Percentage Of Large Granular Lymphocytes Research Articles

NK Cell Homing and Persistence in Bone Marrow after Adoptive Immunotherapy Correlates with Therapy Response

Background: Natural killer (NK) cells are innate lymphoid cells capable of killing leukemic cells without prior sensitization. Adoptive immunotherapy with allogeneic NK cells has been used at the University of Minnesota and was shown to be effective in a subset of patients with acute myeloid leukemia refractory to therapy. The factors determining clinical success of adoptive NK cell immunotherapy are largely unknown. We hypothesized that NK cell homing and persistence in the bone marrow niche may be predictive for the clinical outcomes.Methods: We retrospectively analyzed bone marrow biopsy findings 14 days after NK cell infusion in 109 patients treated at our institution between 2003-2014. To evaluate the homing and persistence of NK cell in the bone marrow, multiple methods were used. Immunohistochemical staining for the NK cell marker CD56 was performed on the core biopsy in available cases. Flow cytometry data performed on aspirate material at the time of the biopsy was re-evaluated to enumerate the NK cell percentage. Finally the percentage of Large Granular Lymphocytes was determined in a semi-quantitative manner from Wright-Giemsa stained aspirate smears. CD56 immunohistochemical stain was quantified by counting the number of CD56 positive cells per microscopic field of view (High Power Field, HPF) (400 x magnifications) and averaging over the 10 microscopic fields with the highest density of CD56 positive cells. Cases with obvious CD56 staining of blasts on the trephine core were excluded from further analysis.Results: 95 bone marrow biopsy cases could be stained for CD56, but 6 cases were excluded from analysis due to CD56 staining leukemic blast population. For the whole study group the average CD56+ cell density per High Power Field (HPF) was 10.1 (SD=17). A control group composed of 10 patients with bone marrow biopsy shortly after chemotherapy, but who did not receive NK cell infusion had an average of 3.8 CD56+ cells per HPF (SD=3.4).Patients who had a higher density of NK cells in the bone marrow compared to the control group (more than 3.8 cells/HPF) were less likely to have persistence of acute leukemia as defined by ≥5% blasts, than patients with an NK cell density similar to the control group (Figure 1, Chi-square test, p=0.037). As well, patients with <5% leukemic blasts had more CD56 positive cells per HPF (Median 5.6, range 0.1-95.8 Versus median 1.8, range 0.1-28.1, p=0.009, Mann-Whitney test) and had a greater proportion of NK cells by flow cytometry (p=0.004, Mann-Whitney test).The clinical factors which may affect NK cell homing and persistence were also evaluated. Total Body Irradiation (TBI) at a dose 200 cGy for 2 days was applied as part of pre-infusion conditioning in a subset of the patients. Patients who received TBI (32 with TBI Vs 55 without) had a significantly higher density of CD56 positive cells (Figure 2, Median 4.75Vs 2.8, p=0.04, Mann-Whitney test). Among those patients who did not receive TBI conditioning, a subset received an anti-CD25 immunotoxin agent to reduce regulatory T cells (Denileukin Difitox, Ontak). The density of NK cells was significantly higher in the patients who received anti-CD25 immunotoxin (Figure 3, Median 9.8 Vs 2.5, p=0.02, Mann-Whitney test).Conclusions: Homing and survival/expansion of adoptively transferred NK cells in the bone marrow 14 days after infusion correlates with morphologic remission of acute leukemia. While the factors influencing NK cell homing and persistence in the BM niche remain to be elucidated, our results suggest a role for TBI as part of the conditioning and anti-CD25 immunotoxin in promoting the persistence of adoptively transferred NK cells in the bone marrow niche. [Display omitted] DisclosuresMiller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Changes in cellular immunity among workers occupationally exposed to styrene in a plastics lamination plant

