BY55/CD160 cannot be considered a cytotoxic marker in cytomegalovirus-specific human CD8(+) T cells

CD160/BY55 is a glucosyl-phosphatidylinositol (GPI)-anchored cell membrane receptor that is expressed primarily in natural killer (NK) cells. Its presence in CD8(+) T lymphocytes is considered to be a marker of cytotoxic activity, although there are few data in this regard. In the present work, we analysed the expression of CD160 in subpopulations of cytomegalovirus (CMV)-specific CD8(+) T cells. Subpopulations were defined by CD28 and CD57 expression and exhibited varying degrees of differentiation and cytotoxic potential, as evaluated by the expression of perforin, interferon (IFN)-gamma and interleukin (IL)-7Ralpha/CD127. We included subjects with different intensities of anti-viral immune response. Results showed that the terminally differentiated CD28(-) CD57(+) subset displaying the highest level of perforin expressed CD160 at a level similar to that of memory CD28(+) CD57(-)perforin(-) cells. A comparison of the expression of perforin in CD160(+) cells versus CD160(-) cells showed that expression was significantly higher in the absence of CD160. Interestingly, the CMV-specific CD8(+) T cell subset from a patient with ongoing CMV reactivation did not begin to express CD160 until day +92 of the follow-up period. Taken together, our data show that CD160 cannot be considered a cytotoxic marker in CMV-specific CD8(+) T cells.     

Co-receptor-independent signal transduction in a mismatched CD8+ major histocompatibility complex class II-specific allogeneic cytotoxic T lymphocyte

The contribution of co-receptors in signal transduction upon T cell receptor (TCR)-mediated recognition of major histocompatibility complex (MHC) class II antigen by mature T lymphocytes expressing TCR derived from the apparently co-receptor-independent, I-A^k-specific allogeneic CD8⁺ CTL clone QM11 has been examined. Mature double-negative, CD8⁺ and CD4⁺ bulk T cell lines and clones expressing TCRQM11 were developed from TCRQM11 transgenic mice. All these T cells, irrespective of co-receptor expression, showed specific lytic activity on cells expressing I-A^k. Furthermore, co-receptorless mutants were obtained from a CD4⁺ and CD8⁺ clone. The responses of these co-receptorless mutants upon specific recognition of the alloantigen, as judged by cytolytic activity, granule exocytosis, lymphokine production, proliferation, and tyrosine phos-phorylation of the ξ chain, were comparable to those of the original clones. Thus, the results proved the co-receptor independence of the recognition of I-A^k by TCRQM11 and further indicated there is no indispensable unique signal transduced by co-receptors. However, when the amount of the available antigen was limited by anti-I-A^k antibody, the CD4⁺ T cell clone showed a remarkable resistance to the inhibition whereas the mismatched CD8⁺ clone was readily inhibitable. The anti-I-A^k-resistant component of the CD4⁺ clone showed dependency on the CD4 molecule. Taken collectively, the results indicate that the role played by a co-receptor molecule in mature T cells is purely quantitative amplification of the signal through the formation of a TCR/MHC/co-receptor ternary complex, and also indicate that the role of co-receptor molecules as TCR-independent adhesion molecules is at best minimal.     

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.     

系统性红斑狼疮患者T细胞TCR/CD3复合物介导的信号传导研究

目的：证实SLE患者T细胞功能异常是否与其生物化学信号转导异常有关。方法：用CD3单抗与羊抗鼠二抗IgG相关联刺激T细胞并用Thapsigargin和EGTA干预后，分别用粘附细胞仪连续观察10min T细胞[Ca^2 ]i的变化，并评价[Ca^2 ]i反应与CD3分子和InsP3生成量的相关性。结果：正常人和SLE患者T细胞[Ca^2 ]i反应的基准上似（P＝0．105）；SLE患者T细胞的[Ca^2 ]i反应高峰值、平台值明显高于正常对照（P＜0．001，P＜0．001）；加入Thapsigargin后二者[Ca^2 ]i反应无显著差异，而加入EGTA后二者[Ca^2 ]i反应有显著差异；二者的T细胞CD3阳性率和InsP3生成量无差异（P＝0．665，P＝0．537）。结论：SLE患者T细胞TCR／CD3介导的信号转导途径存在异常；SLE患者T细胞功能异常可能是因细胞内生物化学信号途径异常所致。     

The lymphocyte surface antigen CD38 acts as a nicotinamide adenine dinucleotide glycohydrolase in human T lymphocytes

