Functional activity of spleen and peripheral blood lymphocytes in stress-induced immunodepression

Study of lymphoid organs and T and B cells in white rats shows that long-term stress causes progressive suppression of the immune response. Blockade of steroid hormone synthesis in the adrenal cortex prevents the development of immunodepression, implying a protective effect of such a blockade against stress-related secondary immunodeficiency. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 7, pp. 64–68, July, 1996     

The role of human spleen interferon on natural killer activity of peripheral blood lymphocytes enriched in large granular lymphocytes

The elimination of monocytes as well as B- and T-lymphocytes by forming rosettes with high affinity for sheep red blood cells yielded an enriched population of both natural killer (NK) activity (cytotoxicity: 65.4 +/- 9.9% with an E/T ratio of 12:1, P less than 0.005) and large granular lymphocytes (LGL: 76 +/- 13%) compared to the untreated lymphocyte population where NK activity is 35.7 +/- 17.3% (E/T 12:1) and the percentage of LGL of 26 +/- 6%. We studied the action of type I interferon (IFN) obtained from human spleens, on NK activity of 9 peripheral blood lymphocyte populations and 9 enriched in LGL. NK activity of the total lymphocyte population is significantly increased (P less than or equal to 0.05) in 6 out of 9 cases after treatment by interferon. Cell populations enriched in LGL showed increased NK activity in only one case after treatment by interferon, but no increased activity was found in the other cases. These results are compatible with the notion of cellular cooperation in increased NK activity by interferon.     

Bleomycin sulfate-induced micronuclei in human,rat, and mouse peripheral blood lymphocytes

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 μg/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37°C in a 5% CO₂ atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species concentration-response curves gave good fits (r² values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]^0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations ≧ 20 μg/ml. The nucleation index was significantly decreased in all species at either 80 or 160 μg/ml. © 1995 Wiley-Liss, Inc.     

Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethylnitrosourea

The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 muMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post-injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non-parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE-forming lesions was found to be dose-dependent for the spleen but not for blood. Within each dose the persistence of SCE-forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; analysis of high frequency cells may be a more sensitive method of detecting exposure than the t-test; and a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.     

Cyr61对外周血NK细胞的增殖和活性调节

目的:体外实验探讨Cyr61对外周血NK(pNK)细胞的增殖和活性的影响。方法:磁珠分选得到纯化的pNK细胞;流式细胞术检测pNK细胞的抑制性和活化性受体、细胞因子IFN-γ的生成和细胞毒性。结果:Cyr61促进外周血淋巴细胞中pNK细胞的增殖,降低CD16的表达;降低淋巴细胞中pNK细胞的活化性受体NKG2D、NKp30和CD244的表达,提高抑制性受体KIR3DL1的表达;能够促进纯化pNK细胞的KIR2DL1表达,降低纯化pNK细胞的胞内IFN-γ生成和细胞毒性。结论:Cyr61对pNK细胞活化的抑制作用,支持了其他学者关于Cyr61在正常妊娠和子痫前期的发病方面可能发挥重要作用的研究结果。     

Telomerase activity of peripheral blood lymphocytes in patients with chronic hepatitis B

Chronic hepatitis B is the immunocompromising condition. The decrease of lymphocyte telomerase is linked to immunosenescence in hosts. To know whether telomerase activity of lymphocytes is involved in immunopathogenesis in patients with chronic hepatitis B, telomerase activity of peripheral lymphocytes was determined in such patients. The results showed that telomerase activity in resting peripheral lymphocytes of healthy subjects was detectable at low level, and obviously increased (P<0.001) after stimulation in vitro with phytohaemagglutinin (PHA). Telomerase activity of lymphocytes decreased with age in both groups with or without PHA stimulation. Telomerase activity of resting lymphocytes in patients with chronic hepatitis B was also observed at detectable level and markedly upregulated after PHA stimulation. The decreased telomerase activity of resting lymphocytes was found in patients with chronic hepatitis B (n=14, 0.32+/-0.27) compared to that in healthy subjects (n=17, 0. 52+/-0.28; P<0.05). However, there was no difference present between these two groups in telomerase activity of activated lymphocytes with PHA. In addition, no effect of recombinant human interleukin-12 (rhIL-12) on telomerase expression was observed in either the patient group or the healthy group. We concluded that the decreased telomerase activity of lymphocytes in chronic hepatitis B patients is present, which may be partly responsible for immunosuppressive condition in such patients.     

