Natural Killer (NK) Cell Activity in Mice with Acute Graft-Versus-Host Reactions: Characterization of a Thy-1+ NK-Like Cell with a Broadened Spectrum of Lytic Activity in the Spleen and Lymph Nodes

We have shown that acute (GVH) reactions produced in the parental-F1 hybrid combination, A/J----(C57BL/6 x A/J)F1 result in the activation of two cytotoxic cell populations: a host-derived Thy-1+/- natural killer (NK) cell with a lytic spectrum confined to YAC-1 targets, and a donor-derived Thy-1+ NK-like cell that has the ability to lyse target cells that are normally insensitive to lysis by NK cells. Cold-target inhibition (CTI) experiments have shown that the greater range of target cell killing seen in the NK-like population is mediated by a single effector cell with a broadened lytic repertoire. Percoll density fractionation studies have revealed that NK and NK-like cells co-fractionate, suggesting that both are large granular lymphocytes. We we have also shown that NK-like cells do not express either Lyt-2 or L3T4 markers. We have also observed that there is a close temporal relationship between elevated levels of interleukin-2 (IL-2) secretion by spleen cell cultures from mice with GVH disease and the subsequent emergence of splenic NK activity in both acute [A/J----(C57BL/6 x A/J)F1] and chronic (A/J----CBA x A/J)F1 GVH reactions. We have also noted that, despite high levels of IL-2 secretion, mice with chronic GVH reactions do not generate NK-like activity. Interferon (IFN) measurements have shown that, although increased IFN activity can be detected in both acute and chronic models, a preponderance of IFN-alpha/beta and some IFN-gamma is produced in the acute reaction, whereas only IFN-gamma can be detected in the chronic model. These results suggest that, although IL-2 may participate in augmenting conventional NK activity, IL-2 by itself does not generate NK-like activity. We suggest that IFN-alpha/beta may be the cytokine that, either alone or in concert with IL-2, triggers the NK-like cell response. The NK-like cell described in our study resembles a phenotypically identical, donor-derived large granular lymphocyte, identified by others, in close proximity to dead or dying epithelial cells in mice with GVH disease [14]. It has been suggested that these cells may mediate tissue injury. If in fact these graft-derived NK-like cells are involved in the pathogenesis of acute GVH disease, our present findings suggest that they must first be activated by an appropriate complement of cytokines that includes IFN-alpha/beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis

Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.     

Identity of effector cells participating in the reverse antibody-dependent cell-mediated cytotoxicity.

Our previous work has shown that antibody-coated mouse spleen cells express enhanced cytotoxic activity against some Fc-receptor-bearing target tumour cells by a mechanism which appears to be similar to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction with reversed polarity of the antibody bridge (R-ADCC). In this report we have shown that (i) the levels of basal natural killer (NK), ADCC and R-ADCC cytotoxic activities in mouse spleen cells are strongly correlated with each other, (b) simultaneous induction of ADCC and R-ADCC reactions does not result in an additive cytotoxic response, and (iii) YAC cells which do not bear Fc receptors and are highly sensitive to lysis by NK cells, can specifically and competitively inhibit the ADCC and R-ADCC reactions. These results suggest that the R-ADCC reaction may be mediated by the same effector cell population as mediates NK and ADCC reactions against tumour target cells.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

The macrophage, target cell of the synthetic adjuvant muramyl dipeptide.

The mechanism of adjuvant activity of the synthetic glycopeptide N-acetylmuramul-L-alanyl-D-isoglutamine or muramyl dipeptide (MDP) was studied using in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC). Addition of MDP to DBA/2 mouse spleen cell cultures resulted regularly in a 2 to 3-fold increase of PFC numbers/10(6) recovered cells (p less than 0.01). Supernates (SPN) from MDP-stimulated cultures added to standard spleen cell + SRBC cultures brought about even more important increases of PFC numbers (p less than 0.01 to p less than 0.001). SPN from cultures supplemented with MDP alone (without SRBC) were more active than those of cell + MDP + SRBC cultures, and SPN removed on day 3 of culture were more active than those of day 5. This activity of SPN was maintained accross an H-2 histocompatibility barrier. Although pretreatment of spleen cells with anti-theta antigen serum entirely suppressed the anti-SRBC PFC response in spite of the presence of MDP, SPN from these cultures were as active as SPN from normal spleen cell MDP-stimulated cultures. In contrast, pretreatment of spleen cells with specific rabbit anti-mouse macrophage serum entirely suppressed both anti-SRBC response and SPN activity. It was concluded that the target cell for MDP is the macrophage which releases factors ultimately acting on B cells through T cell mediation.     

