Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4⁺ T cells. CD4⁺ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4⁺ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1^−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4⁺ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4⁺ T cell activation.     

Both CD8+ and CD16+ human T cell clones can provide B cell help for immunoglobulin production

Summary The functional properties of two CD4⁺CD8⁻CD16⁻, five CD4⁻CD8⁺CD16⁻ and three CD4⁻CD8⁻CD16⁺ human T cell clones were compared. All CD4⁻ T cell clones displayed strong cytolytic activity in the lectin-dependent lytic assay against the P815 murine mastocytoma cell line, but only the CD4⁻CD8⁻CD16⁺ T cell clones exhibited lytic activity against the natural killer-sensitive K562 cell line. Upon activation with anti-CD3 monoclonal antibody, all T cell clones were able to support IgM and IgA synthesis in autologous B cells. Both CD4⁺ and CD4⁻ T cell clones required cell-to-cell interaction with the B cells in order to exert their helper activity for immunoglobulin production. However, unlike CD4⁺, CD4⁻CD8⁺CD16⁻ and CD4⁻CD8⁻CD16⁺ T cell clones provided helper function for immunoglobulin synthesis only when low T/B cell ratios were used in culture. At higher T/B cell ratios, there was a decline in the B cell helper activity of CD4⁻ T cell clones that was probably related to the expression of cytolytic capacity against the antigen-presenting B cell. These data support the notion that under certain experimental conditions even cytotoxic T lymphocytes and natural killer cells may provide B cell helper function.     

Partial inefficiency of T cell receptors γ/δ composed of a heavy (55-kD) γ chain to mediate cell activation upon binding to specific monoclonal antibodies

Summary We analyzed CD8⁺ T cell receptor (TCR) γ/δ⁺ (δ-TCS-1 reactive) cell clones expressing the 55-kD γ chain for their susceptibility to triggering by monoclonal antibodies (mAbs) specific for TCR or CD3 molecules. Clones were derived by limiting dilution from CD3⁺, WT31⁻ FACS-purified peripheral blood populations or CD4⁻CD8⁻ thymocytes (a fraction of the latter cells expressingde novo CD8 surface antigen upon culture in IL-2). Clones were screened according to their reactivity with both anti-CD8 and δ-TCS-1 mAbs. Analysis of CD3-associated molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions which preserve the CD3/TCR association (1% digitonin) showed a predominant 55–60-kD molecule both under reducing and non-reducing conditions. All clones expressing the δ-TCS-1⁺ CD8⁺ surface phenotype derived from either thymus or peripheral blood lysed the Fcγ receptor-bearing P815 target cells in the presence of anti-CD3 mAb. On the other hand, δ-TCS-1 mAb was poorly efficient in triggering the lytic machinery of these clones, while it induced target cell lysis by δ-TCS-1⁺ CD8⁻ clones. This work was supported by grants from theConsiglio Nazionale delle Ricerche (CNR), Roma, Italy,Progetto Finalizzato ‘Oncologia’ to M. C. M. and A. M., and from theAssociazione Italiana per la Ricerca sul Cancro (AIRC).     

Thymic origin of some natural killer cells: clonal proliferation of human CD3−16+ cells from CD3−4−8− thymocyte precursors requires the presence of H9 leukemic cells

Summary Purified CD3⁻4⁻ thymocyte populations were cultured in the presenceof interleukin-2 (IL-2) and peripheral blood lymphocytes (PBL) and/or tumor cell lines as a source of irradiated feeder cells. Maximal cell proliferation was obtained in the presence of a mixture of H9 leukemic cells and normal PBL. More importantly, under these culture conditions 30%–50% of these cells were found to express CD16 surface antigen after 1–2 weeks of culture. Similar proportions of CD16⁺ cells could be detected in CD3⁻4⁻ thymocyte populations that had been further depleted of CD16⁺ cells. Cloning of CD3⁻4⁻16⁻ thymocytes under limiting dilution conditions resulted, in the presence of H9 cells, in more than 50% of CD16⁺ clones (cloning efficiency 3%–8%). Since some of the surface CD3⁻ clones expressed cytoplasmic CD3 antigen, it has been possible to identify four distinct phenotypic groups of clones (CD16⁺ cyCD3⁺, CD16⁺ cyCD3⁻, CD16⁻ cyCD3⁺, CD16⁻ cyCD3⁻). Independently of their phenotype, all thymus-derived CD3⁻ clones expressed a strong cytolytic activity against natural killer (NK)-sensitive and NK-resistant tumour target cells. In addition, following stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (or PHA alone) all clones released interferon-γ and tumour necrosis factor-α, but not IL-2. Taken together, our data provide evidence that cells which share their phenotypic and functional properties with CD3⁻ CD16⁺ NK cells can be derived from thymic precursors.     

