CD20scFv-IgGFc T 淋巴细胞的建立及其对B细胞淋巴瘤靶向杀伤作用的比较

目的：研究CD20scFv嵌合T淋巴细胞靶向杀伤Daudi细胞时杀伤的效果和T细胞活化情况。方法：将两种质粒转染至PA317细胞中，用转染成功的PA317上清液感染外周血T淋巴细胞后经800 mg/L的G418筛选1周后去杀伤Daudi、K562细胞，分别在不同时点用流式细胞仪检测Daudi细胞AnnexinⅤ的阳性率 ，用ELISA检测细胞因子IL-2、IFNγ。结果：Daudi细胞AnnexinⅤ的阳性率在24 h内两实验组与K562组相比明显增高，而实验组之间没有显著差异。72 h时CD20scFv-IgGFc-CD28-ζ组比CD20scFv-IgGFc组IL-2（1 509.00 ng/L比220.54 ng/L）和IFNγ（912.16 ng/L比251.42 ng/L）的分泌量有显著增高。结论：①两种CD20scFv特异的T细胞在引起Daudi细胞早期凋亡无显著差异，说明在CD20scFv的靶向杀伤过程中CD28-ζ基因可能不主导Daudi细胞的早期凋亡。②CD20scFv-IgGFc-CD28-ζ组IL-2、IFNγ增幅更加明显，说明CD3ζ和CD28嵌合的T细胞可以自身活化而不受MHC的限制，从而增强了T细胞的活化和杀伤等功能。     

Genetic analysis of CD28 signaling

T cell activation is central to initiating an immune response. Two signals are required: an antigen-specific signal through the T cell receptor (TCR) and an antigen-independent costimulatory signal, primarily through CD28 in naïve T cells. Although many of the molecules involved in TCR signal transduction have been identified, the signaling pathways downstream of CD28 involved in costimulation are not well-defined. Through mutagenesis, we have generated a panel of Jurkat T cell lines in which CD28 costimulation fails to upregulate the RE/AP composite element of the IL-2 promoter. Biochemical analysis and genetic rescue of the defects in these cell lines will lead to a better understanding of CD28 signal transduction.     

Autonomous induction of proliferation, JNK and NF-alphaB activation in primary resting T cells by mobilized CD28

Induction of proliferation in primary resting T cells requires engagement of both the antigen-specific TCR and the co-stimulatory receptor CD28. Here we report that CD28 functions as an autonomous mitogenic receptor which is mobilized by TCR signaling through cytoskeletal rearrangement. Shortcutting of TCR-dependent CD28 recruitment by stimulation with monoclonal antibodies specific for mobilized CD28 results in maximum proliferation and IL-2 secretion in primary resting T cells without activation of ZAP-70, a central component of the TCR's signal transduction machinery. Engagement of mobilized CD28 fully activates the c-Jun N-terminal kinase cascade and translocation of NF-alphaB, two key targets of signal integration in co-stimulation. We propose a two-step activation model for co-stimulation in primary resting T cells in which antigen recognition recruits co-stimulatory receptors which then autonomously transduce signals promoting T cell proliferation.     

Expression and functional significance of CTLA-4, a negative regulator of T cell activation

The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the antigen-specific T cell receptor (TCR) and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response, characterized by proliferation, differentiation, clonal expansion and effector function, or, in the absence of an appropriate costimulation, to a state of long-lasting unresponsiveness, termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between the costimulatory molecule CD28 on T cells and members of the B7 family on antigen-presenting cell results in upregulation of T cell proliferation and cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of these findings, the two-signal requirement model for T cell activation is generally accepted today. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) in T cell activation has been both obscure and controversial. CTLA-4 was initially supposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is in fact not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in the dynamic interplay of activatory and inhibitory signals.     

