Divergent telomerase and CD28 expression patterns in human CD4 and CD8 T cells following repeated encounters with the same antigenic stimulus

Induction of telomerase, the enzyme that extends telomeres, accompanies human T lymphocyte activation. Nevertheless, high proportions of memory T cells with shortened telomeres are present in vivo during HIV infection and aging. To elucidate the long-term telomerase dynamics in human T cells, longitudinal analyses were performed on T cells subjected to repeated encounters with an allogeneic cell line in long-term culture. Whereas CD4(+) and CD8(+) T cells showed similarly dramatic increases in telomerase activity following primary stimulation, by the fourth stimulation, telomerase activity was nearly undetectable in the CD8(+) subset, but remained high in the CD4(+) subset. In addition, we document the dependence of antigen-specific telomerase inducibility on CD28 and that the decline in telomerase activity parallels the loss of CD28 expression. These findings suggest stringent telomerase regulation in human T cells, a property that may ultimately contribute to telomere shortening, finite replicative potential, and loss of control over certain pathogens.     

CD8hi+CD57+ T lymphocytes are enriched in antigen-specific T cells capable of down-modulating cytotoxic activity

Major expansions of CD8hi+CD57+ T lymphocytes frequently occur during human immunodeficiency virus (HIV) infection and after transplantation. To investigate mechanisms of such cell expansion, we compared the activation and functional status of CD8hi+CD57+ and CD57-peripheral blood lymphocytes (PBL) from normal, bone marrow transplantation (BMT) and HIV+ donors. The CD8hi+CD57+ PBL from BMT and HIV+ donors preferentially displayed CD38 and HLA-DR activation markers without correlation between CD8hi+CD57+ percentages and HIV load, the CD45RA+ isoform in all ex vivo conditions but acquired CD45RO after in vitro expansion, CD11b and CD11c in BMT and HIV+ donors but decreased expression of CD62-L, VLA-2 and VLA-6. The CD8hi+CD57+ cells were positive for perforin and granzyme B and spontaneously mediated cytolytic activity in a CD3-redirected assay. In contrast the inhibitor of cytolytic functions (ICF) produced by CD8hi+CD57+ cells down- modulated the CD3-redirected cytolytic activity but only at low levels of CD3 cross-linking. While CD3-triggering induced a low, if any, short- term proliferation of CD8+CD57+ cells, this subset could be amplified after long-term stimulation either with mitogens or with HIV antigens, thereby enriched in HIV-specific T cells producing tumor necrosis factor-alpha. Altogether these data suggest that CD8hi+CD57+ cells represent a terminal differentiation state of activated effector cytotoxic T lymphocytes which are enriched in antigen-specific T cells and down-modulate their own cytolytic potential, thus participating in a negative control of effector cell functions during persistent viral infections or transplantations.      

A subset of CD8 memory T cells from old mice have high levels of CD28 and produce IFN-gamma

Using carboxyfluorescein diacetate succinimidyl ester (CFSE)-tagged cells to measure proliferation in vivo, we found that only memory CD8(+) cells from mice older than 18 months gave measurable levels of proliferation and that the proportion of memory CD8(+) T cells able to proliferate in a nonirradiated recipient increased with age. CD8 cells that had proliferated in vivo contained higher levels of CD28 when compared to CD8 cells that had not divided. Cells with high levels of CD28 were preferentially able to divide in nonirradiated recipients. Using ex vivo intracellular staining analysis, we determined that most of the CD8(+) T cells that were capable of dividing in vivo produced IFN-gamma after isolation from recipient mice or their original host. These studies thus document the presence in aged mice of a population of CD28(hi) CD8(+) cells whose ability to proliferate in vivo without antigenic stimulation and to produce IFN-gamma may be involved in immune regulation.     

