Normal Large Granular Lymphocytes Research Articles

CD8+,CD57+ T cells from healthy elderly subjects suppress neutrophil development in vitro: Implications for the neutropenia of Felty's and large granular lymphocyte syndromes

To investigate the ability of CD8+,CD57+ large granular lymphocytes (LGL) from normal individuals and from Felty's syndrome (FS) or LGL syndrome patients to suppress allogeneic neutrophil precursor development. Six FS patients, 5 LGL syndrome patients, and 13 elderly controls were studied. CD8+,CD57+ T cells were cocultured with cord blood-derived stem cells, and percentage inhibition was calculated. Recombinant chemokines and Fas-stimulating molecules were used in separate cultures to address possible mechanisms of suppression. Proliferation after stimulation with interleukin-2 (IL-2) and anti-CD3 was assessed. Significant (79%) suppression of colony-forming unit-granulocyte-macrophage (CFU-GM) by the CD8+,CD57+ subset was shown by 1 FS patient. None of the CD8+,CD57+ cells from LGL syndrome patients had any effect. Six of 13 controls studied showed >40% inhibition of CFU-GM, and all but 2 showed at least some suppression. The suppressive effect was not mediated by Fas/Fas ligand interactions or by the chemokines macrophage inhibitory protein 1alpha or IL-8. LGL from both patients and controls were largely CD28- and had reduced proliferative capacity. In a subset of FS patients, expansion of CD8+,CD57+ T cells in the bone marrow may be responsible for neutropenia by suppressing neutrophil precursors. This effect is also seen with normal LGL, which are likely to have an important function in neutrophil homeostasis.

Natural killer cells carry the germ‐line configuration of the T cell receptor δ chain gene and heterogeneously express six distinct δ transcripts

Investigations on T cell receptor delta chain (TcR delta) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six T delta transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known T delta variable regions was expressed and the T delta locus was retained in its germ-line configuration. The origin and significance of these NK-associated T delta transcripts are discussed.

Detection of low and high affinity binding sites with fluoresceinated human recombinant interleukin-2

In this study, we describe a new methodology to detect and quantify lymphokine receptors, using interleukin-2 as a prototype. Human recombinant interleukin-2 (IL-2) was conjugated to fluorescein isothiocyanate. Binding of fluoresceinated IL-2 to different cell types was assessed by flow cytometry analysis, on a FACS 440 calibrated using fluoresceinated Sephadex G-25 beads. This calibration procedure allowed us to quantify the actual number of binding sites for IL-2. Fluoresceinated IL-2 did not bind to normal resting T cells, whereas a highly significant binding was observed on PHA-activated human T cells. The binding was inhibited by an excess of unlabeled IL-2 and by an excess of anti-IL-2 receptor p55 antibodies (anti-TAC). Dose curves of IL-2 showed a two plateau saturation, the first plateau corresponding to the saturation of high affinity binding sites, as assessed by correlation with the biological activity on IL-2-dependent T cells. Among the cell types tested, fluoresceinated IL-2 bound to IL-2-dependent mouse T cells (the binding in that case was not inhibited by anti-IL-2 receptor p55 antibodies), and to different p70 expressing cell lines or normal cells (MLA 144, normal large granular lymphocytes). Taken together, these results indicate that fluoresceinated IL-2 can be used to detect high as well as low affinity IL-2 binding sites.

Modulation of human natural killer cell cytotoxic activity, lymphokine production, and interleukin 2 receptor expression by thymic hormones.

Thymic hormone preparations have been shown to modulate natural killer (NK) activity in vivo in mice. We have investigated the effects of thymosin fraction 5 (TF5) on the in vitro NK cell activity of highly purified human large granular lymphocytes (LGL). The results indicate that TF5 but not kidney fraction 5 (a preparation used as control) is able to enhance the spontaneous NK activity of normal LGL. In addition, TF5 exhibited additive effects with recombinant interferon-alpha in enhancing NK activity in vitro. TF5 also enhanced interleukin 2 production and interleukin 2 receptor expression as well as interferon-gamma production in mitogen-stimulated LGL. Thymosin-alpha 1, a synthetic polypeptide originally isolated in its native form from TF5, also exhibited enhancing effects on LGL activities, suggesting that it is the active species in TF5. These results indicate that thymic hormones might regulate NK activity through the induction of lymphokine production and receptor expression by LGL.

The p75 peptide is the receptor for interleukin 2 expressed on large granular lymphocytes and is responsible for the interleukin 2 activation of these cells.