两种不同HLA-B分子对NK细胞表面受体表达的研究

目的：探讨两种HLA-B分子(HLA-B51和HLA—B39)对NK细胞表面活化性受体CDl6和抑制性受体KIR3DL1表达的调节。方法：采用流式细胞仪检测NK细胞分别与转染HLA-BSl和HLA-B39分子的K562细胞相互作用后，CD16和KIR3DL1的表达情况。结果：外周血淋巴细胞与K562细胞作用24小时后，CD56 CD16 细胞数、KIR3DL1 细胞数均明显增加。与表达HLA-B39的K562细胞相比，表达HLA-B51的K562细胞与外周血淋巴细胞作用后，CD56 CD16 细胞数、KIR3DL1 细胞数均明显下降。结论：NK细胞杀伤靶细胞时，活化性受体CD16表达上调后会伴有抑制性受体KIR3DL1的上调；HLA-B51分子表达在K562细胞后，表达外源性HLA-B51分子的K562细胞与NK细胞作用，NK细胞表面受体KIR3DL1的表达可下调，同时伴有CD16的表达下调。     

Surface markers of small lymphocytes appearing in the mouse Ehrlich ascites tumour, host spleen and blood

Small lymphocytes sampled from intraperitoneally growing Ehrlich ascites tumour in CBA/H-T₆ mice as well as host spleen and blood at different days of tumour development were characterized radioautographically on the basis of two surface markers, IgM for B cells and θ antigen for T cells. A direct binding of ¹²⁵I-labelled anti-IgM detected natural surface IgM, while an indirect binding following a prior exposure to anti-θ antibody detected θ antigen. Cells remaining unlabelled with the latter procedure were considered to lack both markers (double negative). While the incidence of IgM+ve small lymphocytes within the tumour declined, their absolute numbers increased with tumour growth. Low levels of antiglobulin binding shown by these cells were considered to reflect low levels of maturation, because (1) our previous studies indicated that they were newly formed, and (2) the extent of antiglobulin binding by B lymphocytes in the marrow is known to increase with increasing post-mitotic age. The proportions and the absolute numbers of θ+ve as well as the double negative small lymphocytes increased within the growing tumours. Within the host spleen, the incidence of IgM+ve small lymphocytes remained unchanged but their absolute numbers increased because of splenomegaly. The degree of antiglobulin binding by these cells was comparable to that of the normal splenic population. The incidence of θ+ve cells dropped but their absolute numbers remained unchanged in the spleen during tumour growth. In contrast, the incidence as well as the absolute numbers of double negative cells increased markedly. This cell category increased also in the blood, possibly in transit to the tumour site and other lymphoid organs from the bone marrow, where they were most prevalent. Their bone marrow origin was further suggested by a preponderance of marrow derived small lymphocytes at the tumour site as well as in the host spleens found in our earlier studies. Double negative population in the spleen showed a paucity of C′3 and Fc receptors on the cell surface and included cells capable of producing B lymphoid colonies in vitro.     

Depletion of peripheral blood T lymphocytes and NK cells during the course of ebola hemorrhagic Fever in cynomolgus macaques

During the course of an experimentally induced Ebola virus (EBOVA) infection of cynomolgus macaques, peripheral blood mononuclear cells were isolated and characterized by multi-color flow cytometry. Both CD4+ and CD8+ lymphocyte counts decreased 60-70% during the first 4 days after infection. Among CD8+ lymphocytes, this decline was greatest among the CD8(lo) population, which was composed mostly of CD3- CD16+ NK cells. In contrast, the number of CD20+ B lymphocytes in the blood did not significantly change during the course of the infection. Phenotypic analysis of T lymphocyte subsets by flow cytometry failed to show evidence of a robust immune response to the infection. Apoptosis could be detected as early as day 2 postinfection among the CD8+ and CD16+ subsets of lymphocytes. Increased expression of CD95 (Fas) suggests that apoptosis may be induced via signaling through the Fas/Fas-L cascade. In contrast, the number of HLA-DR+ cells increased tenfold in the blood during the course of infection. These data suggest that EBOV may block dendritic cell maturation after infection, thereby inhibiting activation of lymphocytes and eliminating those subsets that are most likely to be capable of mounting an effective response to the virus.     

The immunological significance in the guinea-pig of in vitro transformation of lymph node, spleen and peripheral blood lymphocytes

Cells from lymph nodes or spleen, or peripheral blood leucocytes of immunized guinea-pigs were cultured in the presence of antigens or phytohaemagglutinin. Significant incorporation of tritiated thymidine occurred in a variable proportion of the experiments with lymphocytes from each of the three sources. Cells taken from animals that had been immunized with sheep erythrocytes with adjuvant, and which showed strong delayed hypersensitivity, and from animals immunized intravenously with sheep erythrocytes, which failed to show delayed hypersensitivity reactions, both responded to sheep erythrocytes in vitro.

