Impaired production of interleukin-2 after surgery.

The capacity of peripheral blood mononuclear cells (PBM) to produce interleukin-2 (IL-2) was studied serially before and following operation in patients undergoing various surgical procedures. In patients who had major surgery, significant decrease in IL-2 activity was observed 1, 3 and 6 days after operation as compared to that before surgery, although there was no significant change throughout the post-operative course in patients undergoing minor surgery. IL-2 activity returned to the pre-operative level by the 8th post-operative day. However, it remained significantly depressed 8 days after surgery in patients who had undergone major surgical procedures of more increasing severity. Distribution of T cell subsets, especially OKT4 positive cells, did not differ significantly from the pre-operative value throughout the post-operative course. However, the depressed production of IL-2 3 days after surgery could be abolished when adherent cells were removed from PBM by plastic adherence procedures. These results indicated that adherent cells, but not quantitative change in T cell subsets, might be responsible for the depression of IL-2 production after surgery.     

Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice: natural killer cell activity in cultured spleen leukocytes concomitant with T-cell-dependent immune interferon production

The characteristics and specificities of spleen and peritoneal cytotoxic cells generated during lymphocytic choriomeningitis virus (LCMV) infection of C3H/St mice were examined. Activated natural killer (NK) cell activity was identified in fresh leukocyte populations from the 2nd to 8th days postinfection, whereas virus-specific cytotoxic T-cell activity was detected from the 6th to 14th days. When leukocytes were cultured overnight at 37 degrees C before assay, T-cell activity was still observed, but nonspecific activated NK cell-like cytotoxicity was only detected on the 6th and to a lesser degree the 8th day postinfection. Overnight culture of leukocytes taken earlier in the infection eliminated their NK cell activity. Similar activities were seen with spleen cell, plastic-adherent peritoneal cell, and nonadherent peritoneal cell populations. The virus-specific cytotoxicity observed with adherent peritoneal cells was due to contamination with cytotoxic T cells, as shown by H-2-restricted cytotoxicity and sensitivity to anti-theta antibody and complement. The nonspecific cultured day 6 effector cell from either the spleen or peritoneum displayed killing specificities and other physical properties identical to those of activated NK cells, but had sensitivities to anti-theta antibody and complement intermediate between activated day 3 NK cells and cytotoxic T cells. Culture stable NK-like cells were not found in athymic nude mice, suggesting a T-cell-dependent mechanism. Whereas LCMV spleen homogenates contained 10-fold-higher levels of interferon at day 2 than at day 6 postinfection, substantially more (nearly 20-fold) interferon was made in cultures of day 6 cells than day 2 cells. Spleen interferon was predominantly type I, whereas the culture interferon was predominantly type II, as shown by acid lability studies. Significant levels of interferon were produced by nylon-wool-passed day 6 spleen cells, and virtually all interferon production was eliminated by treatment of either day 2 or day 6 cells with antibody to theta antigen and complement, suggesting that T cells produced the interferon in vitro. Furthermore, athymic nude mice had no culture-stable NK cells 6 days postinfection, and spleen cells from them failed to produce significant levels of interferon in vitro. Addition of interferon (type I, fibroblast) to cultured C3H spleen cells affect the already elevated levels of cytotoxicity in day 6 cultures, suggesting that the NK cells in the day 6 culture were already activated. Our results suggest that T cells responding to LCMV infection secrete interferon type II which causes the continued activation of NK cells in culture. The resulting population of activated NK cells therefore appears to be relatively stable in culture and to express more theta antigen because of this T-cell dependence. Although one could mistakenly or allospecific cytotoxic T cells or cytotoxic macrophages, more careful examination shows that they are most likely activated NK cells...     

The Appearance of Cytotoxic Lymphocytes in Unstimulated and Concanavalin A-Activated Human Peripheral Mononuclear Cells

The presence of a mitogenic lectin during the cytotoxicity assay enables the detection of non-specific cytotoxicity (lectin-dependent cell-mediated cytotoxicity (LDCC]. Human mononuclear cells cultured at 37 degrees C in vitro became cytotoxic after 1 day, as detected by the LDCC. In some individuals, the presence of adherent cells during culture inhibited the development of cytotoxicity. Activation by concanavalin A (Con A) resulted in enhanced cytotoxicity after a few days in culture. There was no clear relationship between the lectin doses required for optimal induction of cytotoxicity and [3H]thymidine uptake. High Con A concentrations that induced a proliferative response often inhibited cytotoxicity in mononuclear cells depleted of adherent cells.     

