a closed conformation creating an auto-inhibitory loop, which is relieved by Lck-mediated phosphorylation of Y315/Y319. Zap70 is differentially expressed in thymic subsets and mature T cells with CD8 T cells expressing the highest amount compared to CD4 T cells. However, the mechanistic basis of differential Zap70 expression in thymic subsets and mature T cells is not well understood. Here, we show that Zap70 is degraded relatively faster in DP and mature CD4 T cells compared to CD8 T cells, and inversely correlated with relative level of activated Zap70. Importantly, we found that Zap70 expression is negatively regulated by Lck activity: augmented Lck activity resulting in severe diminution in total Zap70. Moreover, Lck-mediated phosphorylation of Y315/Y319 was essential for Zap70 degradation. Together, these data shed light on the underlying mechanism of Lck-mediated differential modulation of Zap70 expression in thymic subsets and mature T cells.

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags.

BACKGROUND: A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. METHODS: Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized. RESULTS: Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. CONCLUSIONS: Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Cellular therapy approaches harnessing the power of the immune system for personalized cancer treatment.

Cancer development often implies failure of the immune system to recognize tumor antigens and kill malignant cells. While the whole immune cell repertoire is broad, that of immune cells with the ability to react to individual tumor antigens is usually very limited. The purpose of cancer immunotherapy is to augment the power, quantitative and qualitative, of the immune system such that it readily recognizes and eliminates cancer cells. As immune therapy is shifting toward more personalized medicine, different types of tumor antigens can be used as target antigens to allow T cells to destroy tumor cells. These antigens are mostly defined as tumor associated antigens (TAA), neoantigens or minor histocompatibility antigens. Their clinical usage involve either direct injection of TAA and neoantigens, administration of peptide-loaded dendritic cells in vaccination approaches, or infusion of ex vivo expanded tumor-specific T cells. However, such cellular therapies are facing several challenges including immune suppressive tumor microenvironment, lack of persistence of ex vivo expanded antigen specific T cells and potential off-target toxicity of these therapies. In this review, we will discuss recent advances allowing for better expansion of tumor reactive T cells and novel strategies used to overcome the challenges facing cellular therapy for cancer.

A novel nested polymerase chain reaction targeting the testis-specific protein Y-encoded family of genes for high sensitivity of recent semen exposure detection: Comparison with four other assays of semen detection.

OBJECTIVES: Because self-report of sexual behaviours is prone to biases, biomarkers of recent semen exposure are increasingly used to assess unprotected sex. We aimed to present a novel nested polymerase chain reaction (PCR) assay targeting testis-specific protein Y-encoded (TSPY) genes and to compare its performance in detecting recent semen exposure with that of four other assays. METHODS: Forty-five vaginal samples were selected at baseline of a prospective observational demonstration study of early antiretroviral treatment and pre-exposure prophylaxis among female sex workers in Benin. Semen exposure was assessed with: a rapid prostate-specific antigen (PSA) detection assay, a quantitative PCR targeting the sex-determining region (SRY) gene, a standard PCR targeting SRY, a standard PCR targeting TSPY, and a nested PCR targeting TSPY (n-TSPY). Because we had hypothesized that n-TSPY would be the most sensitive of the five assays while remaining specific, and as our results suggested that it was the case, sensitivity and specificity were calculated for each assay in comparison with n-TSPY. RESULTS: The n-TSPY could detect male DNA at concentration 16 and 64 times lower compared to s-TSPY and s-SRY, respectively. Among the 45 vaginal samples, prevalences of semen exposure according to the different assays varied from 22.2% (95%CI: 11.2%-37.1%) to 70.5% (95%CI: 54.8%-83.2%), with the highest prevalence measured with n-TSPY. The n-TSPY products were of expected size and we observed no false-positive in female DNA controls. The assay that offered the second best performance in detecting semen exposure was the PSA rapid test, with a sensitivity of 61.3% and a specificity of 100% compared to n-TSPY. CONCLUSIONS: Compared to n-TSPY, all other PCR assays had poor performance to detect semen exposure. The n-TSPY is an accessible assay that may have great utility in assessing semen exposure in studies where many factors are expected to accelerate biomarkers' clearance.

