Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Early development of CD8-negative and CD8-positive MHC-unrestricted cytotoxic cells induced by alloantigen inoculation in mice

Peritoneal cells (PC) in C57B1/6 (B6, H-2b) mice receiving an intraperitoneal (i.p.) injection of allogeneic BALB/c (H-2d) spleen cells demonstrated potent cytotoxic activity against syngeneic, xenogeneic, third-party allogeneic tumors as well as H-2d derived tumors. Maximum cytotoxic activity against various tumors other than H-2d derived tumor, B16 (H-2b) was elicited on day 3 post allosensitization and decreased drastically thereafter, whereas cytotoxic activity against P815 (H-2d) peaked 3 days after the inoculation and maintained the peak activity thereafter. Surface phenotype of PC responsible for the cytotoxic activity against B16 was Thy-1+/-, Lyt-2-, L3T4-, asialo GM1 (AGM1)+, and that of PC against P815 was Thy-1+, Lyt-2+ (or Lyt-2+/-), L3T4-, AGM1+. These phenotypes showed similar phenotypes to the counterparts against B16 and against P815 in spleen cells induced by intravenous inoculation of alloantigen. When mice were pretreated i.p. with anti-AGM1 antibody before the allosensitization, anti-P815 cytotoxic-activity in PC was completely diminished. Similar activity in spleen, however, was enhanced by i.v. treatment with the antibody before the i.v. inoculation of alloantigen. The data clearly demonstrate that in vivo inoculation of B6 mice with normal allogeneic cells induces "NK-like" CD8- cytotoxic cells and "anomalous" CD8+ cytotoxic cells in PC.     

Anti-metastatic effect by in vivo administration of concanavalin A through augmentation of T-derived activated killer activity: efficacy to B16 melanoma expressed MHC antigen

We attempted to investigate if the in vivo administration of concanavalin A (Con A), a potent T cell stimulator, would render anti-metastatic activity in hosts. Assays of activity were performed 20 days after iv inoculation of two clones of the B16 melanoma, B16-H (H-2+, highly metastatic), B16-L (H-2-, low metastatic), or 3LL cells into C57BL mice by enumerating lung colonies. In some experiments, hosts treated with anti-asialo GM1 Ab were used to evaluate effector mechanisms other than NK cells. While the injection of Con A alone had no significant effect on anti-metastatic activity, in nonimmunized hosts the effect by Con A was displayed when the mice were preimmunized with B16-H cells but not in those immunized with B16-L cells. Immunization with B16-H or B16-L cells alone resulted in the generation of killer cells with promiscuous lytic activity and induced an anti-metastatic effect against B16-H, B16-L, and 3LL cells. Con A treatment significantly augmented the killer activity of spleen cells of mice preimmunized with B16-H cells but not of those immunized with B16-L cells. The effectors from mice immunized with B16-H alone or given both Con A and B16-H were mainly of Thy 1+ Lyt2+ asialo GM1- cells, on the other hand, those from mice immunized with B16-L cells expressed asialo GM1 antigen. We showed the efficacy of Con A on the anti-metastatic effect in relation to the host immune response.     

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analysis revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1-, Lyt 2- and Thy 1+, Lyt 1-, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1-, Lyt 2- cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2-responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1-, Lyt 1-, and Lyt 2-. On the other hand, a predominant type of the IL 2-responsive cells in BALB/c nu/nu mice were Thy 1-, Lyt 1-, and Lyt 2-, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Characterization of Effector Cells against B16 Melanoma in Mice Inoculated with Allogeneic Spleen Cells

Inbred C57BL/6 (B6) mice which had received an inoculation of allogeneic spleen cells showed remarkable antitumor activity against syngeneic tumor challenge with B16 melanoma cells 3 days after the allogeneic cell inoculation. This antitumor activity was not specific to the inoculated alloantigen, since the challenging B16 cells are syngeneic to B6 mice and since it was induced by BALB/c spleen cells as well as C3H/He spleen cells. The antitumor activity was sensitive to an in vivo treatment with anti-asialo GM1 (AGM1) antiserum or anti-Thy.1 monoclonal antibody (mAb) just before the tumor challenge and was resistant to an in vivo treatment with anti-CD8 (Ly. 2) mAb. These results suggest that AGM1+Thy.1+CD8– activated natural killer (NK) cells were generated by alloantigen inoculation and took an important part in the antitumor effect of the alloantigen inoculation.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

