Number Of Large Granular Lymphocytes Research Articles

Programmed cell death 1-expressing CD56-negative natural killer (NK) cell expansion is a hallmark of chronic NK cell activation during dasatinib treatment.

Dasatinib treatment markedly increases the number of large granular lymphocytes including natural killer (NK) cells in a proportion of Ph+ leukemia patients, which associates with a better prognosis. In‐depth immune profiling of NK cells can predict therapeutic response in these patients. In the present study, we showed that CD56‐negative (CD56neg) NK cells increased exclusively in cytomegalovirus‐seropositive (CMV+) patients treated with dasatinib. The increase longitudinally paralleled with progressive differentiation of CD56dim NK cells during dasatinib therapy driven by CMV reactivation as shown by principal component analysis on 19 NK cell markers. The CD56neg NK cells showed downregulation of NK‐activating receptors, upregulation of PD‐1, and lower cytotoxicity and cytokine production, indicating that these cells are anergic and dysfunctional as seen in chronic infections with HIV‐1 or hepatitis C virus. Moreover, cytolytic activity of CD56dim and CD56neg NK cells against leukemia cells was partially restored by nivolumab in proportion to the frequency of PD‐1+ NK cells. The proportion of patients who achieved deep molecular responses at 2 years was significantly higher in dasatinib‐treated patients with ≥3% CD56neg NK cells than in those with fewer CD56neg NK cells (54.5% vs 15.8%, P = .0419). These findings suggest that CD56neg NK cells may be an exhausted population induced by chronic activation through CMV reactivation during dasatinib therapy. Expansion of CD56neg NK cells is a hallmark of chronic NK cell activation in patients treated with dasatinib and may predict a better clinical outcome. Furthermore, PD‐1 blockade may enhance anti‐leukemia responses of such NK cells.

Analysis of a single-institution cohort of patients with Felty's syndrome and T-cell large granular lymphocytic leukemia in the setting of rheumatoid arthritis

T-cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder characterized by a persistent increase in the number of large granular lymphocytes (LGLs), neutropenia, and splenomegaly. Clinical manifestations of T-LGLL in the setting of rheumatoid arthritis (RA) are often identical to those in which one would suspect Felty's syndrome (FS). These disorders are distinguished by the presence of T-cell clonality, which is present in T-LGLL but not in FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) and 5b (STAT5b) genes can be used as molecular markers of T-LGLL, but their prevalence in FS is unknown.Eighty-one patients with RA and unexplained neutropenia or/and an increase in the number of LGLs above 2 × 109/L were stratified into RA-associated T-LGLL (N = 56) or FS (N = 25) groups based on the presence or absence of T-cell clonality. STAT3 and STAT5b gene mutations were assessed in each group by means of allele-specific polymerase chain reaction assays. Clinical, immunological, laboratory data and the results of immunophenotyping of blood and bone marrow lymphocytes were also evaluated.Mutations of the STAT3 gene and an increase in the number of LGLs above 2 × 109/L were detected in RA-associated T-LGLL, but not in FS (39% vs 0% and 21% vs 0%, respectively). Mutations in the STAT5b gene were not observed in either group. Expression of CD57, CD16, and CD5−/dim on CD3+CD8+ T-lymphocytes was observed in both RA-associated T-LGLL and FS.STAT3 gene mutations or LGL counts over 2 × 109/L in RA patients are indicative of T-LGLL.

Principal component analysis uncovers cytomegalovirus-associated NK cell activation in Ph+ leukemia patients treated with dasatinib.

Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) in a proportion of Ph+ leukemia patients, which associates with a better prognosis. The lymphocytosis is predominantly observed in cytomegalovirus (CMV)-seropositive patients, yet detectable CMV reactivation exists only in a small fraction of patients. Thus, etiology of the lymphocytosis still remains unclear. Here, we identified NK cells as the dominant LGLs expanding in dasatinib-treated patients, and applied principal component analysis (PCA) to an extensive panel of NK cell markers to explore underlying factors in NK cell activation. PCA displayed phenotypic divergence of NK cells that reflects CMV-associated differentiation and genetic differences, and the divergence was markedly augmented in CMV-seropositive dasatinib-treated patients. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis, and was further enhanced after starting dasatinib in virtually all CMV-seropositive patients. Thus, the extensive characterization of NK cells by PCA strongly suggests that CMV is an essential factor in the NK cell activation, which progresses stepwise during leukemia and subsequent dasatinib treatment most likely by subclinical CMV reactivation. This study provides a rationale for the exploitation of CMV-associated NK cell activation for treatment of leukemias.