Critical Role for TCR Signal Strength and MHC Specificity in ThPOK-Induced CD4 Helper Lineage Choice.

Sustained TCR signaling is critical for ThPOK induction in MHC class II (MHCII)-signaled thymocytes leading to the CD4 helper lineage commitment. ThPOK suppresses the cytotoxic program in the signaled thymocytes and is shown to be necessary and sufficient for the CD4 helper lineage choice. Accordingly, loss and gain of ThPOK function redirects MHCII- and MHC class I (MHCI)-signaled thymocytes into the CD8 cytotoxic and CD4 helper lineage, respectively. However, the impact of a defined ThPOK level on the CD4 helper lineage choice of MHCII- and MHCI-specific thymocytes and the role of TCR signaling in this process is not evaluated. Equally, it is not clear if suppression of the cytotoxic program by ThPOK is sufficient in redirecting MHCI-restricted thymocytes into the CD4 helper lineage. In this study, we have investigated CD8 to CD4 helper lineage redirection in three independent ThPOK overexpressing transgenic mouse lines. Our analysis shows that one of the transgenic lines, despite overexpressing ThPOK compared with wild-type CD4 mature T cells and compromising cytotoxic program, failed to redirect all MHCI-signaled thymocytes into the CD4 helper lineage, resulting in the continued presence of CD8+ mature T cells and the generation of a large number of double negative mature T cells. Critically, the same ThPOK transgene completely restored the CD4 helper lineage commitment of MHCII-specific Thpok -/- thymocytes. Importantly, augmenting TCR signaling significantly enhanced the ThPOK-mediated CD4 helper lineage choice of MHCI-specific thymocytes but was still substantially less efficient than that of MHCII-specific thymocytes expressing the same amount of ThPOK. Together, these data suggest that the ThPOK-induced CD4 helper lineage commitment is strongly influenced by TCR signal strength and MHC specificity of developing thymocytes.

Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.

Efficient BST2 antagonism by Vpu is critical for early HIV-1 dissemination in humanized mice.

BACKGROUND: Vpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(ß-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions. RESULTS: Using a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the ß-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to ß-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated ß-TrCP-dependent BST-2 degradation for optimal initial viral propagation. CONCLUSIONS: Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

Role of CD3ε-mediated signaling in T-cell development and function.

Receptor-mediated signal transduction plays an important role in T-cell differentiation and function. The pre-T-cell receptor (pre-TCR) and TCR complexes are the most critical receptors for T-cell biology. Signals induced by pre-TCR and TCR in a ligand-independent or a ligand-dependent manner, respectively, are essential for thymocyte maturation. CD3 proteins, which are γ, δ, ε, and ζ polypeptides, play pivotal role in intracellular assembly, surface expression, and signal transduction via the pre-TCR and TCR complexes. Recent studies have suggested central and multiple roles for CD3ε in T-cell development and function. We review the role of the CD3ε chain in T-cell biology.

HIV-1 Nef disrupts maturation of CD4+ T cells through CD4/Lck modulation.

The HIV-1 Nef protein is a major determinant of HIV-1 pathogenicity. It has been found to induce thymocyte depletion, but the mechanisms involved are not completely understood. Also, nothing is known about its effects on thymocyte selection. We used the CD4C/HIV(Nef) transgenic (Tg) mice, which develop a profound CD4(+) T cell lymphopenia, to study their thymic development. We report that HIV-1 Nef causes depletion of double-positive thymocytes and impairs selection and lineage commitment of CD4(+) single-positive thymocytes. This latter defect could be relieved by increasing the affinity of the TCR-MHC interaction or by allowing CD4(+) T cell maturation to proceed in absence of the CD4 tail, in double-Tg (Nef × CD4(tailless)) mice or in the presence of constitutively active Tg Lck(Y505F). These rescue strategies also resulted in reversal of peripheral CD4(+) T cell lymphopenia. Our data indicate that impairment of Lck-mediated CD4 coreceptor signaling by Nef is an important in vivo mechanism of HIV-1 pathogenesis.