Suppression of B-cell differentiation by natural killer (asialo GM1+) cells in mice

Mice were injected intravenously with rabbit antiserum to ganglio-n-tetraosylceramide (asialo GM1, ASGM1), a neutral glycosphingolipid present at high quantities on the surface of natural killer (NK) cells. Spleen cells prepared from the mice were then examined for NK activity against YAC-1 targets, for phagocytic cells and by flow cytometric analysis for Thy1, Lyt1, Lyt2, ASGM1 and surface Ig (SIg) phenotypes. Administration of anti-ASGM1 in mice resulted in a complete depletion of NK activity and ASGM+1 cells in the spleen, but no changes in the proportions of Thy1+ cells and their Lyt1+ and Lyt2+ subsets and phagocytic cells. Corresponding to this selective depletion of ASGM+1 cells and NK activity, the spleen cells showed an increased number of SIg+ B cells and augmented mitogenic responses to B-cell but not T-cell mitogens. These NK-depleted spleen cells also showed production of pokeweek mitogen (PWM)-driven plaque-forming cells (PFC) to much higher levels than those of control spleens. In the spleens of mice treated with varying concentrations of anti-ASGM+1, a good correlation was found between the decreased NK activity and the enhanced PFC response. To directly test the possible suppressor activity of NK cells on PWM-induced PFC response, NK (ASGM+1) cells were highly purified from the spleen by a combination of Percoll gradients and cytolysis of T cells by monoclonal antibodies followed by indirect panning. When added to NK-depleted spleen cells, they suppressed the augmented PFC response of NK-depleted spleen cells, depending on the number of cells added. These results suggest that NK (ASGM+1) cells in mice exhibit a suppressor property on B cells, which are undergoing spontaneous or mitogen-induced differentiation.     

Establishment of hybridoma cells with natural killer(NK)-like activity against syngenic tumor cells.

The 4D1D4 hybridoma cells were derived from the fusion of spleen cells from BALB/c nude mice with NS-1 mouse myeloma cells. The surface phenotypes of 4D1D4 hybridoma cells were Thy-1.2+, L3T4 (CD4)-, Lyt-2 (CD8)-, Asialo GM1+ and p-55 interleukin-2 (IL-2) receptor (CD25)-. This phenotypic pattern was consistent with the surface phenotype of NK cells. The 4D1D4 cells showed the definite killer activity against a syngenic tumor cell line, RL male-1, but not against an allogenic YAC-1 line. The killer activity of the 4D1D4 cells was not affected by the addition of exogenous IL-2. It was, therefore, suggested that 4D1D4 cells might be representative of resting NK cells with expression of no functional IL-2 receptors. The hybridoma technology might be useful for establishment of the cloned NK cells.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

Generation of lymphokine-activated killer (LAK) cells from tumor-infiltrating lymphocytes

Culture of tumor-infiltrating lymphocytes (TIL) containing about 20% BMC2 tumor cells with recombinant human interleukin 2 (rIL-2) resulted in the diminish of tumor cells and the growth of lymphocytes. These IL-2-activated lymphocytes showed a strong cytotoxic activity against not only syngeneic tumor cells but also allogeneic tumor cells. Such broad-reactive killer cells, termed lymphokine-activated killer (LAK) cells, are also inducible from spleen cells by in vitro activation with IL-2. However, LAK cells generated from TIL (TIL-LAK) showed higher cytotoxic activity against BMC2 than LAK cells generated from spleen cells (S-LAK). Furthermore, it was demonstrated that TIL-LAK cells revealed marginal cytotoxic activity against normal Con A blasts and YAC-1 cells as opposed to S-LAK. Flow cytometric analysis of TIL-LAK indicated that TIL-LAK cells mainly consisted of Thy 1.2+, Ly 2+, asialo GM1+ cells. TIL-LAK cells displayed not only in vitro cytotoxicity but also in vivo anti-tumor activity. Furthermore, it was also confirmed that TIL-LAK cells could be induced in autochthonous mouse tumor systems and human gastric tumor systems.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.     