Natural Killer Cell Activity of Human Fetal Liver Cells after Allogeneic Stimulation

Cells isolated from the liver of human fetuses were confronted with mitomycin C-treated adult allogeneic cells. After this mixed leukocyte culture (MLC)-type reaction, the cytotoxic activity of fetal cells was tested against K562 cell line in a 4-h ⁵¹Cr release assay. Three of the seven fetuses tested (8 to 11 weeks of gestational age) expressed marginal cytotoxic activity before cultivation. Cells from one 8-week-old and one 9-week-old fetus were slightly more cytotoxic when cultured in the presence of allogeneic cells than when cultured in the medium only. Production of gamma interferon (IFN-γ) was not detected in these cultures. Cells of one 18-week-old fetus expressed strong cytotoxic activity against K562 cells after MLC. Thymocytes from the same fetus were not cytotoxic, either before or after MLC against K562 cells. The results indicate that the 'prethymic' human liver contains cytotoxic cells able to spontaneously kill natural killer (NK)-sensitive target cells. Generation of strong NK-like cytotoxicity from noncytotoxic precursor cells was observed only after the thymus becomes lymphoid, suggesting that thymus-processed cells may regulate the generation of NK-like cytotoxic activity. The results suggest a different ontogenity of spontaneous and MLC-induced NK-like cells in the human fetus.     

Augmented Followed by Suppressed Levels of Natural Cell-Mediated Cytotoxicity in Mice Infected with Toxoplasma gondii

The cytotoxic activity of effector cells from mice infected with Toxoplasma gondii was tested in a 4- to 5-hr (51)Cr release assay, using RL 0001000 0011100 0101010 0001000 0001000 0011100 0100010 1000001 1000001 1000001 0100010 0011100 1 and YAC-1 target cells. They showed enhanced cytotoxicity with a peak on the 3rd day postinfection followed by suppression with a peak on the 12th day. The cytotoxicity seemed to be exhibited by natural killer (NK) cells because: (i) pretreatment of the effector cells with antiasialo GM(1) or antiasialo GM(2) plus complement abolished the cytotoxic activity; (ii) the altered cytotoxicity levels were also induced in nude mice; and (iii) the activity was elicited by nonadherent-nonphagocytic cells. The alteration occurred simultaneously in various lymphoid organs with a similar profile. Neither spleen nor bone marrow cells of 12-day-postinfected mice inhibited NK activity of uninfected mice. Culture fluids of the infected mouse spleen and bone marrow cells did not affect the normal mouse NK activity. The proportion of effector cells capable of binding to target cells was constant during the infection. There was no positive correlation between NK activity and serum interferon level; i.e., interferon was detected in the serum of 12-day-postinfected mice but not in that of 3-day-postinfected or uninfected mice. Passively administered interferon or polyinosinic-polycytidylic acid could not restore the suppressed NK activity of 12-day-postinfected mice. Moreover, in vitro treatment of spleen cells from 12-day-postinfected mice with interferon failed to restore the suppressed NK activity. These results suggested that after toxoplasma infection, defective sensitivity to interferon was induced in NK precursor cells, and differentiation to functionally active NK cells might be blocked.     