Background Styrene is a widely used industrial chemical. Immune and hematological parameters were examined in 29 hand laminators and sprayers exposed to styrene for an average of 14 years and in 19 in-factory unexposed controls. The workers performed hand lamination procedures in a production area with an average area airborne styrene level of 139.5 mg/m3. Mean concentration of styrene in the blood of exposed workers was 945.7 μg/L and the mean styrene in exhaled air was 38.8 μg/L. Methods Parameters of internal and external exposure, immune function assays, immunoglobulins, acute phase reactants and hematology were evaluated in exposed and non-exposed populations. Results Using multifactorial analysis of variance we found a significant decrease in proliferation of lymphocytes stimulated by Concanavalin A but not by pokeweed mitogen (PWM) in workers occupationally exposed to styrene. Proliferative response to PWM was significantly correlated with the levels of styrene in blood. Phagocytic activity of monocytes, levels of IgG, IgA, IgM, IgE and alpha-2-macroglobulin in serum were indistinguishable in the two groups. The population exposed to styrene had increased levels of C4-component of complement. Levels of C3-component of complement were positively correlated with duration of exposure. A significant elevation in the percentage and number of monocytes and a significantly decreased number of lymphocytes were seen in exposed workers. Styrene concentrations in both blood and exhaled air were associated with decreased percentage of large granular lymphocytes. Conclusions These results suggest immune alterations of cell-mediated immune response of T-lymphocytes and imbalance in leucocyte subsets in peripheral blood of workers exposed to styrene. Am. J. Ind. Med. 38:576–583, 2000. © 2000 Wiley-Liss, Inc.

Anti-C-Reactive Protein Inhibits Cytoskeletal Rearrangement without Altering Calcium Influx in Natural Killer Cell Activation

C-reactive protein (CRP), an acute phase protein in human serum, is present on large granular lymphocytes (LGL). Anti-CRP inhibits natural killer (NK) cell-mediated lysis. Our current study shows that anti-CRP also inhibits antibody-dependent cell-mediated cytotoxicity (ADCC) of LGL. Calcium influx and protein kinase C (PKC) activation are the early signal transduction events in NK activation. In the conjugates formed between LGL and targets (NK or ADCC), 75-90% of LGL respond with a calcium influx. Addition of anti-CRP had no effect on the percentage of LGL which respond to target cell binding or on the magnitude of the calcium response of LGL. This was true for both NK and ADCC effector cells. Crosslinking anti-CRP with a secondary antibody did not alter this result. Next, the effect of PMA, a PKC activator, and calcium ionophore, A23187, on anti-CRP-mediated inhibition of cytotoxicity were studied. PMA alone reversed most of the inhibition of lysis seen with anti-CRP. Based on previous observations that anti-CRP inhibited target cell-stimulated release of lytic factors, the effect of anti-CRP on release of lytic factors stimulated by PMA and calcium ionophore was evaluated. Anti-CRP blocked the release of lytic factors stimulated by PMA and ionophore. Release of lytic factors involves the rearrangement of cyloskeletal element of NK cell toward the target cell. The effect of anti-CRP on cytoskeletal reorganization was studied. In conjugates formed between effector and target cells, the polarization of cytoskeleton at the contact site of NK and target cell was significantly reduced in the presence of anti-CRP. Although anti-CRP inhibits both ADCC and NK lytic mechanisms, it does not alter target cell-induced Ca 2+ influx. CRP interacts with the secretory mechanisms involved in granule exocytosis since anti-CRP inhibits the cytoskeletal polarization and the release of lytic factors and PMA might reverse anti-CRP-mediated inhibition by activating alternative mechanisms of cytotoxicity in effectors.

Qualitative defects in natural killer cell function in ia osteopetrotic rats.

Recent studies have provided evidence that cells of the immune system and their associated cytokines function in the regulation of bone turnover. The incisors absent (ia) osteopetrotic rat represents a model in which a defect in the immune system and bone resorption can be studied. Osteopetrosis in the ia rat is characterized by a generalized excess accumulation of bone as a result of reduced bone resorption by defective osteoclasts that lack a ruffled border and the ability to exocytose their osteolytic enzymes. Previous attempts to identify associated defects in the ia immune system have proven unsuccessful; ia rats demonstrate normal delayed hypersensitivity, mitogenic activity, and macrophage function. Inasmuch as the skeletal manifestations of the ia mutation may be the result of a defect in exocytosis, related defects may be evident in immune cells utilizing exocytosis of granules or enzymes for their cytolytic function. Natural killer (NK) cells function by such a mechanism. Therefore, these studies were undertaken to evaluate the natural immune system in ia rats. NK activity assessed by 51Cr release assays was significantly reduced in ia animals compared to normal littermates. Mononuclear cells isolated from the peripheral blood of ia rats revealed a significantly greater percentage of large granular lymphocytes than normal littermates. Comparison of NK cell phenotypes using two phenotypic parameters for NK cells (OX8+, OX19- cells and 3.2.3+ cells) revealed that the mononuclear isolates of spleen and peripheral blood of mutant animals had significantly greater percentages of OX8+, OX19- and 3.2.3+ cells than normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)

