Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8⁺ lymphocytes a higher proportion of OKT4⁺ lymphocytes was ANAE-positive exibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for T_G and T_M lymphocytes.     

Alpha-Naphthyl Acetate Esterase Activity in Human Lymphocytes: Distribution in Lymphocyte Subpopulations and in Mitogen-activated Cells

The cytochemical demonstration of nonspecific alpha-naphthyl acetate esterase (ANAE) activity in human peripheral blood mononuclear cells was studied. Different staining patterns were found, allowing differentiation of mononuclear cells into macrophages (strong granular cytoplasmic activity), B lymphocytes (negative reaction), Tγ lymphocytes, i.e. bearing IgG Fc receptors (granular scattered reaction), and T non-γ lymphocytes, i.e. devoid of IgG Fc receptors (single cytoplasmic ANAE spot). During the early phases of phytohaemagglutinin (PHA)- and concanavalin A (Con A)-induced activation, the reactivity of most lymphocytes became granular and scattered, similar to that found in T_γ cells. Blast cells generating in successive phases, appeared devoid of detectable enzymatic activity. The hypothesis is put forth that T cells showing granular, scattered reactivity represent a population of activated cells and that the redistribution of enzymatic activity could represent a preliminary step leading to secretion (lymphokine-like?) of enzyme from cytoplasm in the course of cell activation.     

Suppression of B lymphocyte differentiation by newborn T lymphocytes with an Fc receptor for IgM

The IgM (Fc)-binding subpopulation of human newborn T lymphocytes (T_M) suppressed the pokeweed mitogen induced differentiation of adult B lymphocytes. Positively isolated IgG (Fc)-binding T lymphocytes (T_G) also suppressed, but lymphocyte preparations negatively enriched for T_G by the depletion of T_M cells were not suppressive. The Fc receptor specificity of the human newborn suppressor appears to resemble that of the human Con A suppressor.     

A human natural killer cell-associated antigen defined by monoclonal antibody anti-Leu (NKP-15): functional and two-color flow cytometry analysis

A monoclonal antibody, anti-Leu 11a (NPK-15), was generated against human large granular lymphocytes (LGL). Anti-Leu 11a reacted with the majority of Percoll gradient-enriched LGL cells, a subpopulation of peripheral blood lymphocytes (PBL) approx (approximately 10-20%), and most granulocytes, but not with a significant number of monocytes, T lymphocytes, or erythrocytes. Cell sorting experiments demonstrated that the Leu 11a+ population encompassed essentially all functional natural killer (NK) cells in the peripheral blood. Two-color flow cytometry analysis of PBL populations stained with anti-Leu 11a and anti-Leu 7 revealed the existence of four distinct populations: Leu 11a-, 7+; Leu 11a+, 7-; Leu 11a+, 7+; and Leu 11a-, 7-. The Leu 11a+ population did not appear to include cells marked with the T cell-associated antigens Leu 1, Leu 2, or Leu 3. The existence of a cell surface antigen common to granulocytes and NK cells, which is capable of distinguishing subpopulations of Leu 7+ cells, provides a useful probe to analyze the nature of the NK lineage.     

Human large granular lymphocytes and natural killing: Ultrastructural studies of strontium-induced degranulation

Large granular lymphocytes (LGL) are a subpopulation of human peripheral blood mononuclear cells believed to contain the mediators of spontaneous cytotoxicity or natural killing. In the present study, the mononuclear cells were enriched for LGL by differential density centrifugation on Percoll gradients and examined by transmission electron microscopy. The ultrastructure of LGL and of their binding interaction with natural killer-susceptible target cells (K562) is described in detail. Some morphological similarity between LGL and cells of a myelocytic origin was observed. Studies reported elsewhere have shown that Sr²⁺, an alkaline earth ion known to degranulate granulocytes, inhibits NK cell function. Comparison of the morphology of control and Sr²⁺-treated LGL showed that Sr²⁺ caused several characteristic changes in LGL ultrastructure and, indeed, led to their degranulation. The recovery of natural killer function seen following in vitro culture of Sr²⁺-treated effector cells was accompanied by the reappearance of typical intracytoplasmic granules. These data strongly suggest that the granules of LGL are required for and, perhaps, involved in natural killer-mediated cytolysis.     