Distinctive CD3 heterodimeric ectodomain topologies maximize antigen-triggered activation of alpha beta T cell receptors.

The alphabeta TCR has recently been suggested to function as an anisotropic mechanosensor during immune surveillance, converting mechanical energy into a biochemical signal upon specific peptide/MHC ligation of the alphabeta clonotype. The heterodimeric CD3epsilongamma and CD3epsilondelta subunits, each composed of two Ig-like ectodomains, form unique side-to-side hydrophobic interfaces involving their paired G-strands, rigid connectors to their respective transmembrane segments. Those dimers are laterally disposed relative to the alphabeta heterodimer within the TCR complex. In this paper, using structure-guided mutational analysis, we investigate the functional consequences of a striking asymmetry in CD3gamma and CD3delta G-strand geometries impacting ectodomain shape. The uniquely kinked conformation of the CD3gamma G-strand is crucial for maximizing Ag-triggered TCR activation and surface TCR assembly/expression, offering a geometry to accommodate juxtaposition of CD3gamma and TCR beta ectodomains and foster quaternary change that cannot be replaced by the isologous CD3delta subunit's extracellular region. TCRbeta and CD3 subunit protein sequence analyses among Gnathostomata species show that the Cbeta FG loop and CD3gamma subunit coevolved, consistent with this notion. Furthermore, restoration of T cell activation and development in CD3gamma(-/-) mouse T lineage cells by interspecies replacement can be rationalized from structural insights on the topology of chimeric mouse/human CD3epsilondelta dimers. Most importantly, our findings imply that CD3gamma and CD3delta evolved from a common precursor gene to optimize peptide/MHC-triggered alphabeta TCR activation.

Hierarchical role of CD3 chains in thymocyte development.

Assembly, expression, and signal transduction by the pre-T-cell receptor (pre-TCR) and TCR complexes are critical for normal thymocyte development. How environmental cues sensed by these two receptor complexes are translated into biological responses that result in the generation of functionally mature T cells is increasingly better understood. Invariant CD3gamma, delta, epsilon, and zeta polypeptides are central to the function of these two receptor complexes. CD3 chains ensure correct intracellular assembly, surface expression, and signal transduction via the receptors in ligand-independent and -dependent manners. In the present review, the roles of the CD3 chains, particularly CD3gamma, delta, and epsilon, in thymocyte differentiation in mice and humans are reviewed.

Expression of fully assembled TCR-CD3 complex on double positive thymocytes: synergistic role for the PRS and ER retention motifs in the intra-cytoplasmic tail of CD3epsilon.

TCR expression on double-positive (DP) thymocytes is a prerequisite for thymic selection that results in the generation of mature CD4(+) and CD8(+) single-positive T cells. TCR is expressed at very low level on preselection DP thymocytes and is dramatically up-regulated on positively selected thymocytes. However, mechanism governing TCR expression on developing thymocytes is not understood. In the present report, we demonstrate that the intra-cytoplasmic (IC) domain of CD3epsilon plays a critical role in regulating TCR expression on DP thymocytes. We provide genetic and biochemical evidence to show that the CD3epsilon IC domain mutations result in elevated expression of fully assembled TCR on DP thymocytes. We also demonstrate that TCR up-regulation on DP thymocytes in these transgenic mice occurs in a ligand-independent manner. Further, we show that the proline-rich sequence and endoplasmic reticulum (ER) retention motifs in the IC domain of CD3epsilon play synergistic role in regulating TCR surface expression on DP thymocytes.

Critical and multiple roles for the CD3epsilon intracytoplasmic tail in double negative to double positive thymocyte differentiation.