Cells from guinea-pigs immunized with complete Freund's adjuvant alone, which showed strong delayed hypersensitivity to tuberculin PPD, gave more positive responses in vitro than did cells taken from animals which received an intravenous injection of tuberculin PPD before the adjuvant. These animals showed no or weak delayed hypersensitivity reactions (immune deviation).

The immunological significance of the in vitro proliferative reaction is discussed.

     

Increase of natural killer (NK) activity of mouse lymphocytes following in vitro treatment with cytosine-arabinoside

The in vitro influence of cytosine-arabinoside (Ara-C) on mouse NK activity was studied treating effector cells, target cells or effector and target mixture with graded concentrations of the drug. Ara-C increased the NK efficiency of mouse splenocytes without enhancing the susceptibility of target cells or the cytolytic events when added to effector-target mixture. This phenomenon was confirmed with splenocytes collected from congenitally athymic (nude) or conventional donors of different ages, untreated or depressed or boosted for NK activity by various agents. In addition Ara-C increased the NK activity of spleen cells of nude mice deprived of nylon-adherent cells, and did not affect suppressor cells capable of inhibiting the lytic phase of NK process. The drug was able to significantly augment the binding ability of spleen cells to the NK-sensitive YAC-1 target. It was concluded that Ara-C would increase the efficiency of natural cytotoxicity presumably through a direct influence on effector lymphocytes.     

The immunosuppressive potency of various steroids on peripheral blood lymphocytes, T cells, NK and K cells

The immunosuppressive effect of natural and synthetic steroids was tested in vitro on phytohemagglutinin (PHA) stimulated T lymphocytes and peripheral blood lymphocytes (PBL), as well as on NK and K cell activity. Three groups of steroids, significantly different in their immunosuppressive activity, were identified. Fluorohydrocortisone, and methylprednisolone were highly potent in suppressing PHA stimulation of T lymphocytes and PBL. Hydrocortisone was of intermediate potency, whereas cortisone, dihydrocortisol, and tetrahydrocortisol were of low potency. T lymphocytes were more sensitive to the suppressive effect of fluorohydrocortisone and methylprednisolone than were PBL cultures. NK and K cell activity was suppressed only by the high potency synthetic steroids and even then the suppression of K cell activity was not significant except at high in vitro steroid concentrations. The present findings support the conception that different lymphocyte subpopulations have different susceptibility to the effect of highly potent steroids. Thus, lymphocyte heterogeneity must be taken into account when the immunosuppressive potencies of different glucocorticoids are studied. Furthermore, the findings in different lymphocyte populations ranked the relative in vitro immunosuppressive potency of glucocorticoids different from the relative anti-inflammatory potencies reported in the literature.     

Targeting of peripheral blood T lymphocytes

Conclusions In summary, human single-chain gene transduced T cells were shown: to express the scFv on their surface, to recognize their relevant ligand (tumor-associated antigen) on tumor target cells, to produce cytokines and, to lyze tumor cells. In our earlier review on retargeting T lymphocyte specificity [11], we concluded that a number of questions needed to be answered: (1) Is triggering of cytolysis by CTL or lymphokine production most important to generate anti-cancer effects? Both are important, especially to eliminate bystander cells which do not express tumor-associated antigen [35, 75, 83, 106]. (2) Can targeted CTL traffick and home to the tumor site? Yes, they can. (3) Does humanization of mouse mAbs reduce HAMA responses? Our preliminary experiences suggest that this is the case (unpublished data).Significant progress has therefore been made in the laboratory and in clinical tests, and will continue to be made. We are now preparing for the clinical phase I/H testing of in vivo anti-tumor activity of T lymphocytes retargeted by transfer of chimeric receptor genes encoding Ab-type specificity.     

A clonal analysis of human peripheral blood lymphocytes displaying natural killer-like activity