Inhibition of in Vitro Generation of Cytotoxicity Against Tumor Cells by Monoclonal Antibody to Thy-1

The Thy-1 molecule on murine T lymphocytes has been suggested to play a role in cellular activation events leading to a variety of immunologic functions. We present evidence that this molecule may be involved in signals leading to the in vitro generation of cytotoxic T cells against several tumor cell lines used as stimulators in mixed tumor-lymphocyte culture. The presence of monoclonal antibody against a polymorphic determinant on the Thy-1 molecule markedly reduced the generation of cytotoxicity after three days of culture of murine splenocytes with stimulator tumor cells bearing low levels of Ia antigen. In contrast, no effect was seen when the stimulators were either allogeneic splenocytes, or a tumor cell line expressing large amounts of Ia. These results suggest that the Thy-1 molecule is critically involved in events leading to the generation of cytotoxic effectors under some, but not all conditions.     

Cytotoxicity of temporarily adherent human mononuclear blood cells in monocyte monolayers

Adherent monolayers of freshly isolated human peripheral blood mononuclear cells consist mostly (greater than 90%) of monocytes. The initially adherent nonmonocytic cells detach during the first day of in vitro culture. Concomitant with cell detachment the cytotoxicity mediated by adherent monolayers is reduced. This is in part due to medium exchange with removal of initially adherent cells from the monolayers, cells that are potent mediators of cytostasis and cytolysis. The temporarily adherent cells consist of 10% monocytes, 35% T lymphocytes, 10% B lymphocytes, and 45% non-T, non-B lymphocytes. In addition, 45% of these cells stain positively with the monoclonal antibody OKT 10. The reduction in monolayer-mediated cytoxicity from day 0 to day 1 of in vitro culture is less pronounced if the temporarily adherent cells are kept within the culture during the cytotoxic assay time. When the temporarily adherent cells are returned to adherent monocyte monolayers an additive effect on cytolysis is demonstrated with nonactivated, lymphokine-activated and alpha-interferon-activated effector cells, and in antibody-dependent cell-mediated cytotoxicity. The heterogenous population of temporarily adherent cells in freshly isolated monolayers of human mononuclear blood cells seems to be responsible for some of the cytotoxic effects regularly ascribed to freshly isolated human blood monocytes.     

Function of surface-adherent cells in the induction of the human mixed leucocyte reaction. I. The transient surface adherence of responding lymphocytes in the human mixed leucocyte response

The aim of this study was to establish the temporal requirement for adherent cells in the mixed leucocyte reaction (MLR). At various intervals after co-cultivation, mixed leucocytes from two unrelated donors were separated into adherent and nonadherent cells and the MLR response of each population assayed on day 6. After 1 hr of co-cultivation adherent cells had a high MLR response, whereas the nonadherent cells were unresponsive and could not be restimulated by the addition of allogeneic leucocytes or syngeneic adherent cells. In contrast, the MLR was positive when syngeneic adherent cells were added to nonadherent cells which had not been co-cultured prior to the initiation of culture. The MLR of adherent cells declined progressively as a function of time of co-cultivation prior to separation. Conversely, there was a progressive increase in the MLR of nonadherent cells obtained after 12, 18 and 24 hr of co-cultivation. Morphological studies revealed a higher number of lymphocytes in the early adherent cell population than at later times. We conclude that during the first few hours of co-cultivation lymphocytes with the potential of responding to allogeneic stimulation are retained in the adherent cell population. These lymphocytes are released from the adherent cells and can generate a positive MLR without further support from adherent cells. The transient adherence of responding lymphocytes after short exposure to allogeneic cells could provide a mean of separating a subpopulation of lymphocytes capable of reacting to allogeneic stimuli.     