Expansion of Dysfunctional CD56-Negative NK Cells Is a Hallmark of NK Cell Activation in Ph+ Leukemia Patients Treated with Dasatinib

[Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) including NK cells in peripheral blood, which associates with a better prognosis. We previously showed that dasatinib expands CMV-associated highly differentiated NK cells in Ph+ leukemia patients through reactivation of CMV (Ishiyama, et al., Leukemia, 2016). NK cells consist of conventional CD56bright NK cells and CD56dim NK cells, and also rare CD56-negative (CD56neg) NK cells, which have been reported to increase in chronic viral infection such as HIV and HCV. Here, we show that CD56neg NK cells increase in CMV seropositive (CMV+) patients treated with dasatinib (DA), but not in those treated with other TKIs (OT). However, characteristics and clinical implications of CD56neg NK cells in CMV+DA patients remain unknown. Therefore, we sought to examine the phenotypic and functional properties of CD56neg NK cells in these patients.[Methods] We assessed NK cell subsets in 36 DA and 26 OT patients. NK cells were defined as lin-CD16+ or lin-CD56+ cells in peripheral blood. NK-cell marker expression and cytotoxic activity were compared between CD56neg NK cells and CD56dim NK cells in CMV+ DA patients with 5% or more of CD56neg NK cells. In vivo phosphorylation of intracellular signaling molecules in NK cells were evaluated by flow cytometry combined with phospho-specific antibodies (BD PhosflowTM).[Results] CD56neg NK cells exclusively increased in CMV+ DA group, compared to CMV- DA and CMV+ OT groups (median count: 20.9/μl, 1.1/μl, 1.5μl; p < 0.001. Median percentage: 4.5%, 0.8%, 0.4% of total NK cells; p < 0.001). CD56neg NK cell counts strongly correlated with total NK cell counts in CMV+ DA (r = 0.720, p < 0.001). CD56neg NK cells gradually increased over a period of one year after starting dasatinib. When we compared CD56neg NK cells and CD56dim NK cells in CMV+ DA, CD56neg NK cells showed decreased expression of activating NK cell markers including NKG2C, CD57, NKG2D, NKp30 and NKp46. In contrast, expression of PD-1 increased. CD56neg NK cells also showed lower rate of degranulation and IFN-γ production in functional assays. CMV+ DA patients with 4% or more of CD56neg NK cells significantly achieved deep molecular responses at 1 year after starting dasatinib than those with lower CD56neg NK cells (10/14, 3/14; p < 0.05). Phosflow analysis showed enhanced phosphorylation of ZAP70 in NK cells from CMV+ DA compared to CMV+ healthy controls (MFI/Isotype = 24.6 vs. 7.4, p < 0.01).[Discussion] CD56neg NK cells exclusively increased in CMV+ DA, and their increase persisted during dasatinib therapy. This suggests the similarity with the previous findings that CMV-associated differentiation in NK cells is enhanced during dasatinib therapy in CMV+ DA, which reflects the NK cell activation in response to CMV reactivation. In addition, CD56neg NK cells exhibited downregulation of activating receptors, upregulation of PD-1, and lower cytotoxic activity, indicating that these cells are the anergic and exhausted population as seen in chronic viral infection. These findings suggest that CD56neg NK cells accumulate owing to chronic stimulation by reactivated CMV during dasatinib therapy. ZAP70 is an immediate downstream tyrosine kinase of activating NK cell receptors, and is regulated by Src-family kinases, which is potentially inhibited by the kinase inhibitor property of dasatinib. Intriguingly, phosphorylation of ZAP70 in CD56dim NK cells was enhanced in CMV+ DA, although they had taken dasatinib a few hours before collecting blood samples. This finding suggests that strong activation of NK cells by CMV reactivation in CMV+ DA may overcome the direct suppressive effect of dasatinib on NK cell activation.[Conclusion] The accumulation of CD56neg NK cells is a consequence of NK cell activation specifically observed in dasatinib-treated patients. The NK cell activation is likely to be a response to the CMV reactivation induced by dasatinib treatment. Assessing CD56neg NK cells in peripheral blood could be a practical clinical indicator for the immunomodulatory effect of dasatinib, which significantly affects the prognosis. DisclosuresTakaori-Kondo:Chugai Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Pfizer: Research Funding; Toyama Chemical: Research Funding; Takeda Pharmaceutical: Research Funding; Kyowa Kirin: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Mochida Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Speakers Bureau.