The extracellular domain of the lymphocyte surface antigen CD38 has been recently shown to share a high sequence homology with a nicotinamide adenine dinucleotide (NAD⁺)-specific hydrolyzing enzyme cloned from the ovotestis of the gastropod Aplysia (E. States, D. J., Walseth, T. F., Lee, H. C, Trends Biochem. Sci. 1992. 17: 495). In agreement with this finding, we present here evidence that CD38-overexpressingT cells, such as human thymocytes and cells from the human HPB-ALL T cell line, exhibit a NAD⁺-hydrolyzing enzymatic activity present on the outer surface of the cell membrane. In contrast, T lymphocytes with relatively low levels of CD38 marker, such as the human Jurkat cell line, display a lower activity. This suggests a relationship between ecto-NAD⁺ glycohydrolase activity and CD38 expression, as confirmed here when comparing wild-type Jurkat cells and a Jurkat cell variant overexpressing the CD38 molecule. Moreover, CD38 immunoprecipitates from thymocytes behave as an authentic NAD⁺ glycohydrolase enzyme: it transforms NAD⁺ stoichiometrically into nicotinamide plus adenosine 5′-diphosphoribose. Altogether these results strongly support the assumption that CD38 is actually a lymphocyte-specific NAD⁺-hydrolyzing enzyme, a finding that give new prospects to understand the in vivo function of this cell membrane protein.     

Differentiation of human CD8 T cells: implications for in vivo persistence of CD8+ CD28- cytotoxic effector clones

CD8 T cells contain a distinct subset of CD8+ CD28- cells. These cells are not present at birth and their frequency increases with age. They frequently contain expanded clones using various TCRalphabeta receptors and these clones can represent >50% of all CD8 cells, specially in old subjects or patients with chronic viral infections such as HIV-1. Herein, it is shown that a large fraction of CD8+ CD28- cells expresses intracellular perforin by three-color flow cytometry, in particular when this subset is expanded. Together with their known ability to exert potent re-directed cytotoxicity, this indicates that CD8+ CD28- T cells comprise cytotoxic effector cells. With BrdU labeling, we show that CD8+ CD28- cells derive from CD8+ CD28+ precursors in vitro. In addition, sorted CD8+ CD28+ cells gave rise to a population of CD8+ CD28- cells after allo-stimulation. Moreover, ex vivo CD8+ CD28+ cells contain the majority of CD8 blasts, supporting the notion that they contain the proliferative precursors of CD8+ CD28- cells. CD95 (Fas) expression was lower in CD8+ CD28- cells, and this subset was less prone to spontaneous apoptosis in ex vivo samples and more resistant to activation-induced cell death induced by a superantigen in vitro. Thus, the persistence of expanded clones in vivo in the CD8+ CD28- subset may be explained by antigen-driven differentiation from CD8+ CD28+ memory precursors, with relative resistance to apoptosis as the clones become perforin(+) effector cells.     

Impaired T cell signal transduction through CD28 in a patient with idiopathic thrombocytopenia.

We describe an infant whose peripheral blood mononuclear cells were unable to proliferate or synthesize IL-2 in response to a mitogenic combination of antibodies directed against CD2 and CD28. This peculiar defect, which has been stable to date, was attributed to an impairment in CD28-mediated T cell activation, because further comitogenic combinations containing anti-CD28 monoclonals also failed to induce normal proliferation of the patient's T cells. In contrast, proliferation after membrane stimulation (with anti-CD2, recombinant IL-2, or certain lectins) or transmembrane activation (with phorbol ester and calcium ionophore) was normal, suggesting that his lymphocytes did not have a general membrane or intracellular signalling impairment. A T cell line derived from the patient confirmed the existence of a severe defect in CD28-mediated T cell proliferation, but also showed a profound impairment in CD3-induced T cell proliferation. Other cell surface molecules like CD2 and CD25 were, in contrast, capable of transducing normal proliferation signals. As all relevant molecules were detectable by cytofluorography and immunoprecipitation, we conclude that the patient's lymphocytes had an intrinsic defect in the delivery of CD28-mediated signals which, in the absence of monocytes, also affected CD3-mediated proliferation. The study of this novel kind of immunodeficiency may help to unravel the complex interactions that take place among CD2, CD3 and CD28 during T cell activation. The presence of an idiopathic thrombocytopenia in the patient suggests the intriguing possibility of a role for CD28 in the maintenance of peripheral blood platelets levels, although alternative interpretations are not ruled out.     