There are at least two interleukin 2 (IL-2) binding peptides: one is the Mr 55,000 peptide (p55) reactive with the anti-Tac monoclonal antibody, and the other is a Mr 75,000 non-Tac IL-2 binding peptide (p75). Independently existing Tac or p75 peptides represent low-affinity IL-2 receptors, whereas high-affinity IL-2 receptors are expressed when both peptides are present and associated in a receptor complex. It has long been known that normal large granular lymphocytes (LGL) or leukemic cells from the patients with abnormal expansions of LGL can be activated by IL-2 not only to more-potent natural killer cells but also to effectors of lymphokine-activated killer (LAK) activity, although they do not express the Tac peptide. In the present study, using cross-linking methodology, we found that normal LGL and leukemic LGL from all individuals tested expressed the p75 IL-2 binding peptide but did not express the Tac peptide. These LGL leukemia cells made proliferative responses to IL-2 but required a much higher concentration than that required for the proliferation of normal phytohemagglutinin-stimulated T lymphoblasts that express high-affinity receptors. Furthermore, the addition of IL-2 to Tac-negative LGL leukemic cells augmented transcription of the Tac gene and induced the Tac peptide. Neither the IL-2-induced proliferation nor the up-regulation of Tac gene expression was inhibited by the addition of anti-Tac. These results strongly suggest that the p75 peptide is responsible for IL-2-induced activation of LGL and that the p75 peptide alone can mediate an IL-2 signal. Thus, the p75 peptide may play an important role in the IL-2-mediated immune response not only by participating with the Tac peptide in the formation of the high-affinity receptor complex on T cells but also by contributing to the initial triggering of LGL activation so that these cells become efficient natural killer and lymphokine-activated killer cells.

Adult-onset cyclic neutropenia is associated with increased large granular lymphocytes

Human cyclic neutropenia occurs in adults as well as children. Clinical illness is similar in the childhood and adult diseases, but distinctly different modes of onset suggest heterogeneity in its pathophysiology. We studied seven patients with cyclic neutropenia, three with disease acquired in adulthood, and four with the childhood-onset disorder. All three patients with adult-onset cyclic neutropenia had increased numbers of circulating large granular lymphocytes (LGL), whereas the four children with cyclic neutropenia had normal LGL counts. LGL from patients with adult-onset cyclic neutropenia expressed cell surface antigens HNK-1 (three of three patients) and IgG Fc receptors (two of three patients), although natural killer activity was low. Two of these patients were treated with alternate-day steroids, resulting in decreased LGL counts and abrogation of neutrophil cycling. We suggest that adult-onset cyclic neutropenia may be distinguished from the childhood-onset form of the disease by increased numbers of LGL. Furthermore, increased LGL may identify a subset of patients with cyclic neutropenia who respond to steroid therapy.

Adult-Onset Cyclic Neutropenia Is Associated With Increased Large Granular Lymphocytes

Abstract Human cyclic neutropenia occurs in adults as well as children. Clinical illness is similar in the childhood and adult diseases, but distinctly different modes of onset suggest heterogeneity in its pathophysiology. We studied seven patients with cyclic neutropenia, three with disease acquired in adulthood, and four with the childhood-onset disorder. All three patients with adult-onset cyclic neutropenia had increased numbers of circulating large granular lymphocytes (LGL), whereas the four children with cyclic neutropenia had normal LGL counts. LGL from patients with adult-onset cyclic neutropenia expressed cell surface antigens HNK-1 (three of three patients) and IgG Fc receptors (two of three patients), although natural killer activity was low. Two of these patients were treated with alternate-day steroids, resulting in decreased LGL counts and abrogation of neutrophil cycling. We suggest that adult-onset cyclic neutropenia may be distinguished from the childhood-onset form of the disease by increased numbers of LGL. Furthermore, increased LGL may identify a subset of patients with cyclic neutropenia who respond to steroid therapy.