Principal component analysis uncovers cytomegalovirus-associated NK cell activation in Ph+ leukemia patients treated with dasatinib

Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) in a proportion of Ph+ leukemia patients, which associates with a better prognosis. The lymphocytosis is predominantly observed in cytomegalovirus (CMV)-seropositive patients, yet detectable CMV reactivation exists only in a small fraction of patients. Thus, etiology of the lymphocytosis still remains unclear. Here, we identified NK cells as the dominant LGLs expanding in dasatinib-treated patients, and applied principal component analysis (PCA) to an extensive panel of NK cell markers to explore underlying factors in NK cell activation. PCA displayed phenotypic divergence of NK cells that reflects CMV-associated differentiation and genetic differences, and the divergence was markedly augmented in CMV-seropositive dasatinib-treated patients. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis, and was further enhanced after starting dasatinib in virtually all CMV-seropositive patients. Thus, the extensive characterization of NK cells by PCA strongly suggests that CMV is an essential factor in the NK cell activation, which progresses stepwise during leukemia and subsequent dasatinib treatment most likely by subclinical CMV reactivation. This study provides a rationale for the exploitation of CMV-associated NK cell activation for treatment of leukemias.

P.1.f.005 Immune response correlates with behavioral activity, associated with memory processes following chronic stimulation of the medial septum

The study examined cortisol (COR) involvement in stress-related changes in natural killer cell cytotoxicity (NKCC). The relationship between blood COR level, phasic changes in NKCC, and the number of large granular lymphocytes (LGL) was examined in pigs during the course of 4-h immobilization stress (IMB) and for 6 days after its termination. NKCC was determined using 18-h 51Cr-release assay, LGL number was assessed with a standard hematological method, and plasma COR level was measured by radioimmunoassay. The blood level of COR was increasing during IMB (max 446Δ% at the second hour) and decreased after its termination (max −59Δ% on day 2). Changes in NKCC level and LGL number were biphasic; i.e., an initial increase in both measures (NKCC max 24Δ%, LGL max 18Δ%) in an early phase of stress (0–1h) was followed by their subsequent decrease (NKCC max −35Δ%, LGL max −41Δ%) in the late phase (3–4 h) of stress, which persisted for several days after termination of IMB. Thus, in the early phase of stress, there was a positive correlation between NKCC, LGL number, and COR levels (all elevated); a positive correlation between the measures also occurred after termination of IMB (all decreased). A negative correlation between COR and NKCC, which might be indicative of COR-related immunosuppression, was found only in the late (3–4 h) phase of stress. It is concluded that COR may be only one of multiple factors (possibly antagonistic) determining an actual immune response during stress.

Restraint effects on stress-related hormones and blood natural killer cell cytotoxicity in pigs with a mutated ryanodine receptor

A mutation in the ryanodine receptor gene (RYR1) of the calcium release channel is responsible for increased stress susceptibility in pigs. In the present study, the relation of a mutation in RYR1 with the neuroendocrine (stress-related hormone) response and the immune defense represented by natural killer cell cytotoxicity (NKCC) during a 4-h restraint and recovery phase in 60 male pigs was investigated. Blood samples were collected from pigs previously divided into RYR1 genotypes (nn, Nn, NN), based on PCR amplification and restriction analyses. The blood samples collected during the restraint and recovery phases of the experiment were used to determine NKCC (51Cr-release assay), large granular lymphocyte number (hematologic method), and plasma concentrations of prolactin (PRL), GH, ACTH, and cortisol (COR) (by specific RIA). The greatest degree of NKCC response (P < 0.05) to restraint stress relative to controls was observed for the stress-susceptible homozygote group (nn). Measures of stress-related hormones were positively correlated with NKCC during the entire experimental period (P < 0.001 for all investigated hormones) in the nn group. Immunostimulatory effects in the early (0–60 min) phase of restraint were associated with increased hormone responses, especially PRL and GH. In the late (180–240 min) phase of stress and the recovery phase (480 min), a decrease in immune response was accompanied by an elevated COR response in all RYR1 genotypes. Moreover, divergent responses of both PRL (greatest in nn, P < 0.001) and GH (greatest in NN, P < 0.001) to the 4-h restraint were observed. Our results suggest that stress-susceptible RYR1-mutated homozygotes develop a greater level of immune defense, including cytotoxic activity of NK cells, and accompanied by more pronounced stress-induced changes in neuroendocrine response than stress-resistant heterozygous (Nn) and homozygous (NN) pigs.