间充质干细胞对混合淋巴细胞反应体系中T淋巴细胞的免疫调节作用

目的:探讨间充质干细胞(MSC)在体外对T淋巴细胞免疫调节作用的特点。方法:通过Ficoll梯度密度离心法分离出正常人骨髓单个核细胞,体外培养扩增MSC,获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养(MLC)体系中,在第3、5天,采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况,再用流式细胞术分析其与MSC共孵育前后细胞表面标记的变化情况。结果:加入了MSC的MLC体系中,MSC对T淋巴细胞的增殖抑制具有剂量依赖性,且随着时间延长,抑制程度增强;其中,CD4 T细胞亚群受抑不如CD8 T细胞亚群显著;另外,T淋巴细胞表面CD25的表达虽然较对照组有所下降,但是CD4、CD25共表达的细胞却较对照组有明显的上升趋势。T淋巴细胞表面活化抗原HLA-DR的表达较对照组有轻度减低。结论:MSC在体外能够明显抑制T淋巴细胞的增殖,其主要是针对CD8 T细胞(CTL)。另外,它还能下调活化T淋巴细胞上一些较特异的表面标记CD25和HLA-DR的表达。     

Age-associated deficiency in activation-induced up-regulation of telomerase activity in CD4+ T cells

For lymphocytes, the ability to undergo clonal expansion is crucial for effective immune function. Telomerase activity compensates for telomere erosion during cell division and contributes to the capability of lymphocytes to maintain cellular proliferation. In addition, telomerase activity may have a fundamental role in cell growth and survival. To determine whether age-related immune dysfunction is associated with an abnormality in telomerase activity, we investigated telomerase activity in T cell populations from young adult and aged mice. Our data show that the ability of T cells from aged mice to up-regulate telomerase activity after activation was significantly diminished. This age-related deficiency in telomerase induction is restricted to CD4(+) T cells, as CD8(+) T cells retain the capability to up-regulate telomerase activity. These findings reinforce the notion that age-related immune dysfunction results mainly from impairment of helper T cells, and may have important implications for designing novel means to improve immune responses in aged individuals by enhancing CD8(+) T cell functions, which are crucial in both viral and tumour immunity.     

A subset of functional effector-memory CD8+ T lymphocytes in human immunodeficiency virus-infected patients

CD8(+) T cells provide protective immune responses via both cytolytic and non-cytolytic mechanisms in subjects infected with human immunodeficiency virus (HIV). In the present study, we investigated the CD28 expression of CD8(+) T cells present in the peripheral blood lymphocyte subset isolated from chronically HIV-infected subjects. Using flow cytometric analysis, a continuous spectrum of CD28 intensity ranging from negative to high, which could be separated into CD28-negative, intermediate (int) and high, was seen for CD8(+) T cells. Our study focused mostly on the CD28(int) CD8(+) T cells. The CD28(int) CD8(+) T cells are CD57(-) CD27(+) CD45RO(+) CD45RA(-) CCR7(low) CD62L(int). The proliferative capacity of CD28(int) CD8(+) T cells was intermediate between those of CD28(-) CD8(+) T cells and CD28(high) CD8(+) T cells. The CD28(int) CD8(+) T cells are specific for HIV, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) antigens as measured by human leucocyte antigen pentamer binding and produce both intracellular interferon-gamma and tumour necrosis factor-alpha in response to their cognate viral peptides. The CD28(int) CD8(+) T cells have HIV-specific, CMV-specific and EBV-specific cytotoxic activity in response to their cognate viral peptides. These findings indicate that a subset of functional effector-memory CD8(+) T cells specific for HIV, CMV and EBV antigens may contribute to an efficient immune response in HIV-infected subjects.     

Regulatory T-cell expansion and function do not account for the impaired alloreactivity of ex vivo-expanded T cells

CD3- and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens. To investigate whether this impaired alloreactivity is a result of preferential expansion of regulatory T (Treg) cells, we compared peripheral blood mononuclear cells (PBMC) activated with CD3 and CD28 antibodies co-immobilized on beads and expanded for 12 days with interleukin (IL)-2 (Co(CD3/CD28) cells) to the respective unactivated PBMC in terms of proliferation, cytokine production, and expression of Treg markers [cytotoxic T-lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor (GITR) and forkhead box P3 (FoxP3)] after allostimulation. Alloreactive cells were identified by carboxyfluoresceine succinimidyl ester staining dilution. Alloreactive cells in Co(CD3/CD28) cells had a lower proliferative response and a lower potential for IL-2 and interferon-gamma secretion than did those in PBMC, demonstrating a functional impairment of alloreactive cells during ex vivo expansion. Expression of Treg markers transiently increased during ex vivo expansion and was unaffected by depletion of CD25(+) cells (containing Treg cells) before ex vivo PBMC expansion. Such prior CD25(+) depletion did not restore the alloreactivity of Co(CD3/CD28) cells. After allostimulation, expression of Treg markers was restricted to proliferative (alloreactive) cells among PBMC or Co(CD3/CD28) cells. Lastly, CD4(+) CD25(+) cells purified from Co(CD3/CD28) cells lacked suppressive activity when used as a third party, in contrast to CD4(+) CD25(+) cells purified from PBMC. In conclusion, the impaired alloreactivity of T cells expanded ex vivo is not a result of preferential Treg cell expansion and/or enhanced suppressive Treg activity.     