Correlation between Suppression of B Cell Anti-Hapten Response and Generation of Cytotoxic T Cells Induced by Hapten-labelled Syngeneic Lymphocytes

The correlation between suppression of B cell antibody response and generation of cytotoxic T cells induced by hapten (FITC) labelled syngeneic lymphocytes was studied. Primary in vitro sensitization with lymphocytes labelled with the hapten concentrations (FITC 0.5 and FITC 0.05) previously shown to induce specific B cell unresponsiveness generated cytotoxic cells that specifically lysed haptenated syngeneic blast cells. In vivo priming with either SC-FITC 0.5 or SC-FITC 0.05 could be demonstrated after in vitro restimulation with similarly haptenated cells. Thus, cells from primed mice gave a cytotoxic response already after 3 days in culture, whereas primary responses were only observed on day 5. Furthermore, immunization was evident as an increased specific lysis at low effector/target ratios. However, no direct cytotoxicity could be detected in spleen cells from immune animals. Comparison between kinetics of in vivo development of specific antibody suppression and induction of cytotoxic cells detectable after in vitro boosting revealed no correlation between the two phenomena.     

Interleukin-1 and interleukin-2 production in resistant and susceptible inbred mice infected with Trypanosoma congolense.

Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in cell-mediated cytotoxicity. Spontaneous cytotoxicity in a 2-hr assay against K562 target cells was shown to be largely mediated by contaminating natural killer (NK) cells. Treatment of adherent cells with NK-specific monoclonal antibody anti-Leu-11 plus complement abolished almost completely the spontaneous cytotoxicity. Spontaneous cytotoxicity by adherent cells was also reduced when the phorbol ester PMA was present in the assay. On the other hand, PMA induced a cytotoxic response in NK-cell depleted adherent cells after prolonged 18 hr incubation. The cell population responsible for this dichotomous effect of PMA on adherent cell-mediated cytotoxicity was shown to be monocytes, as revealed by monoclonal antibody treatment. Pure NK cell preparations were not affected by PMA in their cytolytic capacities. Reactive oxygen species are not involved in NK-cell mediated cytotoxicity, while PMA stimulated the monocytes to exert cytolysis and suppressed NK cells by the generation of these highly toxic oxygen products. Hydrogen peroxide especially seemed to be the mediator in this oxygen-dependent monocyte-mediated cytotoxicity and NK-cell suppression.     

吗啡和电针对术后淋巴细胞增殖反应和T细胞亚群的影响

为了观察硬膜外吗啡和电针对术后病人免疫功能的影响，对１８例单纯胆囊切除术病人分别于术前和术后第１、３、７天检测淋巴细胞增殖反应和Ｔ细胞亚群的变化。结果表明硬膜外注入吗啡１ｍｇ在有效镇痛的同时却抑制了淋巴细胞增殖反应直至术后第７天，出现Ｔ细胞亚群中ＣＤ＾３＋，ＣＤ＾＋４表达下降。     

Alloantigen-Induced Enhancement and Suppression of Human Cytotoxic Lymphocyte Responses to Autologous Lymphoblastoid Cell Lines

In vitro sensitization of human lymphocytes to autologous lymphoblastoid cell lines (LCL) has been shown to give rise to cytotoxic lymphocytes capable of lysing autologous as well as allogeneic LCL cells. However, allogeneic LCL cells were found to be markedly less effective than autologous LCL cells in terms of generating lymphocytes capable of lysing autologous LCL cells. The addition of allogeneic LCL cells or allogeneic normal lymphocytes to a mixture of responding lymphocytes and X-irradiated autologous LCL cells suppressed the generation of cytotoxic lymphocytes against autologous LCL cells. Furthermore, suppressor T cells generated in allogeneic mixed leucocyte culture (MLC) and supernatants from MLC likewise decreased the generation of cytotoxic lymphocytes to X-irradiated autologous LCL cells. In contrast to the findings that alloantigens suppress the generation of cytotoxicity of X-irradiated autologous LCL cells, which ordinarily induce strong cytotoxic responses, were the findings that allogeneic stimulating cells and supernatants from MLC enhanced cytotoxic responses to autologous ultraviolet light or extensively heat-treated LCL cells that induce weaker cytotoxic responses. The possible mechanisms whereby alloantigens enhance or suppress cytotoxic responses to autologous abnormal cells and the implications of these findings are discussed.     