The preTCR is associated with signal-transducing CD3gamma, delta, epsilon, and zeta polypeptides. It is generally agreed that CD3 chains play redundant roles in the receptor-mediated signal transduction. In the present study, we show that the intracytoplasmic (IC) domain of CD3epsilon is essential for early thymocyte maturation. We demonstrate that the IC domain-deleted CD3epsilon fails to restore the double negative (DN) to double positive (DP) thymocyte development in CD3epsilon-deficient mice. Additional experiments show that the membrane proximal basic amino acid rich sequence in the IC domain of CD3epsilon is sufficient for the DN to DP differentiation, whereas the proline rich sequence is required for efficient proliferation. This is probably due to impaired ligand independent recruitment of Nck to the proline rich sequence motif of CD3epsilon within the context of the preTCR. The data presented in this study elucidates mechanistic basis for the preTCR-induced proliferation of the DN thymocytes and have identified distinct roles for individual motifs of CD3epsilon in the preTCR-mediated differentiation and proliferation. These data provide the first genetic and phenotypic evidence for requirement of the IC domain of a CD3 chain in thymocyte development.

Different role for mouse and human CD3delta/epsilon heterodimer in preT cell receptor (preTCR) function: human CD3delta/epsilon heterodimer restores the defective preTCR function in CD3gamma- and CD3gammadelta-deficient mice.

The majority of T cell receptor (TCR) complexes in mice and humans consist of a heterodimer of polymorphic TCRalpha and beta chains along with invariant CD3gamma, delta, epsilon, and zeta chains. CD3 chains are present as CD3gammaepsilon, deltaepsilon, and zetazeta dimers in the receptor complex and play critical roles in the antigen receptor assembly, transport to the cell surface, and the receptor-mediated signal transduction. That CD3 chains play critical roles in thymocyte development is apparent from the analyses of CD3 deficient mice. PreT cell receptor (preTCR)-mediated CD4(-)CD8(-) (double negative or DN) to CD4(+)CD8(+) (double positive or DP) transition is severely impaired in mice deficient in either CD3gamma, or epsilon, or zeta chain. In contrast, CD3delta deficiency impairs thymocyte maturation at the CD4(+)CD8(+) double positive (DP) stage suggesting that CD3delta is not required for the preTCR-mediated DN to DP transition. However, recent data suggest that a defect in human CD3delta results in impaired development at the DN stage indicating a role for hCD3delta in preTCR-mediated DN to DP transition. To determine if human CD3delta/epsilon (hCD3delta/epsilon) could mediate preTCR-mediated DN to DP transition, we employed a human CD3 transgene that encodes full length CD3delta and a truncated but functional form of CD3epsilon. Surprisingly, the transgene restored the defective preTCR function in not only CD3epsilon- but CD3gamma- and CD3gammadelta-deficient mice as well. A possible role for human CD3delta/epsilon heterodimer in the preTCR-mediated DN to DP transition is discussed.

The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment.

Development of immature T-cell precursors (thymocytes) to either the CD4 helper or CD8 killer T-cell lineages correlates precisely with their T-cell receptor specificity for major histocompatibility complex class II or class I molecules, respectively, indicating that the process is carefully regulated. Although intensively studied owing to its importance in determining the composition of the mature T-cell compartment and as a general model of binary lineage decisions, the underlying molecular pathways remain obscure. We have previously reported a spontaneous mouse mutant (HD (helper deficient) mice) in which lineage commitment is specifically perturbed without affecting positive selection. Here we show that a point mutation in the zinc finger transcription factor Th-POK (T-helper-inducing POZ/Krüppel-like factor) is responsible for redirection of class-II-restricted thymocytes to the CD8 lineage in HD mice. Furthermore, we demonstrate that constitutive expression of this factor during thymic development leads to redirection of class-I-restricted thymocytes to the CD4 lineage, indicating that Th-POK is a master regulator of lineage commitment.

Biochemical evidence for the presence of a single CD3delta and CD3gamma chain in the surface T cell receptor/CD3 complex.

The T cell antigen receptor (TCR) consists of an alphabeta heterodimer and associated invariant CD3gamma, delta, epsilon, and zeta chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCRalpha, TCRbeta, CD3gamma, and CD3delta and two molecules each of CD3epsilon and CD3zeta, is not clearly understood. Although it has been shown that there are two chains of CD3epsilon and CD3zeta, the stoichiometry of CD3gamma or CD3delta chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3delta and CD3gamma were employed to show that the surface TCR complexes contain one molecule each of CD3delta and CD3gamma. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3delta and CD3gamma in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3delta and CD3gamma in the surface TCR/CD3 complex.