BRM (OK-432およびGe-132)によるメチルコラントレン誘発肉腫の発症抑制に関する研究

The suppressive effects of OK-432 and Ge-132 on 3-methylcholanthrene (MC) carcinogenesis have been confirmed. To clarify the mechanism of this suppression, we investigated the effects of anti-asialo GM1 antibody (AGM1), anti-thymocyte antibody (Thy) and carrageenan (CAR), which eliminate NK cells, T cells and macrophages, respectively, on the growth of MC induced sarcoma. The suppressive effect of Ge-132 in mice was abolished by prior AGM1, Thy or CAR treatment; moreover, these compounds enhanced the development of tumors. In mice treated with OK-432, AGM1 or CAR enhanced the development of tumors, as well; however, Thy treatment did not alter tumor incidence.Prior treatment with AGM1 reduced the percentage of large granular lymphocytes and NK activity, which were increased by OK-432 or Ge-132 treatment; however Thy or CAR did not alter these parameters.These results indicate that NK cells and macrophages play an important role in the defense against MC induced sarcoma in OK-432-treated mice, while T cells as well as NK cells and macrophages do so in Ge-132-treated mice.

Mechanisms of depressed natural killer cell activity in recurrent aphthous ulcers

Natural killer (NK) cell activity was studied serially in the peripheral blood obtained from 35 patients with recurrent aphthous ulcers (RAU) and from 46 age/sex-matched normal healthy controls. The NK cell activity was assayed by a 4-hr 51Cr release assay using K562 cells as targets. The results showed that the patients in remission (2 weeks of convalescence) had normal NK cell activity compared to that of normal controls. Four stages of evolution (early, exacerbation, postexacerbation, and convalescence) in these patients were further evaluated. Increased NK cell activity in the exacerbation of major aphthous ulcer was noted. In contrast, depressed NK cell activity in postexacerbation and in 1 week of convalescence was found at different effector/target ( E T ) ratios. The depressed NK cell activity was interpreted as temporary and a secondary phenomenon in the immunopathogenesis of this disease. Partial restoration of depressed NK cell activity by adding recombinant interleukin-2 (rIL-2) suggests that other factors are also involved in the process or that IL-2 deficiency exists in RAU patients. However, no deficiency of plasma IL-2 and interferon-γ (IFN-γ) was noted. The percentage of large granular lymphocytes (LGL, NK K ) was correlated with NK cell activity, with both parameters being depressed in the postexacerbation of RAU patients. NK cell activity of RAU patients was still depressed after the depletion of plastic adherent cells. Surprisingly, in contrast to the remission stage, unresponsiveness to rIL-2 of normal NK cell activity in the exacerbation stage was found. The discrepancy was not associated with different subpopulations of NK heterogeneous cells. Immunopathogenesis of RAU on the NK-IFN-IL-2-IL-2R system needs further clarification.

Fibronectin facilitates the migration of human natural killer cells

The interaction of lymphocytes with the extracellular matrix plays an important role in the immune defence against tumor cells and virus-infected cells. We have examined the effect of matrix proteins on the migration of large granular lymphocytes (LGL) through 3-microns pores in Nuclepore filters in a Boyden invasion chamber. Fibronectin bound on the filter surface significantly increased (p less than 0.001) the capacity of LGL to migrate, whereas soluble fibronectin did not. In addition, a significantly higher (p less than 0.001) percentage of LGL was capable of migration through fibronectin-coated filters than through untreated filters. With fibronectin-coated filters, a strong enrichment of CD16+ and CD56+CD3- cells with LGL morphology and reduction of CD3+ cells was found among migrating cells when the incubation time was 4 h or less. Later agranular lymphocytes, mainly CD3+ T lymphocytes, also started to migrate. Laminin coating of filters also facilitated migration, and when filters were coated with both fibronectin and laminin the increase in migration was equal to the sum of the increases induced by each protein alone. Interactions between cell surface and the Arg-Gly-Asp (RGD) peptide sequence of many matrix proteins had no role in the LGL migration through untreated filters. However, when filters were coated with either fibronectin or laminin, or with both, peptide containing the RGD sequence reduced migration to the level of untreated filters, whereas an Arg-Gly-Glu control peptide had no effect. Our results show that unstimulated LGL/natural killer cells are capable of rapid migration through matrix-coated porous membranes, and that interactions between cell surface receptors and the RGD sequence of fibronectin and probably laminin are utilized in this process.

