Subset Of Large Granular Lymphocytes Research Articles

T Cell Receptor (α, β, γ) Gene Rearrangements and Expression in Normal and Leukemic Large Granular Lymphocytes/Natural Killer Cells

The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.

Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4− phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Expression of CD55 and CD59 on peripheral blood cells in patients with lymphoproliferative disease of granular lymphocytes

Lymphoproliferative disease of granular lymphocytes (LDGL) is a disorder characterized by the clonal expansion of granular lymphocytes. It has recently been shown that the clonal expansion of granular lymphocytes occurs in patients with paroxysmal nocturnal hemoglobinuria (PNH) in a subclinical fashion. To test the possibility that LDGL patients share a PNH phenotype, we obtained peripheral blood cells from 20 patients with LDGL and examined the expression of the glycosylphosphatidyl inositol (GPI)-anchored proteins, CD55 and CD59. Compared with normal controls, however, a defective expression of CD55/59 was not observed on either granulocytes or erythrocytes from LDGL patients. An unexpected finding was the significantly lower CD55/59 expression on granular lymphocytes from patients with CD16(+)CD56(-) phenotype LDGL than from patients with CD16(+)CD56(+) phenotype LDGL, or natural killer (NK) and NK/T lymphocytes from healthy individuals. The positive correlation between the expression of CD56 and CD55/59 might have some relevance to the functional properties of the CD56(+) subset of large granular lymphocytes.

Single Case Reports A Patient with Large Granular Lymphocytosis of Unusual Phenotype and Polymorphic T-Cell Receptor: Beta-Chain Gene Rearrangement

The authors describe a patient with large granular lymphocytosis who presented with fever of unknown origin and jaundice. Immunophenotyping showed that most of the large granular lymphocytes (LGLs) were CD3-, CD16-, and NKH-1 (Leu-19)+ lymphocytes. Lymphocytosis of this subset of LGLs has not been reported. Analysis of T-cell receptor gene showed polymorphic T-cell receptor beta-chain (TCR beta) gene configuration. Functional studies showed reduced natural killer cell function. The clinical course was very aggressive and resistant to chemotherapy. These features again raise the controversial issues of the ontogeny and heterogeneity of LGLs and their relationship to natural killer cells and T-cells.

Characterization of A Phenotypically and Functionally Distinct Subset of Large Granular Lymphocytes (Pit Cells) in Rat Liver Sinusoids

Pit cells, or large granular lymphocytes, with natural tumoricidal activity are found in the sinusoids of normal rat liver. Hepatic large granular lymphocytes are heterogeneous and can be subdivided into two subsets. These subsets were compared with peripheral-blood large granular lymphocytes and were found to differ phenotypically and functionally. Phenotypical differences included lower expression of the asialo-GM1 marker of natural killer cells, lower cellular density and many more small cytoplasmic granules in hepatic large, granular lymphocytes. Low-density hepatic large, granular lymphocytes were five to eight times more cytotoxic than blood large, granular lymphocytes on a per-cell basis as measured against YAC-1 cells and colon carcinoma cells. In addition, hepatic large, granular lymphocytes were able to lyse P-815 target cells, which are resistant to blood natural killer cells. Large, granular lymphocytes isolated from the liver also contained a subset with intermediate phenotypical and functional characteristics, possibly representing a transitional form between blood and "liver-specific" large, granular lymphocytes. The liver thus contains a specific population of highly activated or further differentiated large granular lymphocytes.

In vitro assays to study role of T cell-derived factors on human B lymphocytes should take into consideration inhibiting effect of large granular lymphocyte subset (CD5 −,CD16 +) that can contaminate B cell preparations