Clinical features of dasatinib-induced large granular lymphocytosis and pleural effusion

During follow-up of leukocyte counts in 20 consecutive patients (age range 29-81 years) treated with dasatinib, 9 patients (7 chronic myeloid leukemia in chronic phase, 2 Philadelphia chromosome-positive acute lymphoid leukemia in complete remission) developed lymphocytosis (>3,000/microl). Peripheral blood smears revealed a population of large granular lymphocytes. Large granular lymphocytosis (LGL) was first noted between 1 and 8 months after initiation of dasatinib, and it has persisted up to 33 months from the onset of LGL in one patient. Peak numbers of large granular lymphocytes ranged from 2,915 to 17,425/microl. The occurrence of LGL might interfere with achieving molecular response (MR, real-time quantification of major BCR-ABL1 mRNA less than 50 copies/microg RNA) in our small cohort; 8 (89%) of 9 patients with LGL attained MR, while only 6 (55%) of 11 patients without LGL eventually achieved MR. With respect to the relationship between LGL and pleural effusion (PE), 3 (27%) of 11 patients without LGL developed PE, while 5 (56%) of 9 patients with LGL developed PE. Moreover, the mean peak number of LGL was 9,215/microl, which was much higher than the mean peak number (4,635/microl) of LGL in patients without PE. These results may suggest possible association of both events in our cohorts.

Polyclonal expansion of large granular lymphocytes in common variable immunodeficiency - association with neutropenia.

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency disease, characterized by low levels of circulating immunoglobulins and recurrent bacterial infections, particularly of the respiratory tract. T cell dysfunction is often present, and lymphoproliferative and autoimmune disorders as well as haematological cytopenias are frequently observed. In this study, we report a polyclonal expansion of large granular lymphocytes (LGL) in a substantial proportion of CVID patients, associated with splenomegaly, increased numbers of CD8(+) T cells, inverted CD4 : CD8 T cell ratios and neutropenia. CVID patients who had both increased numbers of LGL and granulocytopenia had elevated levels of soluble Fas ligand (sFasL). Our observations indicate that CVID may be added to the list of inflammatory diseases associated with increased numbers of LGL. Furthermore, our findings suggest common pathogenic mechanisms of granulocytopenia in CVID and lymphoproliferative disease of granular lymphocytes.

Suppression of natural killer cell cytotoxicity following chronic electrical stimulation of the ventromedial hypothalamic nucleus in rats

In our previous study we found that chronic electrical stimulation of the lateral hypothalamus (LH) enhances and its lesion suppresses natural killer cell cytotoxicity (NKCC) and a large granular lymphocyte (LGL) number in conscious, freely behaving rats. Since the ventromedial nucleus of the hypothalamus (VMH) is regarded as behaviorally and physiologically opposite to LH, in our present study we investigated whether this antagonism also holds for the immune functions. Chronic electrical VMH stimulation effect on 1) immune parameters: both spleen and blood NKCC (chromium release assay and single-cell agarose assay) and the number of large granular lymphocytes (LGL; a morphological method), and 2) endocrine parameters: immunosuppressive—corticosterone (COR) and testosterone (TST) and immunostimulative—growth hormone (GH) and prolactin (PRL) plasma levels (RIA) was assessed. Twenty-one days of electrical stimulation of VMH caused significant decrease in both spleen and blood NKCC at the population level (chromium release assay) but not at the single cell level (agarose assay) with a simultaneous fall in the LGL number. Rats responding to the VMH stimulation with behavioral inactivation (BIN) showed a significantly lower depression of NKCC and LGL number than those responding with an aversive reaction (AVE). Depression of NKCC coexisted with various hormonal changes: increase of PRL, increase (AVE) or fall (BIN) of COR, decrease of GH (BIN), and increase of TST (VMH-stimulated and VMH-sham). There were significant differences in all measured plasma hormones between BIN and AVE groups. The results obtained indicate that VMH decreases cell-mediated immune response, represented by NK cell activity. The immunosuppressive effect is dependent on the behavioral outcome of VMH stimulation (BIN/AVE) rather than tested endocrine variables. Moreover, the present results indicate that the VMH and LH are antagonistically engaged in the regulation of NK cell cytotoxicity.

