Dexamethasone induces apoptosis in mouse natural killer cells and cytotoxic T lymphocytes.

Glucocorticoid hormones (GCH) induce apoptotic cell death in immature thymocytes through an active mechanism, characterized by extensive DNA fragmentation into oligonucleosomal subunits. This requires macromolecular synthesis and is inhibited by protein kinase C (PKC) inhibitors, interleukin-4 (IL-4) and heat shock (hs). We performed experiments to analyse the possible effect of GCH on more differentiated lymphocytes, i.e. mouse natural killer (NK) cells and CD8+ alloreactive cytotoxic T lymphocytes (CTL). The results show that dexamethasone (DEX) induces DNA fragmentation and cell death in NK cells and CTL in vitro. In both NK cells and CTL, DEX-induced apoptosis is inhibited by IL-2 and IL-4 but, unlike that induced in thymocytes, is augmented by mRNA and protein synthesis inhibitors, PKC inhibitors and HS.     

人工合成HLA衍生肽对CTL和NK细胞毒功能的影响

目的探讨合成HLA衍生肽对CTL和NK细胞毒功能的影响.方法人工固相合成2种HLA衍生肽(P1HLA-B*0701.75-84和P2HLA-B*2702.75-84),分别用MTT法和LDH释放法,在体外观察HLA衍生肽对CTL和NK细胞毒功能的影响及其等位基因特异性.结果P2可显著抑制CTL的细胞毒效应(P＜0.01),这种抑制作用不具有等位基因特异性,而对NK的细胞毒功能则没有明显影响.结论合成HLA肽对CTL和NK细胞毒功能的影响只与HLA肽的序列有关,P2即HLA-B*2702.75-84,可抑制CTL的细胞毒功能.     

Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo.

We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti-perforin monoclonal antibody. In the present study, more detailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co-expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin-positive cells in this population was greatly reduced after stimulation with anti-CD3 or anti-T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell- and CTL-mediated cytolysis.     

Disparate effect of beige mutation on cytotoxic function between natural killer and natural killer T cells

Beige mice lack natural killer (NK) cytotoxicity, although NK cells are normally present. In recent studies, NK T cells have been newly identified. We therefore examined the number and function of NK T cells in beige mice. The number of NK T cells was at a normal level in the liver of beige mice. NK cytotoxicity was decreased in the liver of these mice, whereas NK T cytotoxicity was intact. When immunochemical staining for perforin was conducted, the majority of NK cells and the minority of NK T cells in beige mice carried a giant granule, containing perforin, in the cytoplasm. In the case of control B6 mice, the majority of NK cells and the minority of NK T cells had multiple, dispersed granules containing perforin. These results suggest that NK T cytotoxicity is unaffected by the beige mutation, owing to their cytotoxicity being mediated without the secretion system of perforin.     

Natural killer, natural killer T, helper and cytotoxic T cells in the decidua from sporadic miscarriage

PROBLEM: The aim of this cohort study was to assess natural killer (NK) cell and natural killer T (NKT) cell populations and cytokine expressions of helper T (Th) and cytotoxic T (Tc) cells in the decidua of sporadic miscarriage (MS) and induced abortion (IA). METHODS: The deciduae were obtained from consecutive 40 women whose pregnancies ended in the first trimester MS, and the fetal chromosome karyotypes were analyzed. The cell populations were measured by flow cytometry. RESULTS: No significant differences in NK cell or NKT cell percentages were found among MS with normal chromosome karyotype (MSNK, n = 14), MS with abnormal karyotype (MSAK, n = 26) and IA (n = 14). Interferon (IFN)-gamma(+) cell percentage and interleukin (IL)-4(+)/tumor necrosis factor (TNF)-alpha(+) ratio of Th cells in MS groups were increased, while IL-4(+) cell percentage, IL-4(+)/IFN-gamma(+) and IL-4(+)/TNF-alpha(+) ratios of Tc cells in MS groups were decreased as compared with those in IA. No significant difference in these parameters between MSNK and MSAK was found. CONCLUSIONS: These results suggested that Tc1 predominance existed in the decidua of MS. Th1 predominance was not found in MSNK.     

Unique lectin-binding characteristics of cytotoxic T lymphocytes allowing their distinction from natural killer cells and "K" cells.