In vivo acute depletion of CD8(+) T cells before murine cytomegalovirus infection upregulated innate antiviral activity of natural killer cells

In this study, we investigated the effect of depletion of CD8(+) T cells on the activity of natural killer (NK) cells at an early phase of murine cytomegalovirus (MCMV) infection. For CD8(+) T cell depletion, mice were intraperitoneally treated with anti-CD8 mAb, purified from 2.43 hybridoma, for 2 consecutive days before or after infection. Three days after infection, we found that an acute depletion of CD8(+) T cells before infection caused a significant decrease in the viral load in liver and spleen. This effect coincided with an increase in numbers of CD3(-) NK1.1(+) cells in spleen and their expression of the early activation molecule CD69. Although cytolytic activity of NK cells increased on day 3 of infection in CD8-depleted mice, the level of IFN-gamma decreased in serum and supernatant of cultured spleen cells. In contrast to the effect of acute depletion of CD8(+) T cells before infection, the depletion after infection had no effect on the viral load or number and cytolytic function of NK cells. Lack of effects of CD8(+) T cell depletion on the viral load and NK cytolytic activity is also observed in CD8(+) knockout mice. In conclusion, the results suggest that an acute depletion of CD8(+) T cells before MCMV infection effectively upregulated the antiviral activity of NK cells. This effect appears to be mediated through an increase in numbers, activation and cytolytic activity of NK cells.     

Reversal of functional defects in highly differentiated young and old CD8 T cells by PDL blockade

Highly differentiated CD8(+) CD28(-) CD27(-) T cells have short telomeres, defective telomerase activity and reduced capacity for proliferation. In addition, these cells express increased levels of inhibitory receptors and display defective Akt(ser(473)) phosphorylation following activation. It is not known whether signalling via programmed death 1 (PD-1) contributes to any of the attenuated differentiation-related functional changes in CD8(+) T cells. To address this we blocked PD-1 signalling during T-cell receptor (TCR) activation using antibodies against PD-1 ligand 1 (PDL1) and PDL2. This resulted in a significant enhancement of Akt(ser(473)) phosphorylation and TCR-induced proliferative activity of highly differentiated CD8(+) CD28(-) CD27(-) T cells. In contrast, the reduced telomerase activity in these cells was not altered by blockade of PDL1/2. We also demonstrate that PD-1 signalling can inhibit the proliferative response in primary human CD8(+) T cells from both young and older humans. These data collectively highlight that some, but not all, functional changes that arise during progressive T-cell differentiation and during ageing are maintained actively by inhibitory receptor signalling.     

Age-related changes in the occurrence and characteristics of thymic CD4(+) CD25(+) T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.     

A clonal model for human CD8+ regulatory T cells: unrestricted contact-dependent killing of activated CD4+ T cells

Previous studies in murine systems have demonstrated that CD8(+) Treg cells down-regulate immune responses in vivo through suppressing activated CD4(+) T cells. Here we describe novel regulatory CD8(+) T-cell clones isolated from healthy human peripheral blood following in vitro stimulation with autologous Epstein-Barr virus (EBV)-specific CD4(+) T cells. TCR activation of CD4(+) target T cells was required for CD8(+) Treg cells to exert suppressive activity, which was mediated through lysis of CD4(+) targets in a cell contact-dependent manner. Suppression was independent of Foxp3 expression in CD8(+) Treg cells, HLA compatibility between CD8(+) Treg cells and CD4(+) target cells and antigen-specificity of CD4(+) target T cells. CD8(+) Treg clones expressed CD3 and a variety of TCR V(β) chains as well as CD56, CD69, CD62L and CD95 but did not express CD16, CD161, CXCR4 and CCR7. When used together, antibodies specific for CD11a/CD18 and CD8 inhibited suppressive activity of CD8(+) Treg clones. The ability to establish clonal CD8(+) T cells that maintain regulatory function in vitro will facilitate further studies to define this population in vivo and to identify the mechanisms used for recognition and suppression of activated target cells.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist

CD4(+)CD25(+) T cells play a central role in the suppression of autoimmunity and inflammation, making their in vivo expansion a highly attractive therapeutic target. By phenotyping with a novel rat CTL antigen-4 (CTLA-4)-specific monoclonal antibody (mAb) and functional in vitro assays, we here first establish that rat CD4(+)CD25(+) T cells correspond to the regulatory T cells (Treg cells) described in mice and humans: they constitutively express CTLA-4, produce IL-10 but not IL-2, and are able to suppress the proliferation of costimulated CD25-negative indicator cells. Furthermore, we show that rat Treg cells respond less well than CD25(-) T cells to conventional costimulation, but are readily expanded in vitro with "superagonistic" CD28-specific mAb which are potent mitogens for all T cells without the need for TCR engagement. In vivo, functional Treg cells are preferentially expanded by CD28 stimulation over other T cell subsets, leading to a 20-fold increase within 3 days in response to a single antibody dose. These data suggest that CD28-driven activation of Treg cells may be highly effective in the treatment of inflammatory and autoimmune diseases.     

Naturally anergic and suppressive CD25(+)CD4(+) T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation

A CD4(+) T cell subpopulation defined by the expression levels of a particular cell surface molecule (e.g. CD5, CD45RB, CD25, CD62L or CD38) bears an autoimmune-preventive activity in various animal models. Here we show that the expression of CD25 is highly specific, when compared with other molecules, in delineating the autoimmune-preventive immunoregulatory CD4(+) T cell population. Furthermore, although CD25 is an activation marker for T cells, the following findings indicate that immunoregulatory CD25(+)CD4(+) T cells are functionally distinct from activated or anergy-induced T cells derived from CD25(-)CD4(+) T cells. First, the former are autoimmune-preventive in vivo, naturally unresponsive (anergic) to TCR stimulation in vitro and, upon TCR stimulation, able to suppress the activation/proliferation of other T cells, whereas the latter scarcely exhibit the in vivo autoimmune-preventive activity or the in vitro suppressive activity. Second, such activated or anergy-induced CD25(-) spleen cells produce various autoimmune diseases when transferred to syngeneic athymic nude mice, whereas similarly treated normal spleen cells, which include CD25(+)CD4(+) T cells, do not. Third, upon polyclonal T cell stimulation, CD25(+)CD4(+) T cells express CD25 at higher levels and more persistently than CD25(-)CD4(+) T cell-derived activated T cells; moreover, when the stimulation is ceased, the former revert to the original levels of CD25 expression, whereas the latter lose the expression. These results collectively indicate that naturally anergic and suppressive CD25(+)CD4(+) T cells present in normal naive mice are functionally and phenotypically stable, distinct from other T cells, and play a key role in maintaining immunologic self-tolerance.     

The CD28/HLA-DR expressions on CD4+T but not CD8+T cells are significant predictors for progression to AIDS

To investigate the changes of CD28 and HLA-DR molecules on CD4+ and CD8+ T cells during HIV infection, we classified 130 HIV-infected Koreans into four groups by the CD4 level as follows: group I (> or = 500 cells/mm3), group II (201-499 cells/mm3), group III (51-200 cells/mm3), and group IV (< or = 50 cells/mm3). In CD4+ T cells, the proportion of CD28 expression decreased significantly with the CD4 level while the proportion of HLA-DR expression increased gradually. In particular, the changes of HLA-DR expressions on CD4+ T cells were parallel to the loss of CD28 molecules from stage III to IV. However, the CD28 expression on CD8+ T cells decreased dramatically in the early stage of HIV infection, and the sum and pattern of CD28 and HLA-DR expressions on CD8+ T cells was stable after the first stage. Even though CD28 down-regulation on CD8+ T cells was very severe from the early stage of HIV infection, it might not influence the survival time of HIV-infected Koreans. The sum of the CD28+ subsets and HLA-DR subsets in each T cell was stable in all stages of disease progression. The sums of the CD28+ subsets and HLA-DR+ subsets in CD4+ T and CD8+ T cells were constant as approximately 100% and 55-60% of each T cell. These results suggested that the changes of CD28/HLA-DR expressions on CD4+ T cells were more predictable than those on CD8+ T cells in the evaluation of the disease progression during HIV-infected periods. However, we need further studies to understand why the sum of two molecules in each T cell are constant.     