Chronic electric stimulation of the midbrain ventral tegmental area increases spleen but not blood natural killer cell cytotoxicity in rats

Previously we found that in conscious, freely behaving rats chronic electric stimulation of the lateral hypothalamus (LH) caused significant augmentation of natural killer cell cytotoxicity (NKCC) and a large granular lymphocyte (LGL) number more pronounced in the spleen than in the peripheral blood. The LH belongs to the so-called “brain reward system”, a collection of the central structures whose activation produce positive emotions. The midbrain ventral tegmental area (VTA) is another prominent reward-relevant structure. In the present work, chronic electric stimulation of VTA (constant current 0.1 ms duration cathodal pulses delivered at frequency 50 Hz during 60 min daily session for 14 consecutive days) caused in rats an increase in the spleen but not in the peripheral blood NKCC (chromium release assay) without simultaneous effect on the number of large granular lymphocytes (LGL) (morphological method) and plasma level of prolactin (PRL), growth hormone (GH), corticosterone (COR), and testosterone (TST). This effect was anatomically specific as no influence of analogous thalamic stimulation on immune and endocrine response was found. The results obtained indicate that both reward-related areas VTA and LH enhance the cell-mediated immune response, represented by natural killer cytotoxicity, especially in the spleen. However, the effect pronounced by VTA is weaker than that of LH, possibly due to additional connections of LH with the hormonal and/or autonomic control systems.

Effects of amphetamine on NK-related cytotoxicity in rats differing in locomotor reactivity and social position

The effect of i.p. administration of 1 mg/kg of amphetamine (AMPH) on natural killer cells cytotoxicity (NKCC) and number of large granular lymphocytes (LGL-NK) together with plasma corticosterone (CORT) level and WBC was evaluated in male Wistar rats differing in two behavioral features: locomotor reactivity to novelty (high, HR and low, LR responders) and social position (dominants, D and subordinates, S). In the majority of animals AMPH evoked (30 min after administration) an increase in NKCC and LGL (NK) number accompanied by lymphopenia, neutrocytosis, monocytosis, and an increase in CORT level. Changes in NKCC (LU 20) showed substantial individual variability: in HR group ∼513Δ%, p<.001 (relative to the control); LR group ∼56Δ%, p>.05; D group ∼441Δ%, p<.001; S group ∼216Δ%, p>.05; HR/D group ∼643Δ%, p<.001; HR/S group ∼414Δ%, p<.001; LR/D group ∼191Δ%, p>.05; and LR/S group ∼−19Δ%, p>.05. The increase in CORT level, lymphopenia, and neutrocytosis indicated a stress-like reaction to AMPH. No significant correlation between NKCC and CORT level was found. The results obtained indicate that AMPH can evoke an increase in NK-related cytotoxic activity quantitatively related to high behavioral reactivity to novelty and social dominance, however NKCC is not related to the AMPH-induced CORT changes.

Chronic electrical stimulation of the lateral hypothalamus increases natural killer cell cytotoxicity in rats

Previously, we found that in rats coagulation of the lateral hypothalamus (LH) caused depression of the peripheral blood natural killer cell cytotoxicity (NKCC) and the number of large granular lymphocytes (LGL). In the present work, we have tested the effects on both spleen and blood NKCC of acute (1 day) and chronic (21 days) electrical stimulation of LH, and LGL number in conscious, freely behaving animals. Five groups of male Wistar rats were used: LH stimulated ( n=22), thalamic (Thal) stimulated control ( n=4), operated but non-stimulated LH sham controls ( n=7), non-operated normal control group ( n=8) and spleen baseline group ( n=10). Chronic stimulation of LH caused significant augmentation of NKCC ( 51Cr-release assay) and LGL number (a morphological method), more pronounced in the spleen than in the peripheral blood. Rats responding to LH stimulation with feeding showed a slightly greater effect than those responding with a locomotor reaction. The observed effects were anatomically specific as no influence of Thal stimulation or the sham procedure was found. The results are discussed in terms of the involvement of LH in reward phenomena and the hormonal control of the immune system.