Cytotoxic T lymphocytes (CTL) can be selectively depleted from in vitro of in vivo alloactivated populations of T cells on Vicia villosa lectin adsorbents through the lectin-specific interaction with the CTL-associated surface glycoprotein T 145 (Kimura, A, Wigzell, H. and Holmquist, G., J. Exp. Med. 1979. 149: 473). Results from these and other experiments have demonstrated the general applicability of this fractionation procedure in which no constraints related to antigenic specificity of the CTL have been observed. When this fractionation procedure was applied to other compartments of cytolytic cells (natural killer cells and "K" cells), no detectable impact could be seen. This differential lectin binding would appear to offer a means of dissecting the activities of CTL from other compartments of cytolytic lymphoid cells.     

Role of natural killer cells in Pichinde virus infection of Syrian hamsters.

Pichinde virus produced a fatal infection in adult MHA hamsters but not LSH hamsters after intraperitoneal inoculation. After footpad inoculation, an 8-day swelling response was observed in LSH but not MHA hamsters; however, both strains survived infection by this route. Examination of the kinetics of viral replication in the two hamster strains inoculated by the two routes revealed a correlation between infectious centers and natural killer activity in cells obtained from spleens and popliteal lymph nodes. A subpopulation of cytolytic and infected cells which sedimented at about 3.0 to 4.5 mm/h in albumin gradients was found in greater numbers in MHA than in LSH hamsters. These data suggest that one factor contributing to the fatal outcome of Pichinde virus infection in MHA hamsters is the presence of excessive numbers of splenic target cells which possess properties of natural killer cells.     

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. II. Separation of natural killer cells and cytotoxic T cells

Treatment of mice with ascitic fluid containing high titres of T24-31.7 monoclonal antibody (rat anti-mouse Thy-1) lead to a rapid loss of T cells from peripheral lymphoid organs. Spleen and lymph node tissue lost all detectable Thy-1+ and mitogen-responsive T cells within 72 hr. These tissues were completely T cell-depleted for more than a week before repopulation with T cells began. Lectin-induced splenic T cell cytoxicity in culture was lost within 72 hr after treatment of mice in vivo. In contrast, treatment of mice with T24-31.7 ascitic fluid was followed by an immediate increase in natural killer (NK) cell-mediated cytolytic activity. After 96 hr, NK activity began to decrease and did not reappear in the T cell-depleted spleens. While purified T24-31.7 antibody was responsible for T cell depletion in vivo, a non-immunoglobulin component of the ascitic fluid stimulated splenic NK cell activity. The presence of phytohaemagglutinin (PHA) on target cells in the lytic assay was shown to inhibit NK activity but enhance T cell-mediated cytotoxicity. The relationship of NK cells and cytotoxic T lymphocytes (CTL) to the T cell lineage is discussed.     

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.     

年龄对人细胞毒性T淋巴细胞和自然杀伤细胞中穿孔素表达的影响

目的　通过研究健康儿童、成人和老年人外周血淋巴细胞 (PBL)中细胞毒效应分子穿孔素 (PFP)的表达水平的变化 ,了解细胞毒性T细胞 (CTL )的杀伤活性随年龄增长而下降的分子机制。方法　利用抗人PFP、抗人CD3和抗人CD5 6单克隆抗体 (mAb) ,采用单标记免疫组化染色法 ,检测外周血单个核细胞 (PBMC)中PFP的表达。采用双标记免疫组化染色法 ,检测上述细胞表面标志CD3或CD5 6的表达和细胞内PFP的表达。结果　儿童组、成人组及老年组PBL中表达PFP阳性细胞的百分率 (% ) ,分别为 (2 6 .4± 2 .7) % ,(2 1.6± 3.2 ) %和 (13.4± 2 .0 ) % ,老年组明显下降 ,与其他两组相比较P <0 .0 0 1。 3个年龄组CD3+ T细胞表达PFP的阳性率 (% ) ,分别为 (30 .1± 4 .8) %、(2 1.4± 7.3) %和 (18.8± 4 .2 ) % ,随年龄的增长而显著下降。3个年龄组CD5 6 +NK细胞PFP的表达差别不显著。结论　健康人PBL中PFP的表达随年龄增长而递减 ,CD3+ T细胞亚群PFP表达水平的下降尤其显著。这可能是老年人CTL杀伤活性下降的重要原因之一     

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.     