Interleukin-2-Induced proliferation of CD4-CD8- human thymocytes.In vitro expression of CD3 and CD8 antigens and cytolytic activity

Human thymocytes lacking both CD4 and CD8 differentiation antigens were prepared by treating total thymocyte suspensions with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies and complement. The resulting populations contained less than 2% CD4⁺, CD8⁺ or WT31⁺ cells and variable percentages (less than 20%) of CD3⁺ cells. These cell populations were cultured in recombinant IL-2 in the presence of peripheral blood mononuclear cells as feeder cells. Cells underwent extensive proliferation accompanied by a progressive increase of CD3⁺ and CD8⁺ cells. On the other hand, appearance of neither WT31⁺, α/β-positive T cell receptor (TCR), nor CD4⁺ cells could be observed in several independent experiments. Functional analyses revealed the appearance and the progressive increase of cytolytic activity against the natural killer (NK)-sensitive K562 cells as well as the NK-resistant fresh melanoma cells. Experiments of T cell cloning indicated that both the expression of CD8 and CD3 antigens and the appearance of cytolytic activity were consequent to cell maturation occurring at the level of CD4^-CD8^- non-cytolytic cell precursors. In these experiments, more than 30% of cells underwent clonal expansion and all the clonal progenies obtained displayed cytolytic activity and expressed the CD3⁺WT31^- surface phenotype. The expression of CD8 was variable, whereas no CD4⁺ clones could be obtained. Cells expressing such surface phenotype are known to belong to the TCR γ-positive T lymphocyte subset lacking the typical α/β TCR and thus appear to be the only T cell type capable ofin vitro proliferation and maturation under easily reproducible culture conditions. This work was supported by grants awarded by the Consiglio Nazionale delle Ricerche (CNR), Roma, Italy, Progetto Finalizzato ‘Oncologia’ to M. C. M. and L. M. A fellowship by Associazione Italiana per la Ricerca sul Cancro (AIRC) was awarded to A. M.     

A novel and efficient method to induce allospecific CD8+ memory T lymphocytes

The aim of the current study was to establish a simple method for effectively inducing memory T lymphocytes by the intraperitoneal injection of spleen lymphocytes into mice. In total, 75 mice were divided into the following groups: an injection group administered three doses of spleen lymphocytes (1 × 10⁶, 5 × 10⁶, and 1 × 10⁷ cells), a transplantation group in which a 0.25‐cm² skin section from C57BL/6 mice was transplanted onto the back of the recipient, and a control group in which an equal volume of phosphate‐buffered saline was injected. At 1, 2, or 3 months following transplantation, the following parameters were evaluated: quantity of T lymphocytes, percentage of cluster of differentiation 8⁺ (CD8⁺) memory T cells, and proliferation index of purified CD8⁺ memory T cells. No significant differences among groups were detected at 1 month (p > .05). However, the injection group administered 1 × 10⁶ cells exhibited the highest proportion of CD8⁺ memory T cells among all groups at 2 months, and the proportions of CD8⁺ T cells were higher in the three injection groups than in the skin transplantation and control groups at 3 months. The proportions of memory T cells were higher in the injection groups administered 5 × 10⁶ or 1 × 10⁷ cells than in the skin transplantation and control groups at 3 months. The newly established method effectively induces memory T lymphocytes via the intraperitoneal injection of spleen lymphocytes in vivo and has potential applications in the field of immunotherapy.     