Cutaneous findings associated with chronic natural killer cell lymphocytosis.

Chronic Natural Killer Cell Lymphocytosis (CNKL) is a recently described rare proliferative disorder. We noted an association of cutaneous disorders with CNKL in our clinical experience. We reviewed the medical records of all known patients with CNKL at our institution. Five of 14 patients (36%) with CNKL had associated chronic cutaneous disease: two had livedoid vasculopathy; one, urticarial vasculitis; one, peripheral T-cell lymphoma; and one had complex recurrent aphthous stomatitis. In each case, except the one with lymphoma, the cutaneous disease was present before CNKL was diagnosed and CNKL persisted for the duration of the cutaneous disease. All five patients had increased numbers of large granular lymphocytes on a peripheral blood smear and three or four in the bone marrow (one patient did not undergo bone marrow biopsy). Although CNKL and the reported skin diseases have occurred in the same patients, a causal link cannot yet be established. With CNKL, dermatologists must recognize associated cutaneous diseases, monitor patients for systemic disorders and cytopenias, and appropriately refer patients to a hematologist.

Features of large granular lymphocytes (LGL) expansion following allogeneic stem cell transplantation: a long-term analysis.

Large granular lymphocyte (LGL) proliferation typically follows a chronic course during which major features are cytopenia and immune abnormalities. Elevated numbers of LGL were reported in a few cases following allogeneic stem cell transplantation (allo-SCT). In this report, we present a retrospective analysis of LGL cases that occurred following allo-SCT in a cohort of 201 consecutive patients transplanted over a period of 7 years. Six cases were identified and LGL expansion occurred more frequently following a reduced fludarabine and anti-T lymphocyte globulin-based preparative regimen (4 cases/49), than after a conventional myeloablative regimen (2 cases/152). Expansion of LGL was seen between 3 and 15 months following allo-SCT. Hematopoiesis, with mild to severe cytopenia, was a favored target for LGL. Autoimmune manifestations including polyarthritis and hypergammaglobulinemia were also observed. LGL proliferation was observed in the context of chronic antigenic stimulation associated with recurrent viral infections especially CMV. Moreover, five out of these six high risk patients achieved a long-term complete remission concomitant or following LGL expansion. These data suggest that LGL might be a subset of effector lymphocytes which may participate to the graft-versus-tumor effect.

Stress-Induced Changes in Peripheral Natural Killer Cell Cytotoxicity in Pigs May Not Depend on Plasma Cortisol

The study examined cortisol (COR) involvement in stress-related changes in natural killer cell cytotoxicity (NKCC). The relationship between blood COR level, phasic changes in NKCC, and the number of large granular lymphocytes (LGL) was examined in pigs during the course of 4-h immobilization stress (IMB) and for 6 days after its termination. NKCC was determined using 18-h 51Cr-release assay, LGL number was assessed with a standard hematological method, and plasma COR level was measured by radioimmunoassay. The blood level of COR was increasing during IMB (max 446Δ% at the second hour) and decreased after its termination (max −59Δ% on day 2). Changes in NKCC level and LGL number were biphasic; i.e., an initial increase in both measures (NKCC max 24Δ%, LGL max 18Δ%) in an early phase of stress (0–1h) was followed by their subsequent decrease (NKCC max −35Δ%, LGL max −41Δ%) in the late phase (3–4 h) of stress, which persisted for several days after termination of IMB. Thus, in the early phase of stress, there was a positive correlation between NKCC, LGL number, and COR levels (all elevated); a positive correlation between the measures also occurred after termination of IMB (all decreased). A negative correlation between COR and NKCC, which might be indicative of COR-related immunosuppression, was found only in the late (3–4 h) phase of stress. It is concluded that COR may be only one of multiple factors (possibly antagonistic) determining an actual immune response during stress.