Role of natural killer cytotoxic factors in the mechanism of target-cell killing by natural killer cells

Studies on the mechanism of cell-mediated cytotoxicity (CMC) have suggested a stimulus-secretion model and implicated a role of soluble cytotoxic mediators. Our studies in the natural killer (NK) system provide several lines of evidence for the involvement of natural killer cytotoxic factors (NKCF) in NK CMC and led to the development of a model for the NK lytic mechanism. This model delineates several interactions between NK cells and targets that are deemed necessary to achieve target-cell lysis. The first stage is the interaction of the effector with the target cell, resulting in contact and adhesion. This is presumably mediated by NK recognition structures and target-cell structures. Following binding, the target cell stimulates the NK cell to release NKCF. This step is functionally distinct from the initial effector-target binding. The trigger mechanism for release of NKCF appears to be dependent on protein kinase C. The released NKCF binds to NKCF binding sites on the target cell followed by processing or internalization and, ultimately, resulting in cell death. This model has been shown to be useful in investigating the mechanism of defective NK activity in certain disease states. Biochemical analysis and comparative studies suggest that NKCF is a distinct molecule from other cytotoxins studied to date. The studies in the NK CMC system supporting a role of cytotoxic mediators also suggest a possible role for cytotoxic factors in other cytotoxic systems. Furthermore, the selective susceptibility to lysis of tumor or infected cells by NKCF suggests a possible role of their effectiveness inin vivo therapy.     

Adipose invariant natural killer T cells

Adipose tissue is a dynamic organ that makes up a substantial proportion of the body; in severe obesity it can account for 50% of body mass. Details of the unique immune system resident in human and murine adipose tissue are only recently emerging, and so it has remained a largely unexplored and unappreciated immune site until now. Adipose tissue harbours a unique collection of immune cells, which often display unusual functions compared with their counterparts elsewhere in the body. These resident immune cells are key to maintaining tissue and immune homeostasis, yet in obesity their chronic aberrant stimulation can contribute to the inflammation and pathogenesis associated with obesity. Anti‐inflammatory adipose‐resident lymphocytes are often depleted in obesity, whereas pro‐inflammatory immune cells accumulate, leading to an overall inflammatory state, which is a key step in the development of obesity‐induced metabolic disease. A good example is invariant natural killer T (iNKT) cells, which make up a large proportion of lymphocytes in human and murine adipose tissue. Here, they are unusually poised to produce anti‐inflammatory or regulatory cytokines, however in obesity, iNKT cells are greatly reduced. As iNKT cells are potent transactivaors of other immune cells, and can act as a bridge between innate and adaptive immunity, their loss in obesity represents the loss of a major regulatory population. Restoring iNKT cells, or activating them in obese mice leads to improved glucose handling, insulin sensitivity, and even weight loss, and hence represents an exciting therapeutic avenue to be explored for restoring homeostasis in obese adipose tissue.     

Assessment of the specificity of cytotoxic T lymphocytes for the nucleoprotein of Pichinde virus using recombinant vaccinia viruses

Summary Pichinde virus (PV) infection of mice results in induction of a strong H-2 restricted, virus-specific cytotoxic T lymphocyte (CTL) response and rapid clearance of the virus. To define the specificities of CTL induced by PV infection, we constructed vaccinia virus recombinants containing cloned cDNAs corresponding to full-length (VVNP) and a truncated form (VVNP_51–561) of the nucleoprotein (NP) gene of PV. Radioimmunoprecipitation analysis of infected cell lysates indicated that VVNP expressed a PV-specific product identical in size to that of authentic NP, while vaccinia virus recombinants containing truncated NP produced a polypeptide consistent with the synthesis of amino acids 51–561 of Pichinde virus NP. Interestingly, cells infected with VVNP synthesized easily detectable, but much lower levels of nucleoprotein relative to both PV and VVNP_51–561. Primary virus-specific CTL induced in three different strains of inbred mice following intravenous infection with PV were able to lyse syngeneic target cells infected with PV but did not markedly lyse syngeneic targets expressing full-length or truncated NP following recombinant vaccinia virus infection. Similarly, secondary anti-PV specific CTL generated following in vitro restimulation by PV or selectively restimulated with vaccinia recombinants did not significantly lyse target cells expressing NP. Further, infection of mice with VVNP and VVNP_51–561 did not induce CTLs specific for PV and did not prime mice for the generation of memory anti-PV CTL in vivo. These results suggest that PV gene products other than NP, such as the GPC or L protein, contain the major target epitope(s) recognized by PV-specific CTL.     