白细胞介素2和黄芪多糖对小鼠NK细胞作用的细胞化学和超微结构观察

本文报道在白细胞介素2(IL-2)及黄芪多糖(PA)作用下,小鼠NK细胞的细胞化学和超微结构变化。实验表明,NK细胞酸性磷酸酶、酸性醋酸酯酶及氯醋酸AS-D酯酶均为阳性。经IL-2和PA的处理,前两种酶的活性增加,阳性细胞百分率增高,并与NK活性相关。IL-2培养后的NK细胞超微结构表现为胞体变大,表面不规则,胞质丰富,细胞器增多等特点。     

Complement Receptor Distinguishes between Two Subsets of Large Granular Lymphocytes with Different Natural Killer Activity and Cytochemical and Ultrastructural Features

Human peripheral blood large granular lymphocytes (LGL)--that is, cells with intracytoplasmic azurophilic (electron-dense) granules, with a positivity for the cytochemical localization of certain acid hydrolases, and with avid surface receptors for the Fc portion of IgG--have been purified on Percoll density gradients. Approximately 30% of these cells expressed receptors for the third complement component (C3R). They were separated into C3R-positive and C3R-negative cells. C3R+ cells had a significantly greater natural killer (NK) activity against K562 target cells than C3R+ cells. This difference was unrelated to the presence in the C3R+ cells of a contaminant cell type incapable of NK activity, since cytochemical and ultrastructural analysis revealed that C3R+ and C3R- fractions contained comparable LGL numbers. Agarose cytotoxicity assays at the single-cell level demonstrated that C3R+ LGL contained a large number of cells that bound to but did not lyse the target. The remaining fully cytotoxic C3R+ LGL had, however, the same killing and recycling properties as the cells from the C3R fraction. Electron microscopy and cytochemical studies showed that C3R+ cells had fewer electron-dense granules than C3R cells and stained more faintly for the localization of alpha-naphtyl acetate esterase. In contrast to C3R cells, C3R+ LGL displayed morphological features suggesting that an active process of granule formation was taking place. Taken together, the data indicate that C3R+ cells represent a discrete subset or a maturational stage of LGL.     

Ultrastructural Localization of Alpha-Naphthyl Acid Esterase in Human TM Lymphocytes

The localization of alpha-naphthyl acid esterase (ANAE) activity in T lymphocytes with receptors for IgM (T_M cells) have been studied at the electron microscope. The electron-opaque product of the cytochemical reaction was detected around, but never inside, single (or groups of) vesicles, which suggested a possible membrane localization of the enzyme activity. These same vesicles were found to contain acid phosphatase by both light- and electron-microscopic examination and were bound by unit membranes; this data indicates that they likely represent primary lysosomes. The presence of such lysosomes in a restricted paranuclear area is a distinctive feature of T_M cells.     

Binding of monoclonal antibody to CD16 causes calcium mobilization in large granular lymphocytes but inhibits NK killing.

A monoclonal antibody (mAb), CLB/FcR gran I, reactive with the CD16 Fc receptor (FcRlo/FcRIII) of human cells, leads to calcium mobilization in large granular lymphocytes (LGL) but not in granulocytes. Identical responses are obtained with F(ab')2 fragments of this antibody, indicating that the response is independent of Fc-FcR binding, and that bivalent cross-linking of this receptor is adequate for optimal calcium mobilization. The calcium response was greater in CD3- LGL compared to CD3+ LGL, although the response was augmented in the latter cells by prior rosetting with sheep red blood cells (SRBC). Calcium mobilization in CD3- LGL induced by CLB/FcR gran I is associated with inhibition of natural killer cell (NK) killing, and inhibition of the enhanced NK killing induced by the anti-CD2 low-density monoclonal antibody, 9.1. This supports the view that the NK-enhancing activity of 9.1 is due to simultaneous binding to CD2 and CD16, and may in fact be transduced through the CD16 molecule. The variable reported effects of anti-CD16 antibodies on NK killing are likely to reflect the epitope bound rather than the isotype of antibody used, since F(ab')2 fragments of CLB/FcR gran I also inhibit NK killing.     