Impaired Cytotoxic Activity of Interleukin 2 (IL 2)-cultured Large Granular Lymphocytes (LGL) in Childhood Acute Lymphoblastic Leukemia. Quantitative Measurement of IL 2 Receptor Expression of IL 2-cultured LGL

Large granular lymphocytes (LGL) of the natural killer (NK) cell lineage were highly purified and their interleukin 2 (IL 2) receptors (IL 2R) were investigated in childhood acute lymphoblastic leukemia (ALL). Not only NK activity but also 3-day recombinant IL 2-cultured lymphokine activated killer (LAK) activity were decreased in ALL, despite normal percentages of LGL, CD16 (Leul1)(+) and CD56 (NKH1)(+) cells. The cytotoxic activity of IL 2-cultured LGL, but not IL 2-cultured T cells, was significantly decreased in ALL, indicating a selective defect of LGL among the killer cells. IL 2R (CD25) numbers/cell of 3-day IL 2-cultured LGL were not decreased in ALL by flow cytometric analysis. Scatchard plot analysis demonstrated that high affinity IL 2R numbers/cell of 3-day IL 2-cultured LGL were normal but the binding affinity level of the receptors in these cells in ALL was one-third of that in the similarly-cultured control cells, suggesting inadequate IL 2-IL 2R interaction is responsible, at least in part, for their reduced cytotoxic activity.

Characterization of the natural killer cell activity in Hashimoto's and Graves' diseases.

Natural killer (NK) cell activity and blood mononuclear cell subpopulations were characterized in patients with Hashimoto's thyroiditis (n = 11), Graves' disease (n = 20), non-toxic goitre (n = 10) and in normal controls (n = 22). NK cell activity against K 562 target cells and the capability of IFN-alpha, Il-2, and indomethacin to enhance NK cell activity in vitro did not differ significantly between the groups. The percentages of large granular lymphocytes, CD5+, CD4+, CD8+ and CD16+ cells were normal in patients with non-toxic goitre, Hashimoto's and Graves' diseases. There was no correlation between NK cell activities and TgAb, MAb and TSAb. Although NK cell activity is suppressed in several autoimmune diseases, NK cell function is normal in patients with autoimmune thyroid disorders.

Effects of Different Sample Preparations on Enumeration of Large Granular Lymphocytes (LGLs), and Demonstration of a Sex Difference of LGLs

Peripheral natural killer (NK) cells were identified by their morphologic appearance as large granular lymphocytes (LGLs) in a cytocentrifuged or spun blood film but not in thin wedge blood film. The authors studied the effect of different methods of sample preparation and staining on the enumeration of peripheral human LGLs as a routine test. In the blood film made from anticoagulated venous blood, LGLs were scattered in a field and sometimes deformed by compression of erythrocytes. In a blood film made from mononuclear cells, LGLs were shrunken, rendering it difficult to always identify cytoplasmic granules. In contrast, LGLs were easily and accurately identified in a blood film made from leukocyte-rich plasma, displaying none of the above artifacts. As well, the percentage of LGLs obtained was similar to that obtained from a blood film made from anticoagulated venous blood. In addition, May-Grünwald-Giemsa (MGG) stained azurophilic granules more clearly than did Giemsa above. As a result of these observations, a blood film that was made from leukocyte-rich plasma and that was stained by MGG was considered to be most suitable for the routine enumeration of LGL. Using this method, the authors found a significant correlation between the percentages of LGLs and Leu-7+ cells in the same subjects (r = 0.71; n = 22; P less than 0.001) and also a significant sex difference in the percentages of peripheral LGLs, which were significantly lower in women (17.0 +/- 3.6%; n = 35; P less than 0.05) than in men (19.5 +/- 5.3%; n = 20). Furthermore, the percentage of LGLs with abundant cytoplasmic granules, which might have greater NK activity, was also significantly lower in women (15.9 +/- 3.1%; n = 35; P less than 0.01) than in men (17.9 +/- 4.0%; n = 20).

