Functional and Vbeta repertoire characterization of human CD8+ T-cell subsets with natural killer cell markers,CD56+ CD57- T cells,CD56+ CD57+ T cells and CD56- CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK-type T cell); CD56 single positive (CD56)-T cells, CD56/CD57 double positive (DP)-T cells and CD57 single positive (CD57)-T cells in the peripheral blood. All NK-type T-cell populations expressed CD122 and intermediate levels of T-cell receptor (TCR; regular CD8+ T cells are CD122- and express high levels of TCR). The number of both DP-T cells and CD57-T cells, but not CD56-T cells, gradually increased with age. All NK-type T-cell populations produced larger amounts of interferon-gamma than did regular CD8+ T cells after stimulation with interleukin (IL)-2, IL-12 and IL-15. However, CD56-T cells and CD57-T cells but not DP-T cells showed a potent antitumour cytotoxity to NK-sensitive K562 cells, whereas only CD56-T cells showed a potent cytotoxity to NK-resistant Raji cells. Furthermore, although NK-type T cells produced large amounts of soluble Fas-ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VbetaT cells were found in each NK-type T-cell population. The non-variant CDR3 region(s) for the TCRbeta chain(s) showed CD57-T cells and CD56-T cells to be derived from distinct origins, while the DP-T cell population consisted of a mixture of the clones seen in both CD56-T cells and CD57-T cells. Our results suggest that CD57-T cells and CD56-T cells are functionally and ontogenically different populations while DP-T cells appear to originate from both CD56-T cells and CD57-T cells.     

Inhibition of anti-GD3-ganglioside antibody-induced proliferation of human CD8+ T cells by CD16+ natural killer cells

The ganglioside GD3 has been described as a membrane component of human T cells which is involved in T cell growth. In the present study the activating function of GD3 for human CD4⁺ and CD8⁺ T cells was analyzed by five different monoclonal antibodies (mAb) directed against the GD3 molecule. Three mAb U5, Z21 and R24 induced strong proliferation of peripheral blood mononuclear cells and purified CD8⁺ and CD4⁺ T cells of normal donors containing less than 5% CD16⁺ natural killer (NK) cells. In contrast to CD4⁺ T cells, CD8⁺ T cells proliferated only weakly in the presence of 15% CD16⁺ NK cells. The proliferative response of purified CD4⁺ and CD8⁺ T cells (<5% NK cells) correlated with the antibody-dependent induction of integral and soluble interleukin-2 (IL-2) receptors and was reduced to 20% by an anti-IL-2 receptor antibody. Our results show, that the GD3 molecule represents an activation molecule for both CD4⁺ and CD8⁺ T cells and that CD16⁺ NK cells selectively inhibit anti-GD3 antibody-induced proliferation of CD8⁺ T cells.     

HIV感染后CD8~+T细胞表面NK相关受体表达的变化

目的深入了解人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染后CD8+T细胞表面NK相关受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV感染者、11例AIDS患者、15例进行高效抗逆转录病毒治疗(highly active antiretroviral therapy,HAART)者和13例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血CD8+T细胞表面NKG2D、NKG2A和KIR3DL1的表达。结果 HIV感染后CD8+T细胞表面NKG2D表达的百分比显著低于健康对照,NKG2D+NKG2A-表达的百分比随疾病进展逐渐下降,且NKG2D+NKG2A-表达的百分比与CD4+T细胞的绝对数量呈正相关。AIDS患者CD8+T细胞表达NKG2A显著高于其它各组,随着疾病的进展CD8+T细胞表达NKG2A+NKG2D-百分比逐渐上升,AIDS患者显著高于其它各组,经抗逆转录病毒治疗后下降至健康对照的水平,且NKG2A+NKG2D-表达的百分比与CD4+T细胞的绝对数量呈负相关。HIV感染后CD8+T细胞KIR3DL1+表达的百分比较健康对照并无显著差异。结论 HIV感染机体后,CD8+T细胞NK相关受体表达变化与疾病进展相关,抗病毒治疗后可恢复其变化。     

The expression of killer cell inhibitory receptors on natural killer cells and activation status of CD4+ and CD8+ T cells in the decidua of normal and abnormal early pregnancies.