CD8+ CD28- and CD8+ CD57+ T cells and their role in health and disease

Chronic antigenic stimulation leads to gradual accumulation of late-differentiated, antigen-specific, oligoclonal T cells, particularly within the CD8(+) T-cell compartment. They are characterized by critically shortened telomeres, loss of CD28 and/or gain of CD57 expression and are defined as either CD8(+) CD28(-) or CD8(+) CD57(+) T lymphocytes. There is growing evidence that the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population plays a significant role in various diseases or conditions, associated with chronic immune activation such as cancer, chronic intracellular infections, chronic alcoholism, some chronic pulmonary diseases, autoimmune diseases, allogeneic transplantation, as well as has a great influence on age-related changes in the immune system status. CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population is heterogeneous and composed of various functionally competing (cytotoxic and immunosuppressive) subsets thus the overall effect of CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell-mediated immunity depends on the predominance of a particular subset. Many articles claim that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells have lost their proliferative capacity during process of replicative senescence triggered by repeated antigenic stimulation. However recent data indicate that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells can transiently up-regulate telomerase activity and proliferate under certain stimulation conditions. Similarly, conflicting data is provided regarding CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell sensitivity to apoptosis, finally leading to the conclusion that this T-cell population is also heterogeneous in terms of its apoptotic potential. This review provides a comprehensive approach to the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population: we describe in detail its origins, molecular and functional characteristics, subsets, role in various diseases or conditions, associated with persistent antigenic stimulation.     

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens

CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.     

Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells

The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.     

Activation of primary T lymphocytes results in lysosome development and polarized granule exocytosis in CD4+ and CD8+ subsets, whereas expression of lytic molecules confers cytotoxicity to CD8+ T cells

Lytic granule exocytosis is the major cytotoxic mechanism used by CD8(+) cytotoxic lymphocytes. CD8(+) T cells acquire this effector function in the process characterized by lysosomal biogenesis, induction of expression of cytolytic molecules, and their selective sorting into the lysosomal vesicles. However, temporal relation of these differentiation stages during T cell activation has not been defined precisely. Also, although CD4(+) T cells typically do not express lytic molecules as a consequence of activation, and therefore, do not acquire granule exocytosis-mediated lytic function, it is not clear whether CD4(+) T cells are able to degranulate. By using in vitro TCR stimulation of primary mouse lymphocytes, we found that polyclonally activated CD4(+) T cells degranulate upon TCR ligation and polarize enlarged lysosomal granules in response to target cell recognition, despite the lack of granule exocytosis-mediated cytotoxicity. Upon TCR stimulation, resting CD8(+) T cells rapidly express lytic molecules and acquire potent lytic function early in activation. Maximal cytolytic potential, however, depends on enlargement of lysosomal granules during the subsequent activation stages. Thus, polyclonal TCR stimulation of resting T cells results in development of lysosomal granules and their release upon TCR engagement in CD4(+) and CD8(+) T cells, but only CD8(+) T cells acquire lytic function as a result of induction of expression of lytic molecules.     

Human TSLP directly enhances expansion of CD8+ T cells

Human thymic stromal lymphopoietin (TSLP) promotes CD4(+) T-cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP-activated DCs induce CD8(+) T-cell proliferation, it is not clear whether TSLP acts directly on CD8(+) T cells. In this study, we show that human CD8(+) T cells activated by T-cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8(+) T cells. Although non-stimulated human CD8(+) T cells from peripheral blood did not express TSLPR, CD8(+) T cells activated by anti-CD3 plus anti-CD28 did express TSLPR. After T-cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8(+) T cells. Interestingly, using monocyte-derived DCs pulsed with a cytomegalovirus (CMV)-specific pp65 peptide, we found that although interleukin-2 allowed expansion of both CMV-specific and non-specific CD8(+) T cells, TSLP induced expansion of only CMV-specific CD8(+) T cells. These results suggest that human TSLP directly enhances expansion of CD8(+) T cells and that the direct and indirect action of TSLP on expansion of target antigen-specific CD8(+) T cells may be beneficial to adoptive cell transfer immunotherapy.