Lymphokine Production in Ty Lymphoproliferative Disorders

We have studied five patients with chronic lymphocytosis consisting of large granular lymphocytes (LGL). The increased numbers of LGL in these patients had little or no natural killer activity, mediated antibody-dependent cellular cytotoxicity, and were induced to kill tumour lines after culture for 3 days with interleukin 2 (IL-2). Patients' LGL showed considerable reactivity with HNK-1 and AB8.28 monoclonal antibodies (MoAb), whereas positivity for OKM1 and N901 was found in only two subjects, and only one patient reacted with B73.1. No appreciable reactivity has been found with anti-Tac MoAb in the four patients tested. In the absence of stimulation, the patients' LGL produced no IL-2 and only minimal amounts of IL-1 and interferon (IFN). On stimulation with lipopolysaccharides (for IL-1) or phytohaemagglutinin A (PHA) (for IL-2 and IFN), they produced IL-1 and IFN in amounts similar to those produced by normal lymphocytes, but only modest levels of IL-2. These results indicated that proliferating LGL, like normal LGL, have a secretory capacity. The lack of constitutive lymphokine production, the lack of Tac receptor expression, and the defect in IL-2 production after PHA stimulation do not support the hypothesis of an autocrine proliferation sustained by a known growth factor.

The effect of RU 41.740 (Biostim) on the production of interleukin-1 by monocytes and enriched large granular lymphocytes in normals and patients with hepatocellular carcinoma.

The effect of RU 41.740 on natural killer (NK) cell cytotoxicity from normals and patients with advanced hepatocellular carcinoma (HCC) was studied. RU 41.740-treated normal enriched large granular lymphocytes (LGLs) showed increased cytotoxic activity against 51Cr-labelled K562 cells. Although the drug increased the cytotoxic activity of LGLs from HCC patients, these results were not statistically significant. As LGLs have been shown to release interleukin-1 (IL-1) after suitable stimulation the effects of RU 41.740 on production of this cytokine from both monocytes and LGLs was studied. The drug stimulated IL-1 release from normal cells but monocyte and LGL IL-1 production from HCC patients was not influenced by RU 41.740 by RU 41.740. It is concluded, therefore, that although RU 41.740 can stimulate natural cytotoxicity and IL-1 production by normal LGLs, it is unable to correct the defect which exists in these functions in patients with large mass tumours.

Defective interleukin-1 production by natural killer cells of patients with cancer.

Large granular lymphocytes (LGLs) from patients with malignant disease and from controls were activated by endotoxin or K562 cells, and the supernatants assayed for interleukin-1 (IL-1) activity. Normal LGLs produced significant amounts of IL-1, the activity of which could be neutralized by anti-human IL-1 antiserum. In patients with advanced cancer depressed IL-1 production was observed, which generally correlated with the degree of cytotoxicity produced by the LGLs. Prior treatment of the LGLs with interferon increased production of IL-1 by both control and patient cells. It is suggested that LGLs coming into contact with K562 cells produce IL-1, which is important in the effector-target cell interaction. The decreased cytotoxic activity of LGLs from cancer patients could be related to a defect in IL-1 production, an effect which can be partially corrected by in vitro interferon treatment.

Lysis of fresh human tumor cells by autologous large granular lymphocytes from peripheral blood and pleural effusions.

Human lymphocytes and their subpopulations from the peripheral blood and pleural effusions of cancer patients were tested for cytotoxicity against fresh tumor cells isolated from carcinomatous pleural effusions of the same patients. Fresh tumor cells were relatively resistant to lysis by autologous unseparated lymphocytes in a 4 h Cr-release assay. Positive reactions were recorded in 10 of 38 blood samples and 10 of 37 effusion specimens. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation resulted in enhancement of cytotoxic activity against autologous tumor cells and K562 cells, with no reactivity in LGL-depleted small T-lymphocyte populations. Significant lysis of effusion tumor cells by autologous LGL was observed in 15 of 22 blood specimens and 15 of 21 effusion samples. Further depletion of high-affinity sheep erythrocyte rosetting cells from Percoll-purified LGL populations gave an increase in autologous tumor-killing activity. Depletion of LGL/K562 conjugates from LGL populations decreased lysis of autologous tumor cells and K562 cells. Effusion tumor cells that were susceptible to lysis by allogeneic normal LGL were also killed by autologous LGL, and effusion tumor cells resistant to lysis by allogeneic NK cells were not lysed by autologous LGL. In a single-cell cytotoxicity assay in agarose, 4-26% LGL bound autologous tumor cells and 0.2-5% LGL killed these target cells, while 12-45% LGL bound K562 cells and 2-20% LGL lysed them. These results indicate that cytotoxic potential for autologous effusion tumor cells is present in the peripheral blood and pleural effusions of cancer patients; it is strongly associated with a minor proportion of LGL and restricted to the cell population that can lyse NK-sensitive K562 cells.