Characterization by Monoclonal Antibodies of the Cytotoxic Effector Cells in Human Peripheral Blood Mononuclear Cells Reactive against Anchorage-Dependent Tumour Cell Lines

The effector cells for spontaneous cytotoxicity against anchorage-dependent human or mouse tumour cell lines in a 72-h iododeoxyuridine-release assay by normal human peripheral blood cells (PBMNC) or monocyte-enriched fractions were analysed by the use of monoclonal antibodies. PBMNC or adherent or elutriated monocyte-enriched populations of PBMNC were depleted of monoclonal antibody-reactive cells by complement-dependent lysis or separated into monoclonal-antibody-positive or -negative subsets by an indirect rosetting technique followed by Ficoll-Hypaque density gradient separation. The experimental data indicated that in both PBMNC and monocyte-enriched populations, an appreciable proportion of the effector cells with cytolytic activity against adherent human or mouse tumour target cells were positive with B73.1.1 (an antibody with a high degree of selectivity for natural killer (NK) cells), B43.4.1 (or OKM1), and with OKT11a (an antibody recognizing the receptors for sheep erythrocytes), and had the morphology of large granular cells, which have previously been shown to mediate NK activity. These effector cells were mostly negative for BRL.1, BRL.2, B52.1.1, B44.1.1, B13.4.1 and DR antigens, unlike classical monocytes. Some cells which are cytotoxic for the adherent mouse, SV-40-transformed kidney tumour line, TU-5, may bear B52.1.1 or other monocyte-like antigens. Taken together, these results indicate that, in monocyte-enriched populations, both NK cells and monocytes have cytotoxic effector activity against various human and mouse adherent target cell lines.     

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...     

Mechanism of action of a Db-specific helper clone and factor in cytotoxic responses to alloantigens.

The evidence that NK cells can recognize and kill allogeneic lymphocytes has hitherto been based mainly on experiments in intact animals. Here we report results from an in vitro assay, showing allogeneic lymphocyte cytotoxicity in cell suspensions enriched for NK activity against tumour cells by Percoll gradient centrifugation of nylon-wool non-adherent cells. The addition of phytohaemagglutinin (PHA) to the NK-target cell cultures greatly enhanced the cytotoxic response against K562 and allogeneic, but not syngeneic, lymphocytes. The effector cells of ALC are present in the spleen of both euthymic and athymic nude rats, and to a lesser extent in the blood. ALC is augmented by interferon pretreatment of the effector cells, and by depleting the effector cell suspensions of all T cells and helper T cells with the monoclonal antibody MRC Ox19 and W3/25, respectively. Conversely, the activity was nearly abolished by depleting the cell suspensions of MRC Ox8+ cells reacting with rat cytotoxic T cells and NK cells. Furthermore, removal of residual B cells (Ox12+ cells) from the effector cells or attempts to block any putative antibody-dependent cellular cytotoxic mechanism in vitro with the monoclonal antibody Ox12 did not inhibit the NK activity against allogeneic lymphocytes nor against tumour cells. ALC in vitro did not discriminate between T and B or large and small lymphocyte targets. These characteristics of the ALC effector cells substantiate that they are present within the thymus-independent population of cells with NK activity, and are dependent on neither B cells nor immunoglobulin for their recognition and destruction of the target.     

Assignment of human natural killer (NK)-like cells to the T cell lineage. Single allospecific T cell clones lyse specific or NK-sensitive target cells via distinct recognition structures

The aim of the present study was to define the cell lineage of mixed lymphocyte culture (MLC)-induced natural killer (NK) effector cells. Human MLC cells were plated under limiting microculture conditions in the presence of irradiated spleen cells and interleukin 2-containing supernatant. After 18 days, microcultures were scored for proliferation and for cytolytic activity against specific lymphoblasts and NK-sensitive K562 target cells. About 1 in 7 and 1 in 5 proliferating microcultures had specific or NK-like cytolytic activity, respectively. Moreover, several microcultures exhibited dual (specific and NK-like) cytolytic activity, even when they had been established at relatively low numbers of responding cells/well (0.5-0.25) to ensure a high probability of monoclonality. Direct evidence for the existence of cytolytic effector cells with dual activity was achieved by using clones derived from single MLC T cells by micromanipulation. Out of 26 cytolytic clones so derived, 16 exhibited specific cytolytic activity, whereas 22 lysed K562 target cells. More interestingly, 12 of these 26 clones were active against both types of target cells. Only one of these clones was able to lyse autologous or unrelated target cells. In contrast, all such clones lysed the NK-sensitive cell lines G11, MOLT-4, Raji, Daudi, Chang and T-24. Addition of saturating amounts of B9-4 monoclonal antibody in the lytic assays resulted in the inhibition of the specific cytolysis, but not the NK-like activity of clones with dual cytolytic activity. It thus appears that (a) alloreactive cytotoxic T lymphocytes can mediate both specific and NK-like cytolysis and (b) two independent recognition structures are involved in this dual activity.     