Lymphokine-activated killer cells in rats. IV. Developmental relationships among large agranular lymphocytes, large granular lymphocytes, and lymphokine-activated killer cells.

The developmental relationships among large agranular lymphocytes (LAL) large granular lymphocytes (LGL) and the activation of these cells into lymphokine-activated killer (LAK) cells by rIL-2 was investigated. Highly enriched populations of LAL were isolated from Fischer 344 spleen cells by a combination of nylon-wool filtration (to remove B cells and macrophages), treatment with a pan T cell antibody plus complement (to remove T cells) and incubation in L-leucine methyl ester (to remove LGL). The resultant cells were highly enriched in morphologically identifiable LAL which expressed asialo GM1 and partially expressed the OX8 surface marker. The enriched LAL did not contain detectable NK cytotoxic activity, did not express pan T cell (OX19), Ia, Ig, or laminin surface markers and contained less than 0.2% LGL. Incubation of LAL in a low dose of rIL-2 (100 U/ml) induced the generation of LGL having NK activity within 24 h of culture. Longer culture periods (48 h) resulted in a continued increase in the percentage of LGL and higher levels of NK activity. However, with this low dose of rIL-2, little or no LAK activity (i.e., reactivity against NK-resistant target cells) was generated. With a high dose of rIL-2 (500 U/ml), LAL responded by first generating LGL with NK activity (within 24 h), with subsequent generation of LAK activity by 48 h. Evidence that the development of granular lymphocytes from LAL was responsible first for NK activity and then LAK activity was demonstrated by depletion of the generated granular NK or LAK effector cells by second treatments with L-leucine methyl ester. Concomitant with the induction of LGL with NK or LAK activity, rIL-2 also caused LGL to proliferate and expand four- to five-fold in 48 h. This occurred in the presence of high or low dose rIL-2. These results indicate that LAL are the precursors of LGL/NK cells, that LAL, LGL/NK cells and LAK cells appear to represent sequential developmental or activation stages and that LAL may comprise major source of LAK progenitors in lymphoid populations having few LGL or mature active NK cells.

Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities.

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.

Large granular lymphocytes in bronchoalveolar lavage fluids from immunocompromised patients with cytomegalovirus pneumonitis.

Natural killer (NK) cells activities had been demonstrated to be depressed in patients with fatal cytomegalovirus (CMV) pneumonitis. NK cells can be identified by morphologic features characteristic of large granular lymphocytes (LGLs). Bronchoalveolar lavage (BAL) cells from 16 immunocompromised patients with CMV pneumonitis were analyzed. Two different groups of patients could be distinguished depending on the course of the CMV pneumonitis: nine patients who recovered (Group A), seven patients with a fatal outcome (Group B). Except for the increase in polymorphonuclear cells (PMN) in Group B (12.4 +/- 11.6%), no significant difference in the macrophage or the total lymphocyte population was observed. A differential count excluding alveolar macrophages specified the percentage of LGLs from the total lymphocyte population. The LGLs in Group A (7.1 +/- 9.9%) were similar to those previously reported in normal lung. A significant increase in LGLs was observed in the BAL cells from patients of Group B (28.1 +/- 22%). The discrepancy between the high percentage of LGLs in patients with a fatal outcome and their expected protective effects is discussed.

Stress depresses interferon production by leukocytes concomitant with a decrease in natural killer cell activity.

This study addressed the effects of a commonplace stressful event on interferon production and natural killer (NK) cell activity and numbers. The quantity of interferons (IFN) produced by concanavalin A stimulated leukocytes obtained from 40 medical students during examinations was significantly lower when compared with IFN levels produced by peripheral blood leukocytes (PBLs) taken 6 weeks earlier (baseline). In addition, three different assays measuring NK cells also showed significant decrements during examinations when compared with baseline samples. These assays included lysis of MOLT-4 target cells, percentage of anti-Leu-7+ (NK) cells, and percentage of large granular lymphocytes. Self-report data documented the significantly greater distress associated with examinations in comparison with baseline samples. The data have implications for immunosuppressive disorders and stress-associated illnesses.