Temporal and spatial characterization of negative regulatory T cells in HIV‐infected/AIDS patients raises new diagnostic markers and therapeutic strategies

BackgroundNegative regulatory T cells (Tregs) not only deplete effector T cells but also inhibit the clearance of HIV during infection, which may allow Tregs to be used as informative diagnostic markers. To facilitate both diagnosis and treatment, a thorough understanding of these regulators by characterizing them on temporal and spatial scales is strongly required.MethodsHundred HIV‐infected/AIDS patients, including 87 males, with an average age of 35.8 years, as well as 20 healthy controls, were enrolled. Flow cytometry was used to analyze CD3+T cells, CD4+T cells, and CD8+T cells to evaluate the immune status of the participants. Then, a group of representative negative regulatory T cells, including CD4+PD‐1+T cells, CD4+PD‐1^highT cells, CD8+PD‐1+T cells, and CD4+CD25^high Tregs was also analyzed to explore their effects on disease progression and intercorrelation.ResultsThe percentages of CD4⁺PD‐1⁺T cells and CD4⁺CD25^highTregs increased in patients with the same ultrahigh significance. Temporally, the patients with both intermediate‐stage and late‐stage disease had higher percentages of CD4⁺PD‐1⁺T cells; however, the percentage of CD4⁺CD25^highTregs only increased in the patients with late‐stage disease. In addition, CD4⁺PD‐1⁺T cells but not CD4⁺CD25^highTregs were negatively correlated with the absolute CD4⁺T cell count. Spatially, no correlations between CD4⁺PD‐1⁺T cells and CD4⁺CD25^highTregs were observed, which suggests these Tregs function differently during immunosuppression.ConclusionsThis study characterized negative regulatory T cells in HIV‐infected/AIDS patients at both temporal and spatial scales and found that CD4⁺CD25⁺Tregs and CD4⁺PD‐1⁺T cells could be used as potential diagnostic markers for identifying different disease stages and monitoring disease progression.     

CD4+ T Cells Prevent Spontaneous Experimental Autoimmune Encephalomyelitis in Anti–Myelin Basic Protein T Cell Receptor Transgenic Mice

Autoimmune diseases result from a failure of tolerance. Although many self-reactive T cells are present in animals and humans, their activation appears to be prevented normally by regulatory T cells. In this study, we show that regulatory CD4⁺ T cells do protect mice against the spontaneous occurrence of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Anti–myelin basic protein (MBP) TCR transgenic mice (T/R⁺) do not spontaneously develop EAE although many self-reactive T cells are present in their thymi and peripheral lymphoid organs. However, the disease develops in all crosses of T/R⁺ mice with recombination-activating gene (RAG)-1 knockout mice in which transgenic TCR-expressing cells are the only lymphocytes present (T/R⁻ mice). In this study, crosses of T/R⁺ mice with mice deficient for B cells, CD8⁺ T cells, NK1.1 CD4⁺ T (NKT) cells, γ/δ T cells, or α/β T cells indicated that α/β CD4⁺ T cells were the only cell population capable of controlling the self-reactive T cells. To confirm the protective role of CD4⁺ T cells, we performed adoptive transfer experiments. CD4⁺ T cells purified from thymi or lymph nodes of normal mice prevented the occurrence of spontaneous EAE in T/R⁻ mice. To achieve full protection, the cells had to be transferred before the recipient mice manifested any symptoms of the disease. Transfer of CD4⁺ T cells after the appearance of symptoms of EAE had no protective effect. These results indicate that at least some CD4⁺ T cells have a regulatory function that prevent the activation of self-reactive T cells.     

Permanent CD8+ T Cell Depletion Prevents Proteinuria in Active Heymann Nephritis

Active Heymann nephritis (HN) is a rat model of human idiopathic membranous nephropathy in which injury is thought to be mediated by membrane attack complex of complement (MAC) activated by antibody (Ab) to glomerular epithelial cells. Recent work has shown that HN develops in C6-deficient rats which cannot assemble MAC, and that infiltration of activated cytotoxic CD8⁺ T cells and macrophages into glomeruli coincides with proteinuria. This study examined the role of CD8⁺ T cells in mediating glomerular injury in HN by permanent CD8⁺ cytotoxic T cell depletion via adult thymectomy (ATx) and anti-CD8 mAb. Groups of rats were depleted of CD8⁺ T cells either before immunization for HN or 6 wk after immunization when Ab responses and glomerular IgG deposition were well established. These were compared with groups of HN, ATx/HN, and complete Freund''s adjuvant (CFA) controls. Neither group of CD8⁺ T cell–depleted rats developed proteinuria, although there was normal development and deposition of Ab. CD8⁺ T cell–depleted rats developed neither T cell or macrophage infiltrates nor their effector cytokines, which are present in glomeruli of rats with HN. Examination of lymph node (LN) draining sites of immunization showed these findings were not explained by altered immune events within these LNs. It was concluded that CD8⁺ cytotoxic T cells are essential to the mediation of glomerular injury in HN and may be relevant to the pathogenesis and treatment of membranous nephropathy.     