Qualitatively distinct signaling through T cell antigen receptor subunits

T cell antigen receptors (TCR) contain several subunits including CD3γ, δ, and ?, and TCRζ and η which are capable of mediating signal transduction. It is unclear whether the signaling function of these subunits is completely redundant. To assess the relative signaling capabilities of TCR subunits, we compared proximal events in signal transduction by wild-type TCR complexes and TCR devoid of functional ζ subunits, as well as chimeric receptors containing the cytoplasmic domains of TCRζ or CD3?. Results demonstrate that in BW5147 wild-type TCR, tail-less ζ TCR, CD3?, and TCRζ transduce signals leading to tyrosine phosphorylation of similar sets of cellular substrates, including the receptor subunits, Fyn, ZAP-70, and phospholipase Cγ1 (PLCγ1). Surprisingly, unlike wild-type TCR, tail-less ζ TCR, and CD3?, TCRζ was incapable of transducing signals resulting in inositol triphosphate (IP₃) generation or intracellular free calcium ([Ca²⁺]i) mobilization. These data indicate that tyrosine phosphorylation of PLCγ1 is not sufficient to drive IP₃ production and [Ca²⁺]i mobilization. Most importantly, data presented indicate that TCRζ and CD3? engage partially distinct signaling pathways.     

Interelationship between CD3 and CD28 pathways in a murine T cell thymoma

It is well known that the CD28 costimulatory signal is important to complement T cell receptor (TCR)/CD3-initiated T cell activation, but the mechanism by which these two distinct signaling pathways are integrated is not clearly understood. In our laboratory, we dispose of a murine T cell hybridoma transfected with human CD28 molecule which is able to produce IL-2 in response to stimulation, suggesting that the signal transduction machinery coupled to the CD28 molecule is capable of triggering effector functions. Nevertheless, the action of three immunosuppressive agents previously shown in our model, suggested an interaction between the CD3 and CD28 pathways. We confirmed here this hypothesis by transfecting the cDNA of the human CD28 molecule in the BW5147 thymoma which lacks CD3 surface expression. Stimulation of the human CD28 did not lead to IL-2 secretion while the restoration of the TCR/CD3 complex re-established the functionality of this costimulatory molecule. These data demonstrate that the IL-2 production induced by the CD28 activation pathway is dependent of the TCR/CD3 complex cell surface expression and suggest the formation of a functional membrane complex between the CD3 and CD28 molecules. The molecular basis of the functional dependence of CD28 signaling on the TCR/CD3 complex is presently unknown. Nonetheless, we showed that some early events induced by CD28 stimulation, such as PI3-kinase association, are independent of the TCR/CD3 complex expression.     

Enhanced surface TCR replenishment mediated by CD28 leads to greater TCR engagement during primary stimulation

When T cells are stimulated with high concentrations of strong TCR agonist, engaged TCR are internalized and degraded, resulting in greatly reduced surface TCR levels for up to several days post-stimulation. It has been noted that surface TCR levels rise subsequently, even in the presence of continuing stimulation, but the role of CD28 co-stimulation in surface TCR replenishment has not been investigated. Here, we have examined the return of surface TCR following activation, the availability of these TCR for antigenic engagement and the role of CD28 in that process. We report that within 24 h of stimulation, the level of surface TCR expression becomes dependent on the degree of CD28 signaling provided during T cell activation. In addition, when cells are removed from stimulus after 24 h, surface TCR expression recovers to a stable level which exceeds that of unstimulated cells and is proportional to the degree of CD28 co-stimulation. TCR that replenish the plasma membrane during T cell activation can be down-regulated by receptor occupancy with the same efficiency as TCR on freshly stimulated cells. Thus, as a result of enhanced surface TCR replenishment, CD28-co-stimulated cells can engage and down-regulate more TCR than co-stimulation-deprived cells in the face of ongoing stimulation. Furthermore, engagement of newly expressed TCR on activated T cells re-induces CD69, suggesting participation of these replenishing TCR in continued T cell signaling. These data identify the augmentation of surface TCR replenishment during activation as a novel mechanism that likely contributes to the enhanced antigenic sensitivity of CD28-co-stimulated T cells.     