Impaired natural killer cell activity in Bloom's syndrome could be restored by human recombinant IL-2 in vitro

Natural killer (NK) activity against K562 tumor cells was evaluated in peripheral blood nonadherent mononuclear cells (PBMNC) obtained from four patients with Bloom's syndrome. NK activity of PBMNC from all patients was found to be depressed to less than half of age-matched control values. However, all patients showed the normal percentages of large granular lymphocytes and of cells expressing the NK-cell-associated determinants (OKM1, Leu-7, and Leu-11). Although recombinant interferon-β or -γ augmented NK activity of patients' PBMNC in a manner similar to their effects on NK activity of normal individuals, impaired NK activity of PBMNC from Bloom's patients could be restored to normal ranges only by recombinant interleukin 2 (IL-2). These results suggested that IL-2 and interferons might participate in boosting NK activity of Bloom's syndrome in different ways.

Changes in number and density of large granular lymphocytes upon in vivo augmentation of mouse natural killer activity.

NK activity of mice as well as humans and rats has been clearly associated with large granular lymphocytes (LGL). To better understand the effects of interferon (IFN) and IFN inducers on natural killer (NK) cells, we have compared the LGL in the spleens of normal and boosted mice. Cells were fractionated by centrifugation on discontinuous Percoll density gradients, and each fraction was tested for NK activity against YAC-1 targets and for the presence of LGL. In vivo treatment with C. parvum (0.7 mg/mouse, i.p., day-3), MVE-2 (25 mg/kg, i.p., day-3), poly I:C (4 mg/kg, i.p., day-3), or IFN (10(5) U/mouse, i.p., day-1) resulted in a marked augmentation and a change of distribution of cytotoxic activity. Most of the NK activity of boosted spleen cells was associated with lower density fractions 1 and 2, whereas active normal spleen cells had somewhat higher density (fractions 2 and 3). In parallel to their increased reactivity, the boosted spleens had a marked increase in the percentage of LGL, particularly in fractions 1 and 2. The augmented activity appeared to be mediated by the LGL, because treatment with anti-asialo GM1 or anti-Thy-1.2 plus complement reduced NK as well as the number of LGL. These results indicate that IFN-mediated boosting of NK activity in the spleen is due to an increase in the lower density LGL, as well as to an increase in the function of preexisting NK cells.

Development of hyporesponsiveness of natural killer cells to augmentation of activity after multiple treatments with biological response modifiers.

Four biological response modifiers (BRMs), MVE-2 (maleic anhydride divinyl ether), Corynebacterium parvum (C. Parvum), PolyICLC (polyinosinic:polycytidylic acid stabilized with poly-L-lysine), and mouse alpha beta-interferon (alpha beta-IFN), were tested to assess whether repeated treatments would repeatedly induce or sustain augmented levels of natural killer (NK) cell activity and/or macrophage (M0)-mediated inhibition of tumor cell growth. In contrast to a significant increase in splenic NK activity obtained with a single treatment with each of the agents, multiple treatments with these BRMs led to a progressive decrease in the degree of augmentation of NK activity. In contrast, multiple injections with these agents resulted in sustained augmentation of M0-mediated reactivity. Separation of the spleen cells by Percoll discontinuous density gradient centrifugation indicated that with mice treated once with each BRM high levels of NK activity were detected in the lower density fractions and that these fractions contained a higher percentage of large granular lymphocytes (LGLs) than that found in comparable fractions from normal mice. In contrast, cells in the lower density fractions from mice that received multiple treatments had decreased NK activity and an appreciably lower proportion of LGLs. These results indicate that the development of hyporesponsiveness to augmentation of splenic NK-cell activity following multiple treatments with BRMs may be attributable to a decreased percentage of LGLs, the effector cell population responsible for NK cell-mediated cytotoxicity.