N-α-Benzyloxycarbonyl-l-Lysine Thiobenzyl Ester (BLT) Serine Esterase in Human Cytolytic Effector Cells and Cell Line Targets

The granules of in vitro primed cytotoxic mouse T cells and cytotoxic cell lines have been shown to contain high levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase. The enzyme activity has been suggested to be associated with the cytotoxic capacity of killer cells. We investigated human leucocytes and found that neutrophils, monocytes, cytotoxic T lymphocytes (CTL), natural killer (NK) cells [large granular lymphocytes (LGL)], and interleukin 2 activated killer (LAK) cells, which all display efficient cytotoxic capacity, show only marginal BLT esterase activity. The low BLT esterase activity in human lymphocytes increases about twofold when cells are stimulated in vitro with interleukin 2 (IL-2), phytohaemagglutinin (PHA), or cultured in mixed lymphocyte culture (MLC). Mouse T lymphocytes have about 20 times more BLT esterase activity than human T lymphocytes. The BLT activity in mouse T cells also increases about twofold in MLC. The human leukaemia cell lines (K562, U937, MOLT-4, Jurkat) and the mouse mastocytoma line (P815), which are frequently used as target cells, contain more BLT esterase activity than human resting or activated lymphocytes. We did not find a direct correlation between the cytotoxic capacity and the BLT esterase activity of killer cells.     

A lymphoproliferative disorder of the large granular lymphocytes with natural killer activity

This paper reports the case of a patient with an abnormally expanded population of circulating lymphoid cells displaying the features of the so-called large granular lymphocytes (LGL). These cells were peroxidase negative and nonphagocytic, formed rosettes with sheep erythrocytes, had receptors for IgG, and contained azurophilic (electron-dense) granules. Like normal LGL, the patient cells were positive for two acid hydrolases (acid phosphatase and β-glucuronidase) but did not stain for α-naphthyl acetate esterase (ANAE), which is present in normal LGL. Ultrastructural studies revealed that the patient cells were rich in Golgi-derived vesicles, coated vesicles, multivesicular bodies, and immature granules, indicating that, unlike normal LGL, they were engaged in granulogenesis. These features, together with the absence of ANAE activity, are suggestive of some degree of cell immaturity. The patient cells displayed natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities comparable to those of normal peripheral blood mononuclear cells, or even higher, and did not respond to T-cell mitogens or allogeneic cells. Furthermore, they were incapable of suppressing normal T-cell proliferation or pokeweed mitogen-induced B-cell differentiation. Analysis of the NK activity at the single-cell level revealed that a large proportion of the patient cells bound to the K562 target cells but could not accomplish the entire lytic process. This finding supports further the possibility that the patient cells were immature LGL. The surface phenotype of the patient cells (as defined by a battery of monoclonal antibodies) was somewhat different from that usually observed in the majority of the normal LGL because, in addition to the HNK-1 marker, the cells were OKT3⁺, aLeul⁺, aLeu4⁺, OKT8⁺, aLeu2a⁺, and 3A1⁺ but were OKM1^? and 4F2^?. This phenotype could correspond to that of maturing LGL.     

Involvement of HLA-DR+ large granular lymphocytes in the induction of tumor necrosis factor by Mycobacterium avium-intracellulare complex.

This study shows that normal human large granular lymphocytes (LGL) secrete tumor necrosis factor (TNF) in response to Mycobacterium avium-intracellulare complex (MAI). Percoll density gradient fractionation of peripheral mononuclear cells showed TNF activity in the fractions corresponding to LGL and not T cells, even when 5% monocytes were added to the T lymphocytes for accessory function. TNF release was not abrogated by treatment of the crude LGL preparations with anti-Leu M3, -CD4, and -CD8 antibodies (Ab) plus complement (C), but was abrogated by anti-CD16 and -CD2 Ab, as expected. Interestingly, anti-HLA-DR monoclonal antibody (mAb) treatment significantly diminished TNF activity from LGL, but maintained natural killer (NK) cell function unmodified as opposed to CD2+ and CD16+ cell depletion. Panning studies demonstrated that TNF secretion upon MAI stimulation resided only in the HLA-DR+ LGL and not the DR- LGL population. These results indicate that normal fresh HLA-DR+ LGL, as well as monocytes, are also responsible for rapid TNF secretion during early MAI infection. These DR+ cells appear to be distinct from those expressing NK function.     

Cytochemistry of Human T-Cell Subpopulations

Acid phosphatase and esterase cytochemistry performed on purified normal human T-cell populations showed that both methods produced distinctive localized dot patterns of reactivity in 60–70% of cells. By examination of rosette preparations formed with ox erythrocytes coated with IgM (EAM), with IgG (EAG), or anti-human κ and λ light chains, it was shown that this pattern of reactivity was largely restricted to small T lymphocytes possessing receptors for the Fc of IgG (Tμ cells). In addition, both B lymphocytes and T cells with receptors for the Fc of IgG (Tλ cells) were larger lymphocytes with more abundant cytoplasm and usually displayed scattered granular acid phosphatase activity; in esterase preparations both cell types were either negative or possessed similar scattered granular positivity. As compared with Tμ cells, Tγ cells were seen to form loose spontaneous rosettes with sheep erythrocytes. Combined esterase and acid phosphatase staining showed that both enzyme activities in the Tμ. cells are localized in the same area, and ultrastructural acid phosphatase cytochemistry established that this was in distinctive lysosomal structures. Tμ staining by both esterase and acid phosphatase cytochemistry was greatly reduced after rosetting with EAG, but not after rosette formation with EAM or sheep erythrocytes.     