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed Streptococcal preparation

Summary More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i. p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i. p. injection of the admixture of the nonadherent PEC (1×10⁷ cells) with BAMC-1 cells (1×10⁵) survived for more than 60 days. When the same nonadherent PEC (1×10⁷ cells) were i. p. transferred adoptively 1 day after the inoculation of 1×10⁵ BAMC-1 tumor cells, again all mice survived.When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations.These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.     

In vivo generation of lymphokine-activated killer cells by sensitization with interleukin 2-producing syngeneic T-lymphoma cells

Culture of C57BL/6 mouse spleen cells with syngeneic EL4 lymphoma cells resulted in no induction of killer cells reactive against EL4 cells. However, in vitro sensitization of C57BL/6 mouse spleen cells with interleukin 2 (IL-2)-producing EL4 lymphoma cells caused the generation of lymphokine-activated killer (LAK) cells, which lyse a variety of tumor cells. Consistent with an in vitro system, we demonstrate that Thy 1.2+, Ly2+, asialo GM1+ LAK cells were successfully induced by in vivo immunization with syngeneic IL-2-producing EL4 lymphoma cells.     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

Partially restorative role of T cells for low interleukin 2 dependent growth of NK cell progenitors from nude mice

The frequency of cells in the spleens of nude mice which could be grown in conditioned medium containing interleukin 2 and of those which developed natural killer (NK)-like activity was evaluated. Although BALB/c nu/nu spleen cells have higher spontaneous NK activity than euthymic mice, they showed a substantially lower frequency of proliferating and cytotoxic cells as compared to BALB/c nu/+ littermates. This defect in cells of nu/nu mice was reversed in part by culturing nu/nu responder cells in the presence of irradiated (3,000 R) splenic or thymic feeder cells that included T cells. In contrast to the dissociation of NK activity and progenitor frequencies in nude mice, the results of parallel studies with spleen cells from euthymic mice indicated that the limiting dilution assay correlated well with previously described features of NK activity. High-NK-reactive CBA/J mice were found to have a considerably higher frequency of interleukin 2 dependent NK cell progenitors than low-NK-reactive strains of mice when assessed against NK-susceptible YAC-1 targets. The frequency of progenitors of cells cytotoxic against YAC-1 was higher in spleens of high-NK-reactive mice than that of cells reactive against the NK-insensitive target P-815. Furthermore, the phenotype of the progenitor cells and of the cultured effector cells was consistent with that of NK cells rather than cytotoxic T cells in that the cells expressed asialo GM1, some Thy-1, but no detectable Lyt-1 or Lyt-2 antigens. Thus, the present observations suggest that the subpopulation of NK cell progenitors in nude mice which can grow and develop cytotoxic reactivity in vitro in the presence of interleukin 2 is small, that it can be increased appreciably in the presence of T cells, but that this does not represent the major pathway for development of NK cells in athymic individuals.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

Generation of nonspecific murine cytotoxic T cells in vitro by purified human interleukin 2

Murine spleen cells developed into nonspecific cytotoxic cells within 72 hr of culture in the presence of highly purified sources of human interleukin 2. In whole spleen cell cultures, human interleukin 2 generated effector cells which were Thy 1.2⁺, Lyt 2.2⁺, resistant to γ irradiation (1000 R), and capable of lysing both H-2 compatible and incompatible targets. The effector cells generated in this manner were not restricted to classical natural killer cell-sensitive targets. If thymus-derived cells (T cells) were depleted from the spleen cell population before culture with human interleukin 2, the effector cells generated were enriched in effectors capable of lysing natural killer cell-sensitive targets. Interferon was not produced in interleukin 2-stimulated spleen cell cultures. In addition, heterologous antibody to murine -γ-interferon did not abrogate the generation of cytotoxic cells by human interleukin 2. These and additional data suggest that human interleukin 2 is capable of stimulating γ-irradiation-sensitive Thy 1.2⁺ cell(s) capable of lysing a variety of target cells regardless of inherent sensitivities to classical natural killer cells. Thy 1.2^? cells were also stimulated by human interleukin 2 and lysed only natural killer cell-sensitive targets. Human interleukin 2 caused some Thy 1.2^? cells to become susceptible to lysis by anti-Thy 1.2 serum and complement.