Induction of suppressor T cells in culture--I. Cell-cell interactions

The present study was designed to examine the cellular requirements for the generation of the suppressor T cells induced in the presence of fetal calf serum in culture. When C57Bl/6 mouse spleen cells were cultured for 4-5 days, these precultured cells were shown in mixing experiments to suppress the generation of cytotoxic effector cells (CTL) against allogeneic P815 cells or the generation of anti-SRBC humoral response by freshly explanted C57Bl/6 spleen cells. Spleen cells cultured in the presence of silica (0.5 mg) for 4 days, did not develop suppressor activity. However, when silica was added 3 days after the start of the suppressor generation culture, the development of suppressor cells was only slightly affected, although the phagocytic activity of these spleen cells was still totally abolished. When plastic or G-10 Sephadex column nonadherent spleen cells were cultured alone for 4-5 days, these cells did not suppress the generation of CTL or anti-SRBC humoral response. When the nonadherent spleen cells were cultured with plastic adherent spleen cells, however, suppressor cells developed and the suppressor activity of these cells was dependent on the number of adherent spleen cells co-cultured with the non-adherent spleen cells. This activity of the adherent spleen cells was insensitive to treatment with anti-Thy 1.2 serum plus complement and to X-irradiation. Furthermore, adherent PEC could not substitute for adherent spleen cells, indicating a possible tissue specificity for the macrophages in the adherent cell fraction which can function in supporting and/or accelerating the differentiation of "immature" suppressor T cells. Finally, culture-induced suppressor T cells were sensitive to X-irradiation and their activity was refractory to IL2 (TCGF), whereas the activity of alloantigen-induced suppressor cells was sensitive to IL2.     

Concanavalin A-mediated in Vitro Activation of a Secondary Cytotoxic T-Cell Response in Virus-primed Splenocytes

In a recent report it was shown that what appeared to be secondary cytotoxic T cells could be obtained from lymphocytic choriomeningitis virus (LCMV)-primed splenocytes after stimulation in vitro with the non-specific T cell mitogen concanavalin A (Con A). The present experiments attempt to characterize further these effector cells and, in particular, to establish whether the Con A-activated cytotoxic effectors are qualitatively different from the secondary cytotoxic T cells induced by restimulation with the homologous antigen. It was found that: (1) in vitro activation with Con A could be obtained with populations harvested between 13 days (the earliest tested) and at least 300 days after priming; (2) cytotoxicity was independent of the presence of carried-over Con A in the cytotoxicity assay; (3) cytotoxicity was dependent on close association between activated T cells and target cells, since no evidence was found to indicate a role for other cell types or soluble (cytotoxic or arming) factors; (4) cytotoxicity was specific with regard to both virus and 'self'. By comparison with previous data on LCMV-induced cytotoxic T cells, it is concluded that Con A induces the generation of cytotoxic T cells from LCMV-primed splenocytes, which, by the criteria used, are indistinguishable from virus-induced secondary cytotoxic T cells. The implications of these findings are discussed.     

Generation of phenotypically different T cell populations by cell-free or cell-bound preparations of varicella zoster virus

We used three different preparations of varicella zoster virus (VZV) to sensitise mononuclear cells obtained from VZV immune donors. These were autologous infected fibroblasts, live cell free virus and heat inactivated cell free virus. After 14 days of in vitro sensitisation and expansion with interleukin-2, the mononuclear cells which had been exposed to autologous infected fibroblasts had generated mainly cells of the cytotoxic/suppressor phenotype (CD8) while those stimulated with cell free virus (live or heat inactivated) had generated cells of the helper/inducer phenotype (CD4). Functional assays showed that the effector cells generated after exposure to autologous infected fibroblasts lysed autologous virus infected target cells but not uninfected cells. Effector cells generated in the same way but lacking HLA identity with the virus infected target cells failed to demonstrate cytotoxicity. None of these effector cells showed any significant natural killer cell activity. No specific cytotoxicity was obtained by effector cells generated after exposure to cell-free virus. We conclude that the way in which VZV antigen is presented to the mononuclear cells influences the cell type responding in tissue culture. These findings would be useful in the generation of T cell clones of different cell surface phenotype and function.     