NKP-15: A monoclonal antibody reactive against purified human natural killer cells and granulocytes

A monoclonal antibody of the isotype IgG₁,k, NKP-15, was produced by immunizing Balb/c mice with highly purified suspensions of human natural killer (NK) cells. By indirect immunofluorescence, NKP-15 reacted with 15 ± 5.0% of the peripheral blood lymphocytes, 90 ± 3.0% of the granulocytes and was non-reactive with T-cells, B-cells, monocytes, platelets and erythrocytes. NKP-15 reacted with 90 ± 6.0% of purified large granular lymphocytes (LGL), which displayed highly enriched NK cell activity. NKP-15, however, showed no reactivity with a panel of human tumor cell lines.Analysis of NK cell-K562 tumor cell conjugates and depletion by panning of NKP-15⁺ cells indicated that all functionally tumorlytic NK cells expressed the NKP-15 antigen. It was also determined by competitive blocking experiments that NKP-15 recognizes a new NK cell surface antigen not delineated by the NK-specific monoclonal antibody, Leu-7, and that the epitope for NKP-15 appears to be associated with the Fc-receptor of NK cells and granulocytes.     

Receptors for the Third Complement Component on a Proportion of Large Granular Lymphocytes from Human Peripheral Blood

Large granular lymphocytes (LGL) are nonadherent cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and a paranuclear localization of alpha-naphthyl acid esterase or acid phosphatase. LGL constitute the bulk of TG cells (cells with receptors for sheep erythrocytes and for IgG molecules) and null cells (non-T, non-B cells). In the present study we demonstrate that 20-33% of the circulating human LGL express receptors for the third complement component (C3R). When TG cell or null cell fractions from normal individuals or non-T cells from a patient with infantile agammaglobulinaemia (which contained almost exclusively LGL) were rosetted with erythrocytes coated with antibody and complement, a variable number of C3R-bearing cells were detected. Such cells were isolated and analysed further; the great majority of them displayed the cytochemical and ultrastructural features of LGL.     

Large granular lymphocytes: Morphological studies

Large granular lymphocytes from normal human blood were enriched by centrifugation on discontinuous Percoll density gradients. Their capacity for natural killing, but not for phagocytosis of yeast cells, was demonstrated. Large granular lymphocytes are characterized in electron microscopy by their fine structure, especially by typical granules and by inclusions of tubular structures in a parallel array. Their lymphocyte nature is supported by activity of acid-α-naphthyl acetate esterase and by the absence of myelo-peroxidase (POX) and of macrophage POX. The Fcγ receptor of their cell membrane is marked by soluble POX-anti-POX-complexes; labeled parts of their membrane are not incorporated into the cytoplasm as in monocytes.     

Spontaneous cell-mediated cytotoxicity (SCMC) associated with lymphocytes negative for acid alpha-naphthyl acetate esterase (ANAE) activity.

A modified histochemical procedure for nonspecific acid alpha-naphthyl acetate esterase (ANAE) activity in human lymphocytes was used to identify a subpopulation of E-rosette forming cells. Performing a one hour reaction at pH 6.5 the distinct dot-like staining pattern was almost exclusively observed on high affinity E-rosettes which sedimented readily in a regular Ficoll-Metrizoate gradient. By combining latex phagocytosis with staining for ANAE activity, a clear-cut distinction between mononuclear phagocytes and lymphocytes could be made. An attempt was undertaken to relate the ANAE marker on human lymphocytes to their functional capacity in spontaneous cell-mediated cytotoxicity (SCMC) reactions. Using as targets two allogeneic (K562,IGR3) and a xenogeneic cell line (L1210) it could be clearly demonstrated that high SCMC activity is mediated by ANAE negative mononuclear cells, whereas enrichment for ANAE positive lymphocytes resulted in a loss of SCMC.     