Differential T cell receptor-mediated signaling in naive and memory CD4 T cells

Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3ζ subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3ζ in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72^syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.     

BY55/CD160 acts as a co-receptor in TCR signal transduction of a human circulating cytotoxic effector T lymphocyte subset lacking CD28 expression

In the present study, we examined the role of the recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface molecule BY55, assigned as CD160, in TCR signaling. CD160 is expressed by most intestinal intraepithelial lymphocytes and by a minor subset of circulating lymphocytes including NK, TCRgammadelta and cytotoxic effector CD8bright+CD28- T lymphocytes. We report that CD160, which has a broad specificity for MHC class Ia and Ib molecules, behaves as a co-receptor upon T cell activation. Anti-CD160 mAb enhance the CD3-induced proliferation of freshly isolated CD160-enriched peripheral blood lymphocytes and CD160+ T cell clones. Further, the engagement of CD160 receptors on normal clonal T lymphocyte populations lacking CD4, CD8 and CD28 molecules by MHC class I molecules results in an increased CD3-induced cell proliferation. Further, we found that CD160 co-precipitates with the protein tyrosine kinase p56lck and tyrosine phosphorylated zeta chains upon TCR-CD3 cell activation. Thus, we demonstrate that CD160 provides co-stimulatory signals leading to the expansion of a minor subset of circulating lymphocytes including double-negative CD4/CD8 T lymphocytes and CD8bright+ cytotoxic effector T lymphocytes lacking CD28 expression.     

Commentary: Regulated equilibrium between opposite signals: a general paradigm for T cell function?

The co-signaling receptors specific for the different members of the B7 molecular family are cell surface glycoproteins that are essential to modulate and tune the TCR-mediated activation of T lymphocytes. The common characteristic is that their function appears to be dependent on the engagement of TCR by antigenic peptides presented in the MHC context by antigen-presenting cells. Interestingly, co-signaling molecules can be distinguished into costimulators and co-inhibitors, the prototype being represented by CD28 and CTLA-4, respectively. In the case of costimulators, the co-signals integrate the signal originated from the TCR resulting in optimal T cell activation (two-signal model). In the case of co-inhibitors, the co-signals would moderate and/or switch off the Ag-dependent T cell activation, thus acting as negative regulators of immune responses. The growing number of novel co-signaling molecules has recently highlighted the need to integrate the two-signal model with the emerging data on the different co-inhibitory interactions. Thus, a model has been proposed based on the idea that the TCR signal alone cannot take a full decision on the nature of the functional outcome following an antigen-specific stimulation and that this final event is governed by the co-signaling molecules.     

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.     

Regulation of T-cell differentiation by CD2 and CD28 accessory molecules.

It was analysed to what extent the functional T-cell responses that result from T-cell receptor (TcR)/CD3 triggering differ from responses that are induced after simultaneous ligation of CD2 and CD28 accessory molecules. To allow a quantitative comparison of these activation pathways, purified lymphocytes were stimulated with either graded densities of immobilized anti-CD3 monoclonal antibodies (mAb) or with increasing amounts of anti-CD28 mAb in the presence of a constant concentration of anti-CD2 mAb. Both activation systems were sensitive to the regulatory properties of CD11a/CD18 molecules. T-cell stimulation via CD2/CD28 molecules induced a more potent release of interleukin-2 (IL-2) and more pronounced T helper (Th) cell responses than T-cell stimulation via the TcR/CD3 complex, whereas CD25 expression was more readily initiated after T-cell activation via the TcR/CD3 complex. Optimal Th cell differentiation was detected under conditions of suboptimal receptor occupancy whereas, in contrast, optimal cytolytic T lymphocyte (CTL) differentiation required optimal TcR/CD3 or CD2/CD28 engagement. The findings indicate that T-cell differentiation can be influenced in a qualitative manner by the strength of the activation signal provided, and suggest that antigen-specific T-cell responses might be regulated in a quantitative manner through CD2 and CD28 accessory molecules.     