In these studies, the inhibitory role of a large granular lymphocyte (LGL) subset (CD5 −,CD16 +) on pokeweed mitogen (PWM)-induced B lymphocyte differentiation was examined. CD5 −,CD16 + LGL cells are the predominant subset of LGL cells and are possibly distinct from other LGL subsets in that they lack B and T cell markers. CD5 −,CD16 + LGL posses abundant FcIgG receptors and previous studies have clearly demonstrated that in the presence of insoluble immune complexes, this LGL subset will inhibit B lymphocyte differentiation in the presence of T cells. In the present studies, we analyzed the inhibiting role of CD5 −,CD16 + LGL cells that had not been activated by immune complexes. B + L preparations obtained by removal of E rosette-forming T cells were further depleted of T lymphocytes by complement-dependent lysis of T cells using a monoclonal antibody reactive to total T cells (Leu-1, CD5 antigen, Becton-Dickinson). B lymphocytes in such B + L preparations failed to differentiate into plasma cells containing intracytoplasmic immunoglobulin (Ig), in the presence of PWM, T cell-derived helper supernatants (THS), and interleukin-2 (IL-2). However, B cells differentiated under these conditions, when B + L preparations were further depleted of CD5 −,CD16 + LGL celld by complement-dependent lysis using a monoclonal antibody (Leu-11) reactive to CD16 antigen of FcIgG receptors present on LGL cells. These studies indicated that CD5 −,CD16 + cells unlike the CD8-positive T suppressor cell, will directly inhibit B lymphocyte differentiation into plasmacytoid cells containing intracytoplasmic Ig when T lymphocytes are not present. However, addition of a few T lymphocytes (< 10%) to purified B + L preparations abrogated the CD5 −,CD16 + LGL cell inhibition of B cell differentiation.

T-cell receptor gene rearrangements and expression in normal human large granular lymphocytes (LGL) and their pathological expansions

The lineage to which normal large granular lymphocytes/natural killer (LGL/NK) cells belong is controversial; in fact they share some surface markers and functional activities with monocytes, but also with T lymphocytes. The relationship of LGL to the T cell lineage by analysis with the T cell receptor (T-rec) gene has been investigated. Pure preparations of human LGL and their CD11(+) CD8(-) and CD11(-) CD8(+) subsets had the Tβ gene in its unrearranged germline configuration. Expression of Tα and Tβ genes was not detectable. The organization of Tγ gene, which is of particular importance because it occurs early in T cell ontogeny, was also found in its germline configuration.A rare type of lymphoproliferative disorder, termed Tγ-LPD, is characterized by expansion of cells very similar to LGL for morphology, phenotype, and functional activity. Of 17 patients with Tγ-LPD studied for T-rec rearrangement, 15 displayed rearrangement of Tβ and Tγ loci and were CD3+ (14/15 had monoclonal rearrangement), while 2 cases were in germline configuration and were CD3-. Similarly to very small subsets of CD3+ LGL recently described, most Tγ-LPD cases are CD3+ and have T-rec genes rearranged. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage; they also demonstrate that, in contrast to previous views, most TγLPD are monoclonal, presumably neoplastic, lymphoproliferative disorders.

Functional analysis of chronic T lymphocytosis with peculiar phenotype

The lymphocytes obtained from a patient with chronic T-cell lymphocytosis were analyzed. The patient has no clinical symptoms or sings during our 8-year observation. The lymphocytes showed the typical feature of large granular lymphocytes (LGLs) morphologically. They had peculiar phenotype different from that of LGLs previously reported. The surface markers of these lymphocytes were CD2+, CD3+, CD4+, CD5+, CD8-, CD11+, Leu7+, CD16- and FcIgG-, but these cells had neither helper nor suppressor action on IgG production by normal B lymphocytes. Natural killer activity of freshly isolated cells could not be detected even after augmentation with interleukin 2 (IL2). Lymphokine activated killer activity could not be detected, either. These cells were able to proliferate upon stimulation with such mitogens as PHA and PWM, and to produce IL2. But they were not induced to proliferate by IL2 even if stimulated with ConA beforehand. Almost all cells were in the resting state according to the cell cycle analysis. The LGLs of this case are a novel subset of LGLs in both phenotype and immunological function. Only a few cases of CD4+ LGL lymphocytosis or leukemia have been reported previously, but the further phenotype and the function of these cases varied. The results show that, even in CD4+ LGLs, there may be heterogeneous subpopulation.

Cryopreservation and short-term storage of human lymphocytes for surface marker analysis Comparison of three methods

Three methods were compared for the cryopreservation and short-term storage of human lymphocytes for cell surface marker analysis with monoclonal antibodies and flow cytometry. These methods were (1) controlled-rate freezing followed by storage in liquid nitrogen (CR), (2) direct placement into liquid nitrogen (LN), and (3) direct placement at −70°C (−70). Our findings show that the LN and −70 methods of cryopreserving lymphocytes are as effective as the CR method for the purpose of determining T cell and large granular lymphocyte subsets within 1 month after freezing.