T lymphocyte development in the absence of Fcϵ receptor Iγ subunit: analysis of thymic-dependent and independent αβ and γδ pathways

During fetal development, early thymocyte progenitors transiently express low affinity Fc receptors for IgG (FcγR) of both FcγRII and III isoforms. Only the FcγRIII isoform requires association of an FcγRIII (CD16) α subunit with an FcϵRIγ homodimer for surface expression. To address the role of FcγR in ontogeny, we studied thymic development in FcϵRIγ^−/− mice. We find that day 14.5 CD4⁻CD8⁻ double-negative (DN) fetal thymocytes of FcϵRIγ^−/− mice express mRNA of both FcγRIIb₁ and FcγRIII. Surface expression of FcγRII/III is readily detected on these cells. It appears that FcγRIIb₁, whose surface expression is FcϵRIγ independent, replaces FcγRIII during thymic development in these animals. Moreover, subsequent development into CD4⁺CD8⁺ double-positive and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive subsets appears normal even in the absence of FcϵRIγ. However, alterations were noted in adult animals among the DN αβ TCR⁺ thymocytes and peripheral splenic DN T cells as well as CD8αα⁺ intestinal intraepithelial lymphocytes (iIEL). In contrast to conventional T lymphocytes, which do not express either FcγRIII or FcϵRIγ, DN αβ TCR⁺ thymocytes and extrathymically derived αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL express TCR which incorporate FcϵRIγ as one of their subunits. Consistent with this, the TCR levels of these cells are lower than the TCR levels on cells from wild-type C57BL/6 mice. Despite the reduction in the level of surface TCR, the development of these cells was unaltered by the absence of FcϵRIγ. Thus, we observed alterations in adult DN αβ TCR⁺ thymocytes, splenic DN αβ TCR⁺ and DN γδ TCR⁺ large granular lymphocytes (LGL), and αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL, but no detectable changes in their major fetal thymic developmental pathways. Cultivation of peripheral DN αβ TCR⁺ and DN γδ TCR⁺ cells from FcϵRIγ^−/− mice with interleukin-2 generates LGL which mediate natural killer activity. Unlike LGL from wild-type C57BL/6 mice, LGL from FcϵRIγ^−/− mice lack FcγRIII expression and could not mediate antibody-dependent cellular cytotoxicity through FcγRIII.     

Are “natural killer” cells involved in allograft rejection?

"Natural killer" (NK) effector cells and large granular lymphocytes (LGL) are found inside rat renal allografts during rejection. Their appearance in situ precedes the appearance of cytotoxic T lymphocytes, and concomitantly with their influx in the allograft, the NK activity and the LGL are depleted from the recipient spleen. This suggests that the NK effector cells and the LGL are involved in allograft rejection, although their role(s) among the other in situ inflammatory effector pathways remains to be clarified.     

Expression of the CMRF-35 antigen, a new member of the immunoglobulin gene superfamily, is differentially regulated on leucocytes.

A new monoclonal antibody, CMRF-35, has been generated that recognized a 224 amino acid cell surface protein which is a novel member of the immunoglobulin gene superfamily. The antibody, raised against large granular lymphocytes (LGL), stains LGL, monocytes, macrophages and granulocytes but not platelets or erythrocytes. In addition, a subset of peripheral blood T lymphocytes (26.6 +/- 13.4% CD5+ cells) and B lymphocytes (13.7 +/- 6.8% CD20+ cells) stained with CMRF-35 but tonsil T and B cells were essentially negative. Expression of the CMRF-35 antigen (Ag) on different leucocyte populations was markedly influenced by stimulation of the cells with mitogens and cytokines. Activation of peripheral blood T cells with phytohaemagglutinin (PHA), or phorbol myristate acetate (PMA) and calcium ionophore (CaI) led to a decrease in the proportion of CMRF-35+ T lymphocytes. In contrast, PHA activation of tonsil T lymphocytes resulted in an increase in CMRF-35 Ag expression (47.1 +/- 1.5% CD5 cells at 6 days). An increase in CMRF-35 Ag was also seen on phorbol ester and CaI-activated tonsil B cells. No change in CMRF-35 expression on natural killer (NK) cells occurred following activation with interleukin-2 (IL-2) but the CMRF-35 Ag was down-regulated following Fc receptor stimulation. A moderate increase in CMRF-35 expression occurred during monocyte-macrophage differentiation and the expression of the Ag on monocytes was differentially regulated by interferon-gamma (IFN-gamma). This regulation of the CMRF-35 Ag on the leucocyte surface suggests that the molecule has an important function common to diverse leucocyte types.