An Essential Role for Interleukin 10 in the Function of Regulatory T Cells That Inhibit Intestinal Inflammation

A T helper cell type 1–mediated colitis develops in severe combined immunodeficient mice after transfer of CD45RB^high CD4⁺ T cells and can be prevented by cotransfer of the CD45RB^low subset. The immune-suppressive activities of the CD45RB^low T cell population can be reversed in vivo by administration of an anti-transforming growth factor β antibody. Here we show that interleukin (IL)-10 is an essential mediator of the regulatory functions of the CD45RB^low population. This population isolated from IL-10–deficient (IL-10^−/−) mice was unable to protect from colitis and when transferred alone to immune-deficient recipients induced colitis. Treatment with an anti–murine IL-10 receptor monoclonal antibody abrogated inhibition of colitis mediated by wild-type (WT) CD45RB^low CD4⁺ cells, suggesting that IL-10 was necessary for the effector function of the regulatory T cell population. Inhibition of colitis by WT regulatory T cells was not dependent on IL-10 production by progeny of the CD45RB^high CD4⁺ cells, as CD45RB^low CD4⁺ cells from WT mice were able to inhibit colitis induced by IL-10^−/− CD45RB^high CD4⁺ cells. These findings provide the first clear evidence that IL-10 plays a nonredundant role in the functioning of regulatory T cells that control inflammatory responses towards intestinal antigens.     

Antigen-independent pathways of T-cell activation are functional in human immature thymocytes

Summary The signal requirements for proliferation of CD1⁺CD3⁻ immature thymocytes have been studied in order to define whether this immature cell population could function despite the lack of the CD3/T-cell receptor complex. We found that CD1⁺CD3⁻ cells proliferate upon stimulation with anti-CD28 monoclonal antibody as well as with a pair of anti-CD2 monoclonal antibodies in the presence of low doses (0.5 ng/ml) of phorbol-13-myristate-12-acetate and/or recombinant interleukin-2. A minor fraction of CD3⁺ cells (15%–20%) was also present in the proliferating cell population originating from CD1⁺CD3⁻ thymocytes stimulated with phorbol-13-myristate-12-acetate and recombinant interleukin-2, either in the presence or in the absence of specific monoclonal antibodies. We further observed that the anti-CD3 monoclonal antibody did not induce the proliferation of CD1⁺CD3⁻ cells, as expected, and efficiently triggered unfractionated or CD1⁺CD3⁺ thymocytes only if exogeneous recombinant interleukin-2 was provided. Unexpectedly, we noted that highly purified (>99%), CD1⁺CD3⁻ immature thymocytes could mobilize calcium via CD3, besides CD2 and CD28 surface molecules, suggesting that a minor undetectable fraction (<1%) of CD3⁺ cells was still present in the purified CD3⁻ population. Nevertheless, the preferential expansion of CD3⁻CD8⁺ cells (about one-third of proliferating cells) after triggering via CD28, and to a lesser extent via CD2, support the notion that the alternative pathways of T-cell activation are actually functional in CD1⁺CD3⁻ immature thymocytes.     

The Peripheral Deletion of Autoreactive CD8+ T Cells Induced by Cross-presentation of Self-antigens Involves Signaling through CD95 (Fas,Apo-1)

Recently, we demonstrated that major histocompatibility complex class I–restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8⁺ T cells. In these studies, naive ovalbumin (OVA)-specific CD8⁺ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8⁺ T cells in the whole animal.     

CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4⁺CD25⁺ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4⁺ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4⁺CD25⁺ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4⁺CD25⁻ cells, the CD4⁺CD25⁺ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4⁺CD25⁺ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25⁻ T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4⁺CD25⁺ T cells in normal mice may represent a distinct lineage of “professional” suppressor cells.     