Lymphocyte subpopulations in man: enhancement and inhibition in generation of cytotoxic T lymphocytes in vitro

The requirements for generation of allospecific CTLs in vitro have been studied by "three cell" experiments, with two allogeneic T cell suspensions as cocultured responders, stimulated by irradiated B cells. This study describes enhancement as well as inhibition of the response, functionally defining T amplifier and T depressor cells regulating the differentiation of CTLs. Enhancement is the result of an amplifying effect of cytotoxic precursor T cells. Amplification is due to HLA-D region incompatibility between the responding T cell donor and the stimulating donor resulting in strong proliferation. Inhibition is the result of a depressing effect on cytotoxic precursor T cells mediated by cocultured T cells. The depression seems to be due to HLA-A, -B, -D identity between one of the responder T cell donors and the stimulator cells. The induction of depression is radiosensitive, accompanied by strong proliferation and CTL generation with cytotoxic specificity against the cocultured and depressed donor target cells. It is suggested that the functionally defined T depressor cells are cytotoxic precursors mediating cytostatic functions before strong cytotoxicity is detectable.     

Cell-mediated cytotoxicity against Sendai-virus-infected cells.

After injection of Sendai virus, a parainfluenza virus type I, mice generate cytotoxic lymphocytes which lyse specifically Sendai-virus-infected target cells in vitro. Their action is not inhibited by specific antibody in vitro. Killer cell activity appears 4 days after infection, reaches a maximum on the 7th day and disappears on the 14th to 16th day. Decrease of cytotoxic cell activity is correlated with an increase of haemagglutinating antibodies. The cytotoxic effector cell could be characterized as a thymus-derived cell, there is no specific activity in antibody-dependent cell-mediated cytolysis (ADCC). The degree of cytotoxic effector cell activity is only slightly influenced by the dose of injected infective virus. Using different syngeneic Sendai-virus-infected cells as targets for cell-mediated cytotoxicity, a tumor line was not lysed by cytotoxic lymphocytes in spite of viral surface antigens. Preliminary experiments were performed to demonstrate the H-2 gene restriction of the cytotoxic interaction. Using macrophages and tumor cells as targets only syngeneic infected target cells were lysed.     

Augmentation of natural killer (NK) cell activity by a streptococcal preparation,OK-432, in patients with malignant tumors

Recently, a streptococcal preparation, OK-432 has been used successfully as an immunopotentiator for immunotherapy in patients with malignant tumors in Japan. In this paper, we report that the administration of OK-432 augments the cytotoxic activity of peripheral blood lymphoid cells against a natural killer (NK) cell-sensitive erythroleukemic cell line, K562, in tumor patients. In patients before or after surgery, sufficient amounts of OK-432 strongly augmented the cytotoxic activity within 3 days after the initial administration of OK-432. Thereafter the levels of cytotoxicity declined rapidly. The administration of a lower dose of OK-432 gave a lower increase in cytotoxicity. Enhanced cytotoxicity occurred with the reintroduction of OK-432 but remained at lower levels of activity. Characterization and fractionation of OK-432-induced effector cells revealed that the augmented cytotoxicity seemed to be carried mainly by NK cells. A low titer of interferon was detected in 3 of 10 patients within 72 hr after the first inoculation of the agent. Furthermore, we discuss the potency of OK-432 for the induction of interferon in detail.     

Cellular Changes in Lungs of Mice Infected with Influenza Virus: Characterization of the Cytotoxic Responses

Transpleural lavage of lungs from uninfected C3H mice yielded an average of 300,000 leukocytes per mouse. This number increased eightfold within 6 days after intranasal inoculation with virulent influenza A/Hong Kong/68 (H3N2) virus. Macrophages and lymphocytes in approximately equal numbers comprised 90% or more of the leukocytes both before and during infection. B, T, and null lymphocytes comprised, respectively, 9, 21, and 18% of the leukocytes before infection and 7, 26, and 5% by day 6. In absolute numbers, macrophages and T lymphocytes provided the major increments during infection. Cytotoxic activity of mononuclear cells from lung lavages was compared in a chromium release assay using syngeneic L929 target cells with the activity of mediastinal lymph nodes, spleens, and peripheral blood of uninfected and infected C3H mice. Nonspecific cytotoxicity for target cells infected with H3hkNeq1 or B/Lee influenza virus was found with mononuclear cells from uninfected mice. This activity tended to be highest with lavage leukocytes and was associated with adherent cells, presumably macrophages. Increased virus-specific cytotoxicity was detected with lavage cells by day 6 and persisted through day 9, the period of maximal pneumonia. Similar cytotoxic activity also appeared in cells from the nodes and spleen at this same time but was not detected in peripheral blood cells. The virus-specific cytotoxicity of lavage cells was due largely to a nonadherent cell possessing Fc receptors and theta antigen but lacking C3 receptors; these properties are compatible with actively cytotoxic T lymphocytes. The cytological characteristics of the infiltrating leukocytes and the cytotoxicity data suggest that the local T cell response to influenza virus infection in the lung is a major contributor to the pneumonia observed in this mouse model.     