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer cells within the maternal decidua. These NK cells have an unusual phenotype, CD3- CD16- CD56(bright), distinguishing them from peripheral blood NK cells. They may control trophoblast migration and placentation. Using a panel of monoclonal antibodies to several members of the KIR family and flow cytometry, we found that KIRs are expressed on decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. In anembryonic pregnancy, the proportions of decidual NK cells with a particular KIRs (GL183 and EB6) decreased significantly when compared with normal pregnancy (p = 0.01 and 0.01, respectively), raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site. In the decidua, more CD4+ and CD8+ T cells expressed CD69 and HLA-DR than in blood, indicating that T cells are regionally activated during early pregnancy. When compared with normal pregnancy, decidual HLA-DR+CD4+CD3+, CD69+CD8+CD3+ and HLA-DR+CD8+CD3+ T lymphocytes are significantly increased in anembryonic pregnancy. The over-activation of decidual T cells during anembryonic pregnancy may thus contribute to the increased NK cytotoxicity activity.     

Inverse relationship of CD8+CD11+ suppressor T cells with human immunodeficiency virus (HIV)-specific cellular cytotoxicity and natural killer cell activity in HIV infection.

Profiles of CD8+CD11+ T suppressor cells, human immunodeficiency virus (HIV)-env-specific cytotoxic T-lymphocyte (CTL) activities, and natural killer (NK) cell activity were studied in 12 asymptomatic untreated HIV-infected patients. These patients were followed for 4-7 months. NK activity, HIV-env-specific CTL activities mediated by CD4+, CD8+ T cells and CD8+CD11+ T-suppressor cell number remained stable in seven patients during the study period. Alternatively, NK and HIV-specific CTL activities decreased and CD8+CD11+ cell number increased in five patients whose CD4+ T-cell number fell, and in four of these five patients serum p24 antigen level increased, and they developed minor clinical signs of disease progression during the study period. CD8+CD11+ cells are present in higher percentage (10-45% of peripheral blood mononuclear cells) in these HIV-infected patients as compared to those in normal individuals (3-5%). Our results suggest that CD8+CD11+ cells, NK, and HIV-specific cytotoxic activities may be helpful in monitoring prognosis of HIV infection. These observations also suggest that CD8+CD11+ cells may play an important role in the failure of host immune defences against HIV.     

Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.     

Identification and characterization of CD8+ suppressor T cells

It has long been appreciated that certain subsets of T cells are capable of suppressing immune reactions. Initially, such T cells were described as CD8⁺ suppressor T cells (Ts) and there is a vast body of research spanning 30 years that describes the immunobiology of CD8⁺ Ts. However, studies on CD8⁺ Ts have suffered from the inability to distinguish CD8⁺ Ts from CD8⁺ T cells of other phenotypes. Here we present a brief history of CD8⁺ Ts, a review of recent progress distinguishing CD8⁺ Ts as a unique subset of CD8⁺ T cells, and an overview of the evolving immunological context in which CD8⁺ Ts function.     

Identification and characterization of CD8+ suppressor T cells

It has long been appreciated that certain subsets of T cells are capable of suppressing immune reactions. Initially, such T cells were described as CD8+ suppressor T cells (Ts) and there is a vast body of research spanning 30 years that describes the immunobiology of CD8+ Ts. However, studies on CD8+ Ts have suffered from the inability to distinguish CD8+ Ts from CD8+ T cells of other phenotypes. Here we present a brief history of CD8+ Ts, a review of recent progress distinguishing CD8+ Ts as a unique subset of CD8+ T cells, and an overview of the evolving immunological context in which CD8+ Ts function.     

Type 2 diabetes mellitus is associated with altered CD8+ T and natural killer cell function in pulmonary tuberculosis