Functional Fas-ligand expression on T cells from HIV-1-infected patients is unrelated to CD4+ lymphopenia

Recent studies have demonstrated that the expression of Fas by peripheral T cells from HIV-1⁺ patients is deregulated and increases the susceptibility of these cells to undergo apoptosis. Here, we show that secretion of Fas-ligand (L), the complementary agonist of Fas, is abnormally upregulated in CD4⁺ cells from HIV-1-infected individuals, particularly during the non-lymphopenic stages of the disease. An increase of soluble Fas-L occurred in T cell cultures from 26 patients with a number of CD4⁺ cells higher than 400/μl, whereas it was almost undetectable in cultures from 21 severely lymphopenic patients (CD4⁺ <200/μl). The MTT test, cytofluorimetric analysis of cellular DNA, cytotoxicity, and proliferative assays using the Fas-transfected WC8 mouse lymphoma confirmed the cytocidal capability of T cell supernatants from non-lymphopenic patients. Double-fluorescence analysis revealed that the majority of CD4⁺ cells (approximately 90%) in these cultures secreted Fas-L in the presence of high intracellular γ-interferon and low Bcl-2. In contrast, the CD8⁺/Fas-L⁺ population was comparably decreased (approximately 55%). Molecular cloning of Fas-L revealed a substantial expression of Fas-L mRNA in cells from non-lymphopenic patients compared with patients with advanced disease and healthy controls. Since CD4⁺ cells of Th1 phenotype are impaired during HIV-1 infection and show high cellular expression of Fas-L, it is conceivable that excess Fas-L during the early or non-lymphopenic phase of the disease increases the extent of apoptosis in these cells by the Fas/Fas-L pathway. The defective expression of the ligand in severely lymphopenic stages could be explained by exhaustion of this mechanism as the disease progresses.     

B Cells Directly Tolerize CD8+ T Cells

This report investigates the response of CD8⁺ T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the K^b-restricted ovalbumin (OVA) determinant OVA_257–264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro–activated CD8⁺ T cells from the K^b-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8⁺ T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.     

新型共刺激分子CD137抗白血病作用的体外研究

本研究探讨新型共刺激分子CD137（4—1BB）在人T淋巴细胞上的表达特点及其单克隆抗体hCD137mAb在刺激T淋巴细胞增殖、促进细胞因子分泌、增强细胞杀伤作用的抗白血病功能。应用FACS及间接免疫荧光法分别检测正常人T淋巴细胞上加入植物血凝素（PHA）前后不同时相CD137的表达。在HL-60细胞和T淋巴细胞体外培养体系中,用MTT比色法测定hCD137mAb、PHA对T淋巴细胞增殖作用的影响,用FACS及间接免疫荧光法检测其对T细胞表面上IFN-1和IL-4分泌水平的影响。在体外混合淋巴细胞肿瘤细胞培养（MLTC）体系中,研究不同效靶比例时hCD137mAb增强T细胞杀伤白血病细胞的抗白血病作用。结果表明：①T细胞未经PHA刺激几乎不表达hCD137,经PHA活化后开始表达,且随着时间延长表达率逐渐增高,第7天为高峰（FACS法为56．4％±1．98％,间接免疫荧光法为52．8％±2．01％）。②MTT法检测提示,单独使用hCD137mAb不能刺激T细胞增殖（增殖指数1．002±0．011）,但其与PHA伍用可增强后者刺激活性（增殖指数2．161±0．102）约2倍（增殖指数4．705±0．133）。⑧在PHA存在时,hcD137mAb可使IFN-γ在T细胞上表达增加到大约2倍,而对IL-4几无影响。④hCD137mAb明显增强T细胞对白血病细胞株HL-60的杀伤活性,且共刺激活性在40：1效靶比时最强,杀伤百分比增加到大约2倍。结论：新型共刺激分子CD137具有显著的抗白血病作用,应用hCD137mAb是一种有效排斥白血病的免疫策略,且安全、简便,本研究为其用于临床免疫治疗提供了实验依据。     

CD8+ T-cell counts: an early predictor of risk and mortality in critically ill immunocompromised patients with invasive pulmonary aspergillosis

Introduction

Critically ill immunocompromised (CIIC) patients with pulmonary infection are a population at high risk for invasive pulmonary aspergillosis (IPA). The host defenses are important factors to consider in determining the risk and outcome of infection. Quantification of changes in the status of host immunity could be valuable for clinical diagnosis and outcome prediction.