CD3/CD28共刺激调节T细胞多重信号转导通路的基因mRNA表达

目的:观察激活T细胞中信号转导通路的基因变化情况。方法:小鼠T细胞体外经CD3/CD28共刺激4小时,提取RNA,以信号转导通路发现者PCR芯片分析18个信号转导通路相关的84个关键基因的mRNA改变情况。结果:CD3/CD28共刺激调节了43个基因,其中上调24个,下调19个,涵盖检测的所有18条信号转导通路。结论:多重信号通路共同参与了T细胞的激活过程。     

Characterization of a subset of antigen-specific human central memory CD4+ T lymphocytes producing effector cytokines

CCR7(+ )central memory (T(CM)) CD4(+) T cells play a central role in long-term immunological memory. Recent reports indicate that a proportion of CD4(+) T(CM) is able to produce effector cytokines. The phenotype and the role of this subset remain unknown. We characterized cytokine-producing human CD4(+) T(CM) specific for cleared protein and persistent viral Ag. Our results demonstrate that the type of Ag stimulation is a major determinant of CD4(+) T(CM) differentiation. CMV-specific T(CM) were significantly more differentiated than protein Ag-specific T(CM) and included higher proportions of IFN-gamma-producing cells. The expression of killer cell lectin-like receptor G1 (KLRG1) by protein Ag- and CMV-specific T(CM) was associated with increased production of effector cytokines. KLRG1(+) T(CM) expressed high levels of CD127, suggesting that they can survive long term under the influence of IL-7. The induction of KLRG1(+) T(CM) may therefore represent an important target of vaccination against pathogens controlled by cellular immune responses.     

Fas-FasL和caspase-3信号通路在启动SLE患者T细胞亚群凋亡中的作用

目的：探讨SLE患者T细胞亚群的Fas-FasL信号传导通路与T细胞凋亡紊乱的关系。方法：应用流式细胞术测定T细胞亚群表面Fas、FasL的表达率及细胞质中活化caspase-3的表达率。结果：与健康对照组相比较，活动期及稳定期SLE患者组CD4^＋T细胞表面Fas的表达率均显著增加（P〈0．05），CD8^＋T细胞表面Fas的表达率略有增加但无统计学意义（P〉0．05）。稳定期和活动期SLE患者组T细胞亚群表面FasL的表达率均显著增加（P〈0．05），但两疾病组问T细胞亚群表面Fas、FasL的表达率无显著性差异（P〉0．05）。活动期SLE患者组T细胞亚群细胞质中活化caspase-3的表达率，明显高于稳定期SLE患者组和健康对照组（P〈0．05）。稳定期SLE患者组T细胞亚群细胞质中活化caspase-3的表达率略高于健康对照组，但无统计学意义。结论：SLE患者外周血T细胞亚群凋亡加速，CC4^＋ T细胞的凋亡活跃，其中Fas-FasL信号传导途径可能起重要的作用。T细胞凋亡紊乱的程度与SLE的活动程度密切相关。     

Novel human CD4+ T lymphocyte subpopulations defined by CD300a/c molecule expression

The CD300c (CMRF-35A) and CD300a (CMRF-35H) molecules are leukocyte surface proteins that are part of a larger family of immunoregulatory molecules encoded by a gene complex on human chromosome 17. The CMRF-35 monoclonal antibody binds to an epitope common to both molecules, expressed on most human leukocyte populations, apart from B lymphocytes and a subpopulation of CD4(+) and CD8(+) T lymphocytes. We describe the CMRF-35(pos) and CMRF-35(-) fractions of CD4(+) T lymphocytes. The CMRF-35(pos) fraction can further be divided into CMRF-35(++) and CMRF-35(+)CD4(+) T lymphocyte subpopulations. Resting peripheral CD4(+) T lymphocytes express CD300a mRNA and very low amounts of CD300c. Activation results in an initial decrease in CD300a gene expression before an increase in both CD300a and CD300c gene expression. The up-regulated expression of these genes was associated with increased CMRF-35 binding to activated T lymphocytes. The CMRF-35(-) fraction of CD4(+) T lymphocytes proliferated to a greater extent than the CMRF-35(pos) fraction, in response to mitogens or allogeneic antigen. The poor proliferation of the CMRF-35(pos) CD4(+) in response to mitogens was explained by increased apoptosis within this subpopulation. The recall antigen, tetanus toxoid, stimulated the CMRF-35(++)CD4(+)CD45RO(+) but not the CMRF-35(-)CD4(+)CD45RO(+) subpopulation. Resting CMRF-35(++) CD4(+) lymphocytes express low levels of IFN-gamma mRNA. Within 18 h following in vitro activation, CMRF-35(++) CD4(+) lymphocytes express more IFN-gamma mRNA and protein compared with the CMRF-35(-)CD4(+) lymphocytes, however, after 24 h, both the CMRF-35(+) and CMRF-35(-)CD4(+) T lymphocytes were able to produce IFN-gamma. The CMRF-35(++)CD4(+) T lymphocyte population contains the Th(1) memory effector cells.     

Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites

The heterogeneous expression of lymphocyte homing receptors (HR) by the (CD45RA^low/RO^high) memory/effector T cell population in the human is thought to define subsets with tissue-selective recirculatory potential. To investigate further the localization characteristics of these T cells, we used multiparameter flow cytometry to quantitate T cell subsets defined by expression of the skin-selective HR called the cutaneous lymphocyte-associated antigen (CLA), the peripheral lymph node (PLN) HR L-selectin, the mucosal-associated HR α4β7-integrin, and the mucosal-associated adhesion molecule αeβ7-integrin in either cutaneous or pulmonary immune effector sites and corresponding peripheral blood. Compared to peripheral blood, skin T cells were highly enriched for the CLA⁺/L-selectin⁺/αeβ7-integrin^? memory/effector subset, whereas lung memory/effector T cells were predominantly CLA^{?to low} L-selectin^?, and almost half were αeβ7-integrin⁺. α4β7-integrin expressing memory/effector T cells were diminished in both skin and lung, suggesting that this HR is not a major participant in determining localization specificity in either of these sites. The characteristic pulmonary T cell HR phenotype did not significantly differ between the normal subjects and those with pulmonary inflammatory disease, and did not correlate with markers of T cell activation. Induction of a rapid up-regulation of pulmonary inflammation via intrabronchial allergen challenge in asthmatic patients tended to decrease localization specificity, resulting in a more general importation of memory/effector subsets. Taken together, these results suggest that tissue microenvironments play a major role in determining the character of local T cell infiltrates via their ability to import and retain memory/effector subsets selectively or, more generally, depending on the intensity of local inflammatory stimuli.     

Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop.

CD19 and CD22 are B lymphocyte cell-surface molecules that positively and negatively regulate antigen receptor signal transduction, respectively. Biochemical studies with B cells from CD19-deficient and CD22-deficient mice indicated that these two regulatory molecules influenced each other's functions: CD22 expression negatively regulated CD19 tyrosine phosphorylation, while optimal CD22 function was dependent on CD19 expression. Functional CD19 and CD22 interactions were also assessed in vivo by generating CD19/CD22 double-deficient mice. Remarkably, the CD19 mutation was dominant to the CD22 mutation in most instances. B lymphocytes from CD19/CD22-deficient and CD19-deficient mice were functionally equivalent despite the negative influence normally provided by CD22 expression. These data collectively suggest that CD19 activates the CD22/SHP1 inhibitory pathway that then acts primarily on CD19.     

Effect of hypertonicity and monensin on CD3/TCR surface expression in human T cells

Pretreatment of T lymphocytes with anti-CD3 or anti-TCR mAb results in the disappearance of the T cell receptor complex (TCR/CD3) from the cell surface. The mechanisms of this down-regulation have not been fully elucidated. We demonstrate here that the modulation of the CD3/TCR complex can be completely inhibited by hypertonic medium, a condition known to block receptor-mediated endocytosis. Consequently, the sequestration of the CD3/TCR complex associated to relevant mAb in acid vesicles did not occur under hypertonicity condition. Moreover, monensin, which is known to interfere with receptor recycling, was found to amplify the CD3/TCR modulation induced by specific mAb and decrease the uptake of 125I-labeled anti-CD3/TCR mAb by T cells. We therefore propose that mAb-CD3/TCR complexes are internalized via coated pits by a receptor-mediated endocytosis and then transported to acid compartments. MAb are degraded in lysosomes during this process, whereas CD3/TCR molecules recycle back to cell surface.     

