Purification and target cell range of in vivo elicited blast natural killer cells

Blast natural killer (NK) cells were elicited in the spleens of mice by treatments with the interferon inducers lymphocytic choriomeningitis virus (LCMV) or polyinosinic-polycytidylic acid (poly I:C). The blast-NK cells, separated on the basis of size by centrifugal elutriation, were compared with blast cytotoxic T lymphocytes (CTL) generated during infection with LCMV. In vivo treatments with antibody to asialo GM1 (AGM1) blocked the appearance of blast-NK cells but not blast-CTL. Antibody and complement depletion experiments indicated that the blast-NK cells were AGM1+, NK 1.2+/-, Lyt-5+/-, Thy+/-, Qa-5/NK 1.1+, Lyt-2-, B23.1-, and J11d-. Blast-NK cells could be unequivocally distinguished from blast-CTL, because the blast-CTL were completely sensitive to treatments with anti-Lyt-2 and complement, whereas the blast-NK cells were completely resistant. The blast-NK cells were purified from populations of large-size cells by antibody and complement treatments that depleted the co-eluting monocyte/macrophages and polymorphonuclear leukocytes. The population resulting after separation from dead cells over Percoll gradients represented approximately 1% of the total spleen cells, contained greater than 60% large granular lymphocytes and mediated greater than 15% killing of YAC-1 target cells in a 4-hr 51Cr release assay at an effector to target cell ratio of 1:1. The purified blast-NK cells lysed a broad range of target cells at relatively low effector to target cell ratios. The order of sensitivity of the target cells was YAC-1 much greater than K562 approximately equal to L-929 much greater than P815, consistent with that reported for NK cell-mediated lysis. The ability of the blast-NK cells to mediate lysis of NK cells also was examined. The purified NK cells mediated significant levels of lysis against the NK-like cloned line, NK1B6B10, in a 51Cr release assay. Furthermore, the purified blast-NK cells mediated lysis of bound blast-NK cells in a single-cell agarose assay. These results indicate that highly purified blast-NK cells are exceptionally efficient at mediating lysis and suggest that NK cells may act to negatively regulate the proliferation of NK cells by lysing other NK cells.     

A comparative analysis of the cell surface properties of activated vs endogenous mouse natural killer cells

Mouse natural killer (NK) cells can be activated by interferon and by interferon inducers, such as viruses. In the present report we compared the cell surface properties of the nonactivated "endogenous" NK cells from normal mice with NK cells activated in vivo by acute infection with lymphocytic choriomeningitis virus (LCMV) or in vitro by interferon. Several striking differences were found: 1) in vivo LCMV-activated as well as in vitro interferon-activated NK cells were more adherent to nylon wool columns than the endogenous NK cells; 2) activated NK cells were more EA-monolayer adherent than endogenous NK cells; this adherence could be blocked by streptococcal A protein, indicating that activated NK cells expressed greater Fc receptor mediated adherence; 3) LCMV-activated NK cells that passed through nylon wool columns expressed relatively low EA adherence properties; 4) the sensitivity of NK cells to anti-theta serum + complement treatment was increased in spleen cells late after LCMV infection (day 6 to 7) but not in spleen cells early after infection (day 2 to 3); 5) as shown by centrifugal elutriation, LCMV-activated spleens contained a population of large NK cells that were not present among endogenous spleen cells. In contrast to previous reports, these findings show that there are several distinct changes as a result of both long-term in vivo activation or of short-term in vitro activation of NK cells. These changes might functionally be involved in the increased lytic ability of activated NK cells.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