Type 2 diabetes mellitus (DM) is associated with expanded frequencies of mycobacterial antigen-specific CD4⁺ T helper type 1 (Th1) and Th17 cells in individuals with active pulmonary tuberculosis (TB). No data are available on the role of CD8⁺ T and natural killer (NK) cells in TB with coincident DM. To identify the role of CD8⁺ T and NK cells in pulmonary TB with diabetes, we examined mycobacteria-specific immune responses in the whole blood of individuals with TB and DM (TB-DM) and compared them with those without DM (TB-NDM). We found that TB-DM is characterized by elevated frequencies of mycobacterial antigen-stimulated CD8⁺ T cells expressing type 1 [interferon-γ and interleukin-2 (IL-2)] and type 17 (IL-17F) cytokines. We also found that TB-DM is characterized by expanded frequencies of TB antigen-stimulated NK cells expressing type 1 (tumour necrosis factor-α) and type 17 (IL-17A and IL-17F) cytokines. In contrast, CD8⁺ T cells were associated with significantly diminished expression of the cytotoxic markers perforin, granzyme B and CD107a both at baseline and following antigen or anti-CD3 stimulation, while NK cells were associated with significantly decreased antigen-stimulated expression of CD107a only. This was not associated with alterations in CD8⁺ T-cell or NK cell numbers or subset distribution. Therefore, our data suggest that pulmonary TB complicated with type 2 DM is associated with an altered repertoire of cytokine-producing and cytotoxic molecule-expressing CD8⁺ T and NK cells, possibly contributing to increased pathology.     

Functional characterization of OX40 expressed on human CD8+ T cells

In the present study, we investigated the expression of OX40 on human CD8(+) T cells with regard to expression induction, costimulatory function and possible involvement in cytotoxicity. Human CD8(+) T cells were purified from peripheral blood mononuclear cell (PBMC) of healthy donors and cocultured with allogeneic monocyte-derived dendritic cells. Flow cytometric analysis showed that expression of OX40 was induced on CD8(+) T cells within 1 day and increased to the maximum levels on day 3. An addition of anti-OX40 ligand (OX40L) mAb suppressed CD25 expression, proliferation and IFN-gamma production of CD8(+) T cells, suggesting that OX40 functions as a costimulatory molecule not only for CD4(+) T cells but also for CD8(+) T cells. In parallel, coculture of pre-activated CD8(+) T cells with OX40L-transfected murine epithelial cells (MMCE-OX40L) resulted in an increase in CD25 expression, proliferation and IFN-gamma producing cells, compared with that with the mock control (MMCE-mock). Finally, non-specific cytotoxic activity of preactivated CD8(+) T cells was examined using OKT3 hybridoma as target cells after coculture with these transfectants. Coculture with MMCE-OX40L induced slightly higher cytotoxicity of CD8(+) T cells than that with MMCE-mock. These results indicate that OX40 is induced transiently on CD8(+) T cells upon activation and its signals contribute to both clonal expansion and functional reinforcement.     

HIV感染后CD4~+ T细胞表面NK相关受体表达的变化

目的研究人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染后CD4+T细胞表面NK相关受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV感染者、11例AIDS患者、15例进行高效抗逆转录病毒治疗(highly activeantiretroviral therapy,HAART)者和13例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血CD4+T细胞表面NKG2D、NKG2A和KIR3DL1的表达。结果 AIDS患者CD4+T细胞表面NKG2D表达的百分比显著高于其他各组,且NKG2D表达的百分比与CD4+T细胞的绝对数量呈负相关(R=-0.352,P<0.05),与HIV病毒载量呈正相关(R=0.426,P<0.05)。AIDS患者CD4+T表面NKG2A表达的百分比显著高于其他各组,NKG2A+NKG2D-表达的百分比显著高于其他各组,且NKG2A表达的百分比与CD4+T细胞的绝对数量呈负相关(R=-0.432,P<0.01)。结论 HIV感染机体后,CD4+T细胞NK相关受体表达变化与疾病进展相关。     

Immunelectronmicroscopic characterization of T4 and T8 lymphocytes and natural killer cells in neuronal ceroid-lipofuscinosis

CD4+, CD8+, and CD56+ cells were isolated with the immunomagnetic separation technique from peripheral blood mononuclear cells (PBMC) of 3 patients with neuronal ceroid-lipofuscinosis: one patient each with infantile (INCL), late infantile (LINCL), and juvenile (JNCL) neuronal ceroid-lipofuscinoses, all studied by light (LM) and electron (EM) microscopy. To compare the pathology of these cells with affected cells in other types of lysosomal diseases, the separation was also performed with PBMC of 1 patient with mucolipidosis (ML) type II, 2 patients with mucopolysaccharidosis (MPS) type I, and 4 patients with MPS type III. Diseasespecific lysosomal inclusions were identified in CD4+, CD8+, and CD56+ cells. Subclass-specific storage products could not be found. T4 and T8 lymphocytes showed nearly the same ratio of affected to nonaffected cells. These results suggest that lysosomal storage can be found in most of the lymphocyte subclasses in these forms of neuronal ceroid-lipofuscinoses and other lysosomal diseases. © 1995 Wiley-Liss, Inc.     