Interleukin-2 induced activation of natural killer cells in rats and mice: a comparative study

Effects of interleukin-2 (IL-2) on the natural killer (NK) activities of BALB/c mouse and Wistar rat spleen cells were compared. While mouse spleen cells cultured alone rapidly lost NK activity, co-culture with IL-2 resulted in a marked enhancement of NK activity. In contrast, the levels of NK activity of rat spleen cells cultured alone increased and remained high for 3 days and declined thereafter. Addition of human recombinant IL-2 or purified rat IL-2 did not influence the NK levels in rat spleen cell bulk cultures. Both IL-2 preparations were however biologically active as shown by their capacities to induced proliferation in rat spleen cells. Rat spleen cells suppressed the IL-2 activation of mouse spleen cells in a dose dependent manner, indicating a suppressor influence generated by rat spleen cells. Culture supernatants of rat spleen cells cultured with or without IL-2 for 3 or 5 days could also suppress the mouse spleen NK activation in response to IL-2. The suppressor activity could be concentrated on a 5K MW cut-off Amicon filter indicating that the molecular weight of the factor is more than 5000. These results indicate that a suppressor of IL-2 induced NK activation of mouse spleen cells is released by cultured rat spleen cells.     

B cells from chronic lymphocytic leukemia (CLL) patients are strong inducers of proliferation and major histocompatibility complex (MHC)-unrestricted [natural killer (NK)-like] cytotoxicity in normal T-lymphocytes

We studied the ability of chronic lymphocytic leukemia (CLL) B cells to stimulate proliferation and major histocompatibility complex (MHC)-unrestricted [natural killer (NK)-like] cytotoxicity in a mixed lymphocyte tumor-cell culture (MLTC). CLL-derived B cells induced a significantly higher proliferative response than did B cells from healthy normal donors. Comparable levels of interferon and interleukin-2 production were detected in the mixed lymphocyte cultures (MLC) set up with normal B cells from healthy donors and in MLTC. Higher levels of NK-like cytotoxic activity were induced after stimulation with CLL than with normal B cells in nonlytic precursors. Inhibition experiments with specific monoclonal antibodies indicate that the NK-like response in MLTC is attributable to HLA class II antigens, which are expressed at comparable levels on CLL and normal B cells. Percoll gradient centrifugation of the MLC and MLTC recovered cells demonstrated that the NK-like effectors in both types of cultures were blast-transformed, highly mitotic cells.     

T cell receptor gene rearrangements in cells with natural killer activity in the mouse

Cell-mediated recognition can operate at different levels of complexity and specificity based largely on the time of appearance of effector mechanisms during the course of evolution. Antigen-specific cytotoxic T lymphocytes require both T cell receptor genes and lectin-like cell adhesion molecules (LFA-1, LFA-2, lymphocyte function-associated) to initiate and maintain stable effector target cell conjugates. Natural killer (NK) cells, on the other hand, do not require expression of T cell receptor genes in the recognition and killing of tumor cells and virally infected cells. Adhesion is mediated by a family of glycoprotein molecules, of which the LFA-1 and LFA-2 molecules appear as the most likely candidates. NK-mediated cytolysis proceeds in the absence of MHC restriction, but nevertheless appears to be triggered by depressed levels of self MHC products on the cell surface of target cells. Finally, interleukin 2-dependent, cloned cell lines with NK-like cytotoxic activity should no longer be considered as bona-fide NK cells but rather reclassified as a subset of T cells which displays NK function.     