Development of hyporesponsiveness to augmentation of natural killer cell activity after multiple doses of maleic anhydride divinyl ether: Association with decreased numbers of large granular lymphocytes

A single injection of mice with maleic anhydride divinyl ether (MVE-2) resulted in significantly augmented natural killer (NK) activity. However, multiple injections with MVE-2 led to a hyporesponsive to boosting of NK activity. Stimulation of prostaglandin E secretion of induction of suppressor macrophages (Mø) or lymphocytes were shown not to be responsible for the depressed NK cell responses. Rather the hyporesponsiveness to NK boosting was associated with a decreased number of large granular lymphocytes (LGLs). Percoll discontinuousdensity-gradient studies showed that the augmented NK activity of spleen cells after a single injection with MVE-2 was associated with an increase in the percentage of LGLs in the lowerdensity fractions (Fraction 1 and 2). In contrast, the NK activity and percentage of LGLs in the lower-density fractions were markedly decreased after multiple injections with MVE-2. Polyinosinic: polycytidylic acid stabilized with poly( l-lysine) in carboxymethylcellulose, another BRM capable of augmenting NK activity, was able to substantially augment NK activity in mice hyporesponsive to MVE-2 and this was accompanied by an increase in the percentage of LGLs in the lower-density Percoll fractions.

Influence of in vivo immunosuppressive drugs on production of lymphokines.

The influence in vivo of immunosuppressive drugs (cyclosporine, azathioprine, and corticosteroids) on the production of various lymphokines (alpha and gamma interferon, interleukin 2), both in organ transplant recipients and in normal volunteers taking 100 mg hydrocortisone orally has been studied. To avoid interference with the rejection process or viral infection, patients were studied in a steady state with low maintenance immunosuppression consisting of prednisolone combined with azathioprine or with cyclosporine. In patients treated with both drug regimens, significant depression of production of the three lymphokines was found. Normal volunteers challenged with 100 mg hydrocortisone showed inhibition of production of interleukin 2 and alpha and gamma interferon in 4 hr, a time corresponding to the nadir of T cell lymphopenia, affecting the OKT4 subset preferentially. The percentage of OKT8 cells remained unchanged. Percentages of large granular lymphocytes increased, but their absolute number was not significantly modified. Changes in lymphocyte markers were fully reversible after 24 hr, but interleukin 2 production remained markedly depressed, showing that the redistribution patterns induced by corticosteroids on lymphocyte subsets may be dissociated from functional consequences.

Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.

Natural killer (NK) activity in the rat and human has been attributed to cells having the morphology of large granular lymphocytes (LGL). However, this association has been less clear in the mouse, largely because of difficulties in obtaining highly enriched populations of LGL from normal spleen and blood. We have previously observed that the administration of the biological response modifier (BRM) maleic anhydride divinyl ether (MVE-2) strongly augmented NK activity in lung and liver, and the augmented NK activity coincided with increased resistance to the formation of experimental metastases in these organs. The degree of NK augmentation was most striking in the liver, an unexpected and previously unreported observation. In the present study, both MVE-2 or Corynebacterium parvum induced a dramatic augmentation of liver NK activity, which reached maximum levels 3-5 d after treatment. This augmentation of NK activity in the liver coincided with a large increase in the number of lymphoid cells with the morphological characteristics of LGL that could be isolated from enzymatically digested suspensions of perfused liver. The yield of LGL per liver following BRM treatment corresponded to a 10-50-fold increase as compared to normal mice. LGL were purified from these enzymatically digested suspensions of perfused liver by depletion of adherent cells on nylon wool columns and subsequent enrichment for low-density lymphoid cells by fractionation on Percoll density gradients. The enrichment of LGL correlated with greatly increased NK activity against YAC-1. Conversely, the higher-density fractions were depleted of both LGL and NK activity. This increase in NK activity in the liver was suppressed by in vivo treatment with anti-asialo GM1 (asGM1) serum. This treatment also resulted in a corresponding reduction in both the total number and percentage of LGL. By flow cytometry analysis, the phenotype of the majority of these highly cytolytic LGL isolated from the livers of BRM-treated mice were asGM1+, Thy-1+, Ly-5+, Qa-5+, Mac-1+, and Gma-1+, whereas these LGL were Ly-1-, Lyt-2-, L3T4-, and surface Ig-. We conclude that the livers of BRM-treated mice can provide a rich source of highly active mouse LGL that could be used for further characterization of this lymphocyte subset. Further, these studies imply a potential for BRM therapy of neoplastic or viral diseases through augmentation of organ-associated immune responses.