Opportunistic infections in acquired immune deficiency syndrome result from synergistic defects of both the natural and adaptive components of cellular immunity.

We evaluated the cellular immunity of 408 clinically stratified subjects at risk for acquired immune deficiency syndrome (AIDS), to define the role of interferon-alpha production deficits in the pathogenesis of opportunistic infections (OI). We followed 115 prospectively for up to 45 mo. Onset of OI was associated with, and predicted by, deficiency both of interferon-alpha generation in vitro, and of circulating Leu-3a+ cells. Interferon-alpha production is an index of the function of certain non-T, non-B, large granular lymphocytes (LGL) that are independent of T cell help. Leu-3a+ cell counts are a marker of T cell function. OI did not usually develop until both of these mutually independent immune functions were simultaneously critically depressed, leading to a synergistic interaction. These data suggest that the AIDS virus affects a subset of LGL, and that cytokine production by these cells is an important component of the host defense against intracellular pathogens that becomes crucial in the presence of severe T cell immunodeficiency.

Immunological and genotypic analysis of human T gamma-lymphoproliferative disorders.

T gamma-lymphoproliferative disorders (T gamma-LPD) are rare diseases characterized by expansion of circulating elements with resemblance to large granular lymphocytes (LGL). We have studied 12 patients with T gamma-LPD. Morphological evaluation revealed 79-88% of LGL in non-adherent peripheral blood lymphocytes as assessed by light and electron microscopy. The most common features of the membrane phenotype included expression of T3, HNK-1 and AB8.28 (anti-Fc gamma); other surface markers of LGL (OKM1, B73.1, N901) were variably expressed or absent. Patients' LGL usually had little or no NK activity, with the exception of two patients who had values comparable to those of normal donors; in addition, cell preparations from all patients mediated antibody-dependent cellular cytotoxicity. The recent availability of the T cell receptor beta chain probes allowed us to investigate the lineage and the clonality of T gamma-LPD. Of the 12 patients analyzed, 10 displayed clonal rearrangements of T beta locus and expression of the T3 antigen, whereas the two remaining cases displayed a germ-line configuration of the T beta gene and no expression of the T3 antigen. We suggest that individual T gamma-LPD cases represent the clonal expansion of cells frozen at different stages of differentiation/activation within an individual hematopoietic LGL/NK lineage. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage.

Ultrastructural characterization of human large granular lymphocyte subsets defined by the expression of HNK-1 (Leu-7), Leu-11, or both HNK-1 and Leu-11 antigens.

A peroxidase-colloidal gold double labeling system in immunoelectron microscopy was used to investigate the ultrastructural features of human large granular lymphocytes (LGL) subpopulations. Three subsets of LGL, Leu-7+-Leu-11-, Leu-7+-Leu-11+, Leu-7- -Leu-11+, were characterized using combinations of the monoclonal antibodies anti-Leu-7 and anti-Leu-11. They showed different ultrastructural patterns. In fact, Leu-7+-Leu-11- cells showed a high nuclear/cytoplasmic ratio (N/C), a round nucleus, a cytoplasm with few organelles, and a rather even surface. Moreover, most of them lacked electron-dense granules. On the other hand, Leu-11+ cells displayed a low N/C, an irregular-shaped nucleus, and a cytoplasm containing a well-developed Golgi apparatus, many mitochondria, vacuoles, vesicles, and numerous electron-dense granules. Moreover, they exhibited an irregular cell surface. Thus, Leu-7+-Leu-11- cells seemed to represent an immature form of LGL, while cells expressing the Leu-11 antigen showed a fine structure specific for functional NK cells. Our findings suggest that the expression of HNK-1 (Leu-7) and Leu-11 antigens respectively represents subsequent stages in NK cell differentiation.

Capacity of human large granular lymphocytes (LGL) to produce multiple lymphokines: interleukin 2, interferon, and colony stimulating factor.

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.

The role of calmodulin in human natural killer cell activity.

Calcium is known to play an essential role in the lytic mechanism of natural killer cells (NK), which form a subset of large granular lymphocytes. Many of the intracellular effects of calcium are mediated through the calcium-binding protein calmodulin. In this study we have demonstrated that the specific calmodulin inhibitors (naphthalene-sulphonamides) inhibit NK activity in humans at IC50's of 6.9 microM for W7 and 5.2 microM for W13. Comparison of the potency of these compounds with their less active counterparts suggests that NK activity is calmodulin-dependent.