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.     

The induction of haemolysin producing cells in vitro: inhibition by antiglobulin antisera

The primary immune response (PFC) of mouse spleen cell cultures to sheep red cells was inhibited by anti-IgG, anti-Fab and anti-IgM, but not by anti-Fc or anti-allotype sera. The inhibition of PFC was independent of complement and could be prevented by addition of normal mouse serum.     

Natural killer and lectin-dependent cytotoxic activities of kurloff cells: Target cell selectivity,conjugate formation,and Ca++ dependency

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca⁺⁺ and entry of Ca⁺⁺ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca⁺⁺ was chelated with EDTA or EGTA, or following treatment with the Ca⁺⁺ channel blockers-verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca⁺⁺, PKC activation and serine esterase production.     

Detection of cytotoxic lymphoid spleen cells from STU-mice with Moloney sarcoma by a3H-proline microcytotoxicity assay

A microcytotoxicity assay with³H-proline prelabeled target cells was used for the detection of sensitized lymphoid spleen cells from STU inbred mice inoculated with Moloney sarcoma virus (MSV-M) or ascitic MSV-M tumor cells. The target cell line was derived from ascitic MSV-M tumor cells. With regard to the specificity of the assay nonimmune spleen cells displayed no or only a weak cytotoxicity against these cells, and this was also the case when³H-proline-labeled secondary cultures of syngeneic mouse embryo cells were exposed to both sensitized and nonimmune spleen cells. The time-course pattern of the development of cytotoxic lymphoid spleen cells in STU mice inoculated intramuscularly either with MSV-M or ascitic MSV-M tumor cells was studied. At the stages of tumor development, peak tumor size, and tumor regression the lymphoid spleen cell preparations were found to have relatively strong cytotoxic activity independent of whether the tumor was induced by MSV-M inoculation or tumor cell transplantation. However, in the latter case effector cells appeared earlier and were demonstrable for a longer period than in MSV-M-inoculated mice. Antitheta serum treatment of lymphoid spleen cells taken at the stage of peak tumor size abrogated the cytotoxic activity or diminished it considerably indicating a T-lymphocyte response.     

Mast cells display natural suppressor activity partially by releasing transforming growth factor-beta.

We have previously reported a method for inducing natural suppressor (NS) cells by long-term culture of normal adult mouse spleen cells. The NS cells were further identified as mucosal or immature mast cells by morphology, cytochemistry, histochemistry and function. A cloned immature mast cell line was also confirmed to have NS activity. As NS cells, the cell line suppressed non-specifically the plaque-forming cell (PFC) response. The NS cell-free supernatant was partially enriched by chromatography and some fractions suppressed the PFC response and thymocyte proliferation. Heat treatment of the fractions failed to deplete the suppressive activity. The fractions were confirmed, by immunoblotting analysis, to contain transforming growth factor (TGF)-beta. Recombinant human TGF-beta was also able to suppress the PFC response and thymocyte proliferation. Neutralizing anti-TGF-beta reversed the suppression by both human TGF-beta and the fraction. From the above results, it is clear that mast cells displayed NS activity, at least partially, through the release of TGF-beta.     

Cloned human T-cell lines with allospecific or natural killer-like cytotoxicity

Cloned human T-cell lines were produced by limiting dilution of lymphocytes alloactivated in mixed leukocyte cultures, followed by expansion of clonal progeny with interleukin 2. Selected clones were analyzed for cell-mediated cytotoxicity (CTX) against a variety of targets susceptible or resistant to lytic attack by natural killer (NK) cells. Clones with classical alloantigen-restricted lytic capacity for normal lymphoid targets were found to be distinct from those mediating natural killer-like CTX against NK-susceptible cell line targets. This was established by direct CTX assays and by cold target cross-competition experiments. Moreover, clones with NK-like activity displayed heterogeneous patterns of lysis on different target cell lines in direct CTX, suggesting a clonal distribution of receptors for NK-like target antigens amongst these cultured human NK-active T-cell clones.