Differential interaction of the CD2 extracellular and intracellular domains with the tyrosine phosphatase CD45 and the ζ chain of the TCR/CD3/ζ complex

T cell activation via CD2 requires interaction of CD2 with several signaling molecules. To investigate the structural requirements for an association of CD2 with the tyrosine phosphatase CD45 and the ζ chain of the T cell receptor (TCR)/CD3/ζ complex, we have expressed in mouse EL4 T cells a series of human CD2 chimeric and mutant proteins. Chimeric proteins in which the CD2 transmembrane and/or cytoplasmic domains were deleted or exchanged with analogous regions of CD4, CD28 or CD58 retained association with high levels of murine CD45 phosphatase activity, suggesting that the CD2 extracellular domain largely controls interaction with CD45. To a lesser extent, the cytoplasmic domain of CD2 was also shown to interact with CD45, as demonstrated by an increase in co-immunoprecipitated phosphatase activity observed following replacement of the CD58 cytoplasmic domain with that of CD2. In contrast, the cytoplasmic domain of CD2 was found to be responsible for the majority of CD2 interaction with the ζ chain of the TCR/CD3/ζ complex. Deletion of the CD2 cytoplasmic domain, excluding the first three amino acids, removed virtually all CD2 associated ζ chain and approximately sevenfold higher levels of ζ chain were found in association with a CD58/58/2 chimera than with control human CD58 wild type. This study suggests that the CD2 extracellular and intracellular domains are differentially involved in regulating T cell activation through interaction with the tyrosine phosphatase CD45 and the ζ chain of the TCR/CD3/ζ complex.     

Deconstructing the form and function of the TCR/CD3 complex

When T cells encounter antigens via the T cell antigen receptor (TCR), information about the quantity and quality of antigen engagement is relayed to the intracellular signal transduction machinery. This process is poorly understood. The TCR itself lacks a significant intracellular domain. Instead, it is associated with CD3 molecules that contain intracellular signaling domains that couple the TCR/CD3 complex to the downstream signaling machinery. The earliest events in TCR signaling must involve the transfer of information from the antigen binding TCR subunit to the CD3 signaling subunits of the TCR/CD3 complex. Elucidating the structural organization of the TCR with the associated CD3 signaling molecules is necessary for understanding the mechanism by which TCR engagement is coupled to activation. Here, we review the current state of our understanding of the structure and organization of the TCR/CD3 complex.     

Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.     

CD28 and CTLA4 coordinately regulate airway inflammatory cell recruitment and T-helper cell differentiation after inhaled allergen

Airway inflammation after inhaled allergen exposure requires the recruitment, activation, and differentiation of antigen-specific T cells into T helper (Th) 2 effector cells. These processes are regulated not only by antigen engagement of the T-cell receptor, but also by specific accessory molecules on the surface of the T cell. We examined how the balance of signals derived through the CD28 and cytotoxic T-lymphocyte antigen (CTLA) 4 receptors modulate the outcome of inhaled antigen exposure in a murine model of allergic airway inflammation. Mice deficient in CD28 have defective Th2 cell development and failed to develop inflammation after sensitization and inhaled challenge with ovalbumin. Prevention of B7-CTLA4 interactions in CD28-deficient mice restored lymphocyte but not eosinophil recruitment to the airway. Analysis of cytokine gene expression revealed that T cells from CD28-deficient mice failed to differentiate into Th2 cells in either the presence or absence of B7-dependent signals, and therefore did not recruit eosinophils to the airway. Thus, the processes of T-cell recruitment to the airway and T-cell differentiation have distinct requirements for signals mediated through the CD28 and CTLA4 receptors, demonstrating that these receptors are important regulatory components in the development of allergic airway inflammation.