Co-stimulation via CD28 induces activation of a refractory subset of MRL-Ipr/Ipr T lymphocytes

Peripheral lymphoid tissues of Ipr mice contain a large proportionof TCRß/CD3⁺CD4^–CD8^– T cells that lacksurface CD2 and express the B cell isoform of CD45, B220. Thissubset of T cells does not proliferate or produce IL-2 in responseto mitogenic signals or TCR–CD3 ligation. At the sametime, these abnormal T cells display several characteristicsof an activated phenotype. Collectively, these properties ofIpr CD4^–CD8^– T cells have functional parallels withanergic T cells. A critical co-stimulatory molecule implicatedin the prevention of or recovery from anergy is CD28, whichbinds the ligand BB1/B7 on certain accessory cells. Ipr CD4^–CD8^–T cells express normal levels of CD28 which is capable of transducinga strong proliferative signal to these cells in co-stimulationwith mitogens. However, proliferation of Ipr CD4^–CD8^–T cells in response to CD28 co-stimulation does not reach thelevels observed in normal T cells stimulated under similar conditions.Stimulation with anti-CD28 mAb in conjunction with phorbol myristateacetate and lonomycin promotes cell cycling in the CD2^–subset of CD4^–CD8^– T cells, and results in a slightinduction of CD2 levels during the course of the culture period.However, the majority of cells obtained at the end of the cultureperiod remain TCRß+ CD4^–CD8^–, CD2^low/–and B220^high, similar to freshly isolated CD4^–CD8^–Ipr T cells. In contrast, if IL-2 is included in the cultures,a strong shift toward a CD2⁺ phenotype is observed by a majorityof the Ipr T cells. Upon repeat stimulation, these Ipr CD4^–CD8^–T cells can now proliferate in an IL-2-dependent manner whenstimulated with only anti-CD3 mAb or mitogens, in the absenceof exogenous IL-2 or anti-CD28 mAb. These data show that thehyporesponsiveness of Ipr CD4^–CD8^– T cells doesnot result from a lack of CD28 expression, that it is not afixed state, and that it can be reversed by the induction ofcell cycling in the presence of IL-2. These observations extendthe parallels between Ipr CD4^–CD8^– T cells and anergicT cells.     

The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function

Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.     

Human CD8 T cells of the peripheral blood contain a low CD8 expressing cytotoxic/effector subpopulation

Heterogeneity of lymphocyte populations demonstrates the diversity of cellular immune responses and provide a better understanding of the immune system. CD3+ CD8+ T cells exhibit a low CD8 expressing (CD8low) population in flow cytometric analysis of peripheral blood T cells. In healthy donors, this population consists of 0.2-7.0% of all CD8 T cells. The majority of the CD8low T cell population showed an elevated expression of CD25, CD45RA, and CD95L, and low levels of CD28, CD62L and CD45RO. Circulating CD8low T cells resemble cytotoxic effector cells because they express cytolytic mediators and are able to execute cytotoxicity. A restricted T cell receptor profile with increased Vbeta9, Vbeta14 and Vbeta23 expression was observed and the CD8low T cell population contain Epstein-Barr virus-specific T cells. Therefore, the CD8low population represent a subset of activated CD8 effector T cells, resulting most probably from a continuous and/or balanced immune response to intracellular pathogens.     

In vitro stimulation and expansion of human tumour-reactive CD8+ cytotoxic T lymphocytes by anti-CD3/CD28/CD137 magnetic beads

Adoptive immunotherapy with tumour-reactive CD8(+) cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen-independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour-reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4-1BB). T cells in PBMCs showed an increased expansion rate of 15- to 17-fold during a 2-week culture period using antibody-conjugated beads with interleukin-2 (IL-2) added versus IL-2 alone. No significant difference between CD3/CD28 beads and CD3/CD28/CD137 beads was observed (P = 0.4). In contrast, expansion of tumour-reactive CD8(+) CTLs over 2 weeks was more efficient using CD3/CD28/CD137 beads (14.4-fold ± 1.2) compared with CD3/CD28 beads (10.6-fold ± 0.7) (P = 0.03) and matched well to the control arm using weekly stimulation with tumour cells. Although all modes of in vitro stimulation decreased the expression of central memory markers CD62L and CCR7 on CTLs, bead-activated cultures expressed consistently higher levels than tumour-stimulated cultures. CTLs analysed after bead-induced expansion versus weekly tumour stimulation showed equal IFN-γ production in ELISPOT assay. Furthermore, cytotoxicity assays demonstrated an either unchanged or slightly reduced capability of tumour cell lysis for antigen-independent stimulated CTLs versus those that maintained on weekly tumour stimulation, regardless of which type of beads was used. Our data suggest that the conjugation of anti-CD137 antibodies to conventional CD3/CD28 beads results in a minor but significant increase in the expansion capacity for tumour-reactive CD8(+) CTLs.