Hypothalamic-immune interactions: neuromodulation of natural killer activity by lesioning of the anterior hypothalamus

Placement of bilateral electrolytic lesions in the preoptic-anterior hypothalamic area (AHT) of Fischer 344 rats results in decreased splenic NK activity as compared to control and normal animals. Animals with AHT lesions have a decrease in NK activity 4 and 7 days after lesion placement, with a return to normal activity by day 14. Fractionation of spleen cells on glass bead columns results in nonadherent and adherent cell fractions with NK activity. AHT lesions affect NK activity only in the adherent cell fraction. The removal of macrophages from this cell fraction did not restore NK activity. Moreover, this NK activity is not the result of cytotoxic macrophages. Hypophysectomy decreases NK activity in lesioned and non-lesioned animals, suggesting the influence of pituitary factors. These data indicate that the anterior hypothalamus is capable of modulating the cytotoxic activity of NK cells. Thus, neuroimmunomodulation may be a potential factor in susceptibility to some disease states such as viral infections and neoplasia.     

The effect of BCG stimulation on natural cytotoxicity in the rat.

The natural cytotoxic activity of lymphoid cell populations from control and BCG-stimulated rats was examined using four target cell lines, K562, CCRF/CEM, Bri8 and Mc40. In control rats, the cytotoxicity of peritoneal cells was below that of spleen cells but above that of peripheral lymph node cells. Intraperitoneal injection of BCG induced a significant dose and time-dependent augmentation of cytotoxicity by peritoneal cells from W/Not and PVG/c rats, against all four cell lines. The increased activity reached a peak on days 4 and 5 after injection and returned to control levels by day 12. Spleen and lymph node cells from stimulated rats did not show increased cytotoxicity. K562 and CCRF/CEM target cells were considerably more susceptible to killing than Bri8 and Mc40 target cells. Separation of peritoneal cells from BCG-treated rats by density-gradient centrifugation gave an interface population, enriched with mononuclear cells showing high cytotoxic activity and a pellet population enriched with polymorphs showing very low activity. Nylon-fibre column filtration gave non-adherent and adherent cytotoxic populations. Cytotoxic activity was not diminished by removing cells adhering to Sephadex G10 or cells phagocytosing carbonyl iron, suggesting that much of the activity in this system was due to non-phagocytic mononuclear cell populations.     

Liver regeneration and immune system--morphological and cytochemical studies of activated spleen cells during the liver regeneration after partial hepatectomy

Morphological and cytochemical changes were investigated in spleen cells after 70% hepatectomy in mice. During liver regeneration after the hepatectomy, the spleen weight gradually rose to a peak at 6 day. In the spleen, the number of POD-positive myelocytic cells and NCAE-positive granulocytic cells also reached a peak 4 days after the operation and then decreased. On the other hand, ANBE-positive monocytic cells gradually increased up to 9 day and didn't decrease for this period. On day 9 of the culture, the boundary between the red and the white pulps of the spleen became unclear. In this spleen, the clusters of blast cells were sporadically observed. Splenic T cells cocultured with nonparenchymal adherent normal liver cell or nonparenchymal adherent normal liver cell supernatant developed into granulocyte colonies in earlier periods and monocyte colonies in later periods. These findings suggest that the factors released from liver cells may regulate strictly spleen cell activation. Blast cell formation in the culture with nonparenchymal adherent normal liver cell supernatant was amplified by anti interferon (alpha + beta) antibody. These facts indicate that the functional network of cytokines is formed by the interaction of various cytokines (IL-1, IL-6, CSF, IFN, etc.) in nonparenchymal adherent normal liver cell supernatant.