Effect of a Recombinant Lectin, Viscum album Agglutinin on the Secretion of Interleukin-12 in Cultured Human Peripheral Blood Mononuclear Cells and on NK-Cell-Mediated Cytotoxicity of Rat Splenocytes in vitro and in vivo

A plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to increase the number and cytotoxic activity of natural killer (NK) cells in animal models, but the mechanisms underlying these effects are poorly understood. We investigated the effects of the recombinant form of this lectin (rVAA) on secretion of interleukin (IL)-12 and on NK-mediated cytotoxicity against K562 target cells in cultures of human peripheral blood mononuclear cells (PBMC) as well as against YAC-1 target cells in cultured rat spleen cells. In 24-hour cultures of PBMC, 10 ng/ml plant VAA-I and 50 ng/ml rVAA induced significant increases in the secretion of total IL-12. Its biologically active heterodimeric form, p70, was also significantly induced by rVAA. Preincubation of PBMC or splenocytes for 48 h with rVAA in concentrations ranging between 10 pg/ml and 100 pg/ml resulted in moderate enhancements of NK-mediated cytotoxicity. However, coincubation of a low dose of rVAA (100 pg/ml) together with IL-2 and IL-12 (60 U/ml and 2 U/ml, respectively) led to additive stimulation of NK activity. In in vivo experiments, rVAA showed an enhancing effect on NK activity with a bell-shaped curve of efficacy. Forty-eight hours after a single intravenous injection of its most effective doses, 0.5 and 1 ng/kg, into Wistar rats, the NK cytotoxicity of splenocytes against YAC-1 targets doubled, and the frequency of large granular lymphocytes in peripheral blood showed 2.1- and 3-fold increases as compared to control animals. Twenty-four hours following these low lectin doses, the number of large granular lymphocytes was also significantly elevated. After 48 h, 0.5 ng/kg rVAA induced a significant augmentation in the percentage of peripheral Mac-1+ mononuclear cells, including activated monocytes and NK cells. The present results suggest that rVAA augments the secretion of an active form of IL-12 and potentiates the cytokine-induced NK activation. These effects of rVAA may be related to its stimulatory effects on MHC-unrestricted cytotoxicity in vivo.

Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

Using a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+ CD3- NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+ CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

AbstractUsing a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+CD3−NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

NKR-P1+ Cells in the Rat Uterus: Granulated Metrial Gland Cells are of the Natural Killer Cell Lineage1

An intriguing component of the maternal response to pregnancy is the differentiation of large numbers of large granular lymphocytes, termed granulated metrial gland (GMG) cells, in the decidua and then in the metrial gland, a structure in the mesometrial triangle unique to rodent pregnancy. We have used the monoclonal antibody 3.2.3 to NKR-P1, a surface molecule involved in triggering natural killer (NK) cells for lysis, to determine the numbers and distribution of NK cells in the nonpregnant rat uterus and during the dramatic changes at implant sites during pregnancy. NKR-P1+ cells were abundant in the nonpregnant uterus, especially in the endometrium. These cells also expressed CD8, CD2, and AsialoGM1. In the subepithelial stroma, the numbers were greatest during proestrus and estrus; in ovariectomized animals, they were severely decreased, but returned to normal with estrogen supplementation. At the time of blastocyst attachment (Day 6), NKR-P1+ cells were few around the implant and in the decidualizing stroma. However, on Day 8, substantial numbers were present in the mesometrial decidua only, and many of these cells expressed the cytolytic protein perforin. By Day 10, NKR-P1+ cells were common within the inner muscle at the base of the mesometrial triangle and in the developing metrial gland, often containing perforin. Larger numbers of perforin+ cells were present in the central decidua towards the ectoplacental cone, and many were weakly NKR-P1+ only. On Day 12, NKR-P1+ cells were almost completely restricted to the metrial gland, with few in decidua, and many were weakly positive. Substantially more perforin-containing cells were seen, indicating that many had lost detectable NKR-P1. This distribution pattern from Days 6-12 is similar to that described for GMG cells and demonstrates that in the rat they belong to the NK cell lineage. These cells were also CD8+ and AsialoGM1+ but negative for CD2 and class II histocompatibility antigens, which is very different from interleukin-2-activated NK cells which they resemble morphologically. The loss during differentiation of NKR-P1 and CD2, which are involved in target adhesion and triggering of NK cells, is consistent with the poor cytolytic capacity reported for these cells.