CD8+CD11b+ peripheral blood T lymphocytes contain lymphokine-activated killer cell precursors

CD3+CD8+ cells, purified from peripheral blood T cells by depletion of CD4+ lymphocytes, were tested for their cytotoxic activity against K-562 or HL-60 cells. Freshly prepared cells had no cytotoxic function, but upon exposure to recombinant interleukin 2 (rIL2) in vitro they acquired a lymphokine-activated killer (LAK) activity. Fractionation of CD3+CD8+ cells into CD11b+ and CD11b- cells demonstrated that all the cells with the potential of developing LAK cell functions were within the CD8+CD11b+ subset. These cells lacked natural killer cell markers such as CD16 or NKH1, but a proportion of them stained for Leu-7. Furthermore, they expressed the alpha/beta chains, but not the gamma/delta chains, of the T cell receptor, as could be determined by staining with the appropriate monoclonal antibodies. CD8+CD11b+ cells had a large granular lymphocyte morphology, as shown by both cytochemical and electron microscopy analyses. They proliferated in response to IL2 in vitro and developed cytotoxic functions against a number of natural killer resistant targets. Their response to phytohemagglutinin or pokeweed mitogen was very weak or absent. By contrast, CD8+CD11b- cells failed to generate LAK cells in response to rIL2, did not show a large granular lymphocyte morphology, but responded vigorously to phytohemagglutinin or pokeweed mitogen. Purified CD8+CD11b+ cells were cloned by limiting dilution in the presence of rIL2. The observed cloning efficiency of 19 +/- 0.3% indicates that a fraction of the cells only could respond to IL2. Furthermore, only 50% of the clones obtained had a LAK cell function. These data show that CD8+CD11b+ cells represent a heterogeneous cell population. Nevertheless this cell subset probably represents the major source of LAK cell progenitors within the circulating T cells.     

Low-dose cyclophosphamide combined with IL-2 inhibits tumor growth by decreasing regulatory T cells and increasing CD8+ T cells and natural killer cells in mice

Interleukin-2 (IL-2) benefits some cancer patients by promoting the proliferation of cytotoxic effector T cells, but this process is limited by the expansion of regulatory T cells (Tregs). Low-dose cyclophosphamide (CTX) can inhibit the number and function of Tregs. We treated carcinoma-bearing mice with Vehicle, CTX, IL-2 and CTX + IL-2 to investigate the effects of low-dose CTX combined with IL-2 in antitumor treatment. In comparison to monotherapy, CTX + IL-2 significantly limited tumor growth, via tumor cell proliferation inhibition and increased apoptosis. The infiltration of CD8⁺ T cells in tumor tissues was significantly increased in the CTX + IL-2 group. CTX + IL-2 safely increased CD8⁺ T and natural killer cells in the spleen, lymph nodes and peripheral blood, and CTX attenuated the increase in Tregs induced by IL-2 in the spleen.     

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis

Cytotoxic activity mediated by CD8⁺ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56^dim CD57^bright) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8⁺ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8⁺ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8⁺ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56⁺ CD57⁺) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8⁺ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.     

2B4 (CD244) is a non-MHC binding receptor with multiple functions on natural killer cells and CD8+ T cells

2B4 (CD244) is expressed by memory-phenotype CD8(+) T cells and all natural killer (NK) cells. The ligand for 2B4, CD48, is expressed on hematopoietic cells. 2B4 is conserved in humans and mice, and a number of reports have linked 2B4 with activation of lymphocytes. We have employed 2B4-deficient mice and antibody blocking to analyze 2B4 function both in vitro and in vivo and found that 2B4 is a receptor with multiple functions. 2B4 is required for optimal activation of CD8(+) T cells and NK cells--in this context 2B4 requires interaction with CD48 on neighboring lymphocytes, demonstrating that homotypic interaction within NK cell or T cell populations augments immunity. When 2B4 is engaged by CD48 on a target cell, 2B4 conversely inhibits NK effector function. As an inhibitory receptor, 2B4 is unconventional as it is not regulated by MHC class I molecules. In this review we will discuss the significance of these multiple functions and the events that may regulate differential 2B4 signaling outcome.