Human Fc gamma receptor-bearing lymphocytes and T cells, prepared by sorting peripheral blood lymphocytes using the B73.1 monoclonal antibody, have been cloned by limiting dilution. Although quiescent lymphocytes of either cell type were unresponsive to interleukin 2 (IL 2), following induction with phytohemagglutinin and/or the BSM B lymphoblastoid cell line they could be expanded in IL 2 utilizing a mixed irradiated feeder system. Clones originating from B73.1+ lymphocytes displayed a characteristic large granular lymphocyte (LGL) morphology but were otherwise functionally and phenotypically heterogeneous. Of 36 clones analyzed 19 displayed significant natural killer (NK)-like activity, each clone having a target cell repertoire identical to uncloned NK effectors. Furthermore, only a minority of clones (i.e. 5) displayed significant antibody-dependent cellular cytotoxicity, while levels of lectin-induced cellular cytotoxicity were normally commensurate with a clones level of NK-like activity. No correlation was evident between the phenotype of a clone and its cytotoxic activity since of 12 cytotoxic clones phenotyped, 8 expressed the OKT3 antigen but lacked the B73.1 antigen; 2 lacked the OKT3 antigen but expressed the B73.1 antigen and one lacked both OKT3 and B73.1 antigens. In addition the expression of OKT8 and OKT4 antigens was not in any way predictive of the cytotoxic capacity of a given clone. Several clones expressing T cell associated antigens bore a phenotype that distinguished them from T cell clones insofar as T cell subset antigens were expressed in the absence of the OKT3 antigen and vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunophenotypic study of atypical lymphocytes. Generated in peripheral blood and spleen of nude mice After MHV-72 infection

Inbred athymic nude mice (BALB/c) were injected subcutaneously with the wild-type murine gammaherpesvirus 72 (MHV-72), which has been shown to induce the infectious mononucleosis (IM)-like syndrome in immunocompetent mice. The mice were also injected with UV-irradiated MHV-72. We studied the pattern of acute and chronic infection in the blood cells of the nude mice and detected viral DNA sequences in the infected leukocytes by polymerase chain reaction (PCR) technique up to when the animal died, close to 1 month postinfection. Using the UV-irradiated virus that induces an increase in mouse survival time, the viral sequences were present in the blood up to 3 months postinfection, then disappeared. We detected atypical lymphocytes in the blood of mice infected with both wt and UV-irradiated viruses. These atypical cells were similar in shape to those present in the blood of patients with IM induced by Epstein-Barr virus (EBV). Via Unscheduled DNA Synthesis (UDS), DNA synthesis was demonstrated in the atypical cells whose phenotype is identical to that of B cells, as shown with a panel of monoclonal antibodies. By double immunofluorescence staining, using an hyperimmune anti-MHV-72 serum and an anti-IgG + IgM + IgA monoclonal antibody, we demonstrated that these atypical B cells express some viral antigens. Contrary to the immunocompetent mice, the nude mice did not develop splenomegaly after infection with wt virus, probably due to the lack of T cell subsets. However, we observed an increase of nude mice B cells in the spleen. The nude mice died 1 month postinfection showing a high frequency (40%) of atypical lymphoblast-like B-cells in the blood; the increase in natural killer (NK) cell number was not detected after infection. Such findings suggest that NK cells probably did not play an important role in immune response to the MHV infection in nude mice. Finally, this mouse model could play an important role in antigammaherpesviral therapy of immunocompromised patients.     

Effect of hydrocortisone on spontaneous and mitogen-dependent activity of peripheral blood lymphocytes in some vertebrates

The effect of hydrocortisone on functional activity of peripheral blood lymphocytes in different vertebrates during ontogeny was studied by the blast transformation test. Hydrocortisone in a dose of 100 mg/kg inhibited functional activities of T and B lymphocytes in vitro stimulated by mitogens. This inhibitory effect was observed in both young and adult animals. However proliferative activity of concanavalin A-dependent blood T suppressors in rats increased, especially in adult animals (by 3.5 times). Hence, hydrocortisone produced different effects on functional activity of peripheral blood lymphocytes in the studied species, and these effects are dose- and species-dependent.     

人外周血T淋巴细胞胀亡现象的观察

目的观察人外周血T淋巴细胞的胀亡现象,探讨建立T细胞胀亡检测方法.方法密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞,分空白组及地塞米松组,培养后观察细胞光镜、荧光镜及电镜形态学,并用流式细胞仪检测胀亡细胞比例变化.结果①人外周血T淋巴细胞经96小时体外培养,可自然出现典型细胞胀亡形态学改变.②经72小时培养,不同浓度地塞米松组(1×10-6、1×10-5、1×10-4、1×10-3 mol/L)T细胞的胀亡率分别为(3.49±0.42)%、(5.17±0.48)%、(8.44±0.72)%、17.93±1.50)%.③在1×10-5mol/L地塞米松作用下,不同培养时间(48、72、96、120小时)T淋巴细胞的胀亡率分别为(0.53±0.10)%、(6.36±0.80)%、(20.60±1.59)%、25.56±1.76)%.结论人外周血T淋巴细胞存在胀亡现象,地塞米松可诱导人外周血T淋巴细胞胀亡.