Methods

We evaluated the quantitative changes in key humoral and cellular parameters in CIIC patients with pulmonary infection and their potential influence on the risk and prognosis of IPA. We monitored the evolution of these parameters in 150 CIIC patients with pulmonary infection on days 1, 3 and 10 (D1, D3 and D10) following ICU admission. The primary outcome was 28-day mortality. Follow-up included 60- and 90-day mortality.

Results

Among the 150 CIIC patients included in this study, 62 (41.3%) had microbiological evidence of IPA. Compared with patients without IPA, CD3⁺, CD8⁺, CD28⁺CD4⁺ and CD28⁺CD8⁺ CD28⁺CD8⁺ T-cell counts (D1, D3 and D10) and B-cell counts (D1 and D3) were significantly reduced in patients with IPA (P < 0.05). Multivariate regression analysis revealed that CD8⁺ (D3 and D10) (odds ratio (OR) 0.34, 95% confidence interval (CI) 0.23 to 0.46; OR 0.68, 95% CI 0.56 to 0.80), CD28⁺CD8⁺ (D3) (OR 0.73, 95% CI 0.61 to 0.86) and CD3⁺ (D10) (OR 0.81, 95% CI 0.63 to 0.98) T-cell counts were independent predictors of IPA in CIIC patients. Receiver operating characteristic analysis of immune parameters predicting 28-day mortality revealed area under the curve values of 0.82 (95% CI 0.71 to 0.92), 0.94 (95% CI 0.87 to 0.99), and 0.94 (95% CI 0.85 to 0.99) for CD8⁺ T-cell counts (D1, D3 and D10, respectively) and 0.84 (95% CI 0.75 to 0.94), 0.92 (95% CI 0.85 to 0.99) and 0.90 (95% CI 0.79 to 0.99) for CD28⁺CD8⁺ T-cell counts (D1, D3 and D10, respectively). Kaplan-Meier survival analysis provided evidence that CD8⁺ and CD28⁺CD8⁺ T-cell counts (<149.5 cells/mm³ and <75 cells/mm³, respectively) were associated with early mortality in CIIC patients with IPA (logrank test; P < 0.001).

Conclusions

CD8⁺ and CD28⁺CD8⁺ T-cell counts were significantly lower in CIIC patients with IPA than in non-IPA patients. Lower CD8⁺ and CD28⁺CD8⁺ T-cell counts in CIIC patients with pulmonary infection were associated with higher risk and early mortality in IPA and may be valuable for clinical diagnosis and outcome prediction.     

CD133+ cells isolated from various sources and their role in future clinical perspectives

Background. CD133 is a member of a novel family of cell surface glycoproteins. Initially, the expression of CD133 antigen was seen only in the hematopoietic derived CD34⁺ stem cells. At present, CD133 expression is demonstrated in undifferentiated epithelium, different types of tumors and myogenic cells. CD133⁺ neurosphere cells isolated from brain are able to differentiate into both neurons and glial cells. These data suggested that CD133 could be a specific marker for various stem and progenitor cell populations.

Objectives. The main goal would be to describe the role for CD133 as a marker of stem cells able to engraft and differentiate, to form functional non-hematopoietic adult lineages and contribute to disease amelioration via tissue regeneration.

Results/conclusion. In conclusion, since the rise of CD133 antigen as a suitable stem cell marker, the possible use of CD133⁺ stem cells in therapeutic applications has opened a new promising field in the treatment of degenerating diseases. The human circulating cells expressing the CD133 antigen behave as a stem cell population capable of commitment to hematopoietic, endothelial and myogenic lineages. CD133 cell therapy may represent a promising treatment for many diseases.     

Distinct Methylation of the Interferon γ (IFN-γ) and Interleukin 3 (IL-3) Genes in Newly Activated Primary CD8+ T Lymphocytes: Regional IFN-γ Promoter Demethylation and mRNA Expression Are Heritable in CD44highCD8+ T Cells

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-γ and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8⁺ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-γ and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-γ promoter was widespread across >300 basepairs in clones expressing high levels of IFN-γ mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-γ mRNA exhibited symmetrical methylation of four to six of the IFN-γ promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-γ gene methylation and mRNA expression in families of clones derived from resting CD44^lowCD8⁺ T cells or from previously activated CD44^highCD8⁺ T cells. The patterns of IFN-γ gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44^high cells, but variable in clones derived from CD44^low cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.