Human invariant valpha24+ natural killer T cells activated by alpha-galactosylceramide (KRN7000) have cytotoxic anti-tumour activity through mechanisms distinct from T cells and natural killer cells

Human Valpha24 + NKT cells, a subpopulation of natural killer cell receptor (NKR-P1A) expressing T cells with an invariant T-cell receptor (TCR; Valpha24JalphaQ) are stimulated by the glycolipid, alpha-galactosylceramide (KRN7000), in a CD1d-dependent, TCR-mediated fashion. Little is known about Valpha24 + NKT-cell function. The murine counterpart, Valpha14 + NKT cells, appear to have an important role in controlling malignancy. There are no human data examining the role of Valpha24 + NKT cells in controlling human malignancy. We report that Valpha24 + NKT cells have perforin-mediated cytotoxicity against haemopoietic malignancies. Valpha24 TCR, CD1d and alpha-galactosylceramide may all play a role in cytotoxicity but are not absolute requirements. The greatest cytotoxicity was observed against the U937 tumour cell line (95 +/- 5% lysis). THP-1, Molt4, C1R cells and allogeneic mismatched dendritic cells were also sensitive to Valpha24 + NKT cytotoxicity but neither the NK target, K562, nor lymphokine-activated killer-sensitive Daudi cells, were sensitive. These results indicate a killing pattern distinct from conventional major histocompatibility complex-restricted T cells, NK cells and other cytotoxic lymphoid cells previously described. We conclude that human Valpha24 + NKT cells have cytotoxic anti-tumour activity against haemopoietic malignancies through effector mechanisms distinct from conventional T cells and NK cells and that their specific stimulator KRN7000 may have therapeutic potential.     

Cloned human cytotoxic T lymphocytes develop anomalous killer cell function.

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein-Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes; CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these, 13 (7%) retained specific function, 29 (16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy.     

Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors

Toxic epidermal necrolysis (TEN) is a rare life-threatening adverse drug reaction characterized by a massive destruction of the epidermis. Immunohistological studies of skin biopsies of TEN showed infiltrates of predominantly CD8+ T lymphocytes even though other authors reported a prominent involvement of cells of the monocyte-macrophage lineage. The aim of this study was to characterize phenotypically and functionally the cells present in the cutaneous blister fluid of four patients with TEN. We first determined that lymphocytes were predominant in blister fluid obtained early, while monocytes/macrophages later became the most important population. We then showed that this lymphocyte population, mainly CD3+CD8+, corresponded to a peculiar cell subset as they expressed cutaneous leucocyte antigen, killer inhibitory receptors KIR/KAR and failed to express CD28 molecule. Functionally, we determined that blister T lymphocytes had a cytotoxic T lymphocyte (CTL)- and NK-like cytotoxicity. The role of this cytotoxic lymphocyte population present at the site of lesions during TEN remains to be understood.     

Interferon-γ Expression in Natural Killer Cells and Natural Killer T Cells Is Suppressed in Early Pregnancy

Recent study has suggested that innate immune system might play an important role in pregnancy progression. In this study, to investigate whether NK cells and NKT cells, instead of T cells, are the dominant populations of peripheral blood in early pregnancy, flow cytometry was used to detect the percentage and intracellular cytokine expressions of T cells, NK cells, NKT cdls in peripheral blood of non-pregnant women and early pregnant women. In our result, the percentages of NK calls and NKT calls were significantly increased in pregnancy compared to non-pregnancy. However, the percentage of T cells was not changed. We did not detect the Th2-dominance of total lymphocytes or T cells in peripheral blood of early pregnant women and there were also no significant changes of type 1 and type 2 cytokines in T cells, but IFN-γ production in both NK and NKT cells was decreased in early pregnancy. These results suggest that the innate immune system including NK cells and NKT cells should play a pivotal role in pregnancy progression. Type 1/type 2 shift mechanisms in innate immune system during the human early pregnancy should be paid more attention.