Generation of large granular T lymphocytes in vivo during viral infection

Cytolytic lymphocytes were isolated from the spleens of lymphocytic choriomeningitis virus (LCMV)-infected mice and were characterized in regards to function, cell size, antigen phenotype, and cell morphology. Only 2% of the Lyt-2+ cells from uninfected mice were large granular lymphocytes (LGL), whereas 21% of the Lyt-2+ cells isolated 7 days postinfection were LGL. The day 7 Lyt-2+ populations contained all of the LCMV-specific, class I histocompatibility antigen-restricted cytotoxic T lymphocyte (CTL) activity, but no natural killer (NK) cell activity. The NK cell activity was consistently recovered in Lyt-2- populations isolated from both control mice and mice on day 7 postinfection. The LGL isolated on day 7 postinfection were concluded to be predominantly T cells and not NK cells because 1) the proportions of LGL in fractionated cell populations 7 days postinfection correlated with levels of CTL-mediated lysis but not NK cell-mediated lysis, 2) they were recovered in the Lyt-2+ population, and 3) antibody to asialo GM1, known to eliminate NK cell-mediated lysis but not T cell-mediated lysis, dramatically reduced NK cell LGL numbers in vivo on day 3 postinfection but only marginally affected LGL numbers on day 7. Virus-induced inflammation elicited a 50-fold increase in LGL numbers in the peritoneum on day 7 postinfection. The peritoneal exudate LGL were also associated with CTL activity and were resistant to treatment with antibody to asialo GM1. These results indicate that in vivo-generated CTL have the morphology of LGL and that the appearance of cytoplasmic granules correlates with the ability of cells to mediate lysis. To focus on cells being stimulated during infections, activated blast cells were separated from small resting cells by centrifugal elutriation. Coincidental with the peak in overall spleen leukocyte cytotoxic activity, the peaks of blast NK cells and CTL were at days 3 and 7 postinfection respectively. More than 50% of the blast lymphocytes isolated on either day 3 or day 7 postinfection were LGL. The CTL activity in the blast populations on day 7 postinfection was mediated by Lyt-2+ cells, and 37 to 64% of these Lyt-2+ blast cells were LGL. Cytolytic NK cell and CTL LGL could not be distinguished by morphology or by cell densities, because they overlapped in low density Percoll gradient fractions. Since this technique has been used to enrich for LGL, these data indicate that heterogeneity in LGL populations may result from the presence of both CTL and NK cell LGL.     

Pathogenesis of murine cytomegalovirus infection in natural killer cell-depleted mice.

The effect of natural killer (NK) cells on the course of acute and persistent murine cytomegalovirus (MCMV) infection was examined by selectively depleting NK cell activity by inoculation of mice with antibody to asialo GM1, a neutral glycosphingolipid present at high concentrations on NK cells. The dose of MCMV required to cause 50% mortality or morbidity in control C57BL/6 mice dropped 4- and greater than 11-fold, respectively, in mice first treated with anti-asialo GM1. NK cell-depleted mice had higher (up to 1,000-fold) virus titers in their lungs, spleens, and livers at days 3, 5, 7, and 9 postinfection. Spleens and livers of control mice were virus-free by day 7 postinfection, and their lungs showed no signs of active infection at any time. In contrast, MCMV had disseminated to the lungs of NK cell-depleted mice by day 5, and these mice still had moderate levels of virus in their lungs, spleens, and livers at day 9. Markedly severe pathological changes were noted in the livers and spleens of NK cell-depleted, MCMV-infected mice. These included ballooning degeneration of hepatocytes and spleen necrosis. MCMV-infected, NK cell-depleted mice had severe spleen leukopenia, and their spleen leukocytes exhibited a significantly lower (up to 13-fold) response to the T cell mitogen concanavalin A when compared with those of uninfected and MCMV-infected controls. It appeared that NK cells exerted their most potent antiviral effect early in the infection, in a pattern correlating with interferon production and NK cell activation; treatment with anti-asialo GM1 later in infection had no effect on virus titers. The relative effect of NK cell depletion on MCMV pathogenesis depended on the injection route of the virus. NK cell depletion greatly augmented MCMV synthesis and pathogenesis in mice inoculated either intravenously or intraperitoneally but had no effect on the course of disease after intranasal inoculation, at any time point examined. One month after intraperitoneal inoculation of virus, NK cell depletion resulted in a six- to eightfold increase in salivary gland virus titers in persistently infected mice, suggesting that NK cells may be important in controlling virus synthesis in the salivary gland during persistent infection. This treatment did not, however, induce dissemination of virus to other organs. These data support the hypothesis that NK cells limit the severity, extent, and duration of acute MCMV infection and that they may also be involved in regulating the persistent infection.     

The role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1

The relative roles of interferon (IFN) and natural killer (NK) cells in herpes simplex virus type 1 (HSV-1) infection of mice were examined. Adoptive transfer of adult mouse leukocytes into 4- to 6-day-old suckling mice protected the recipients from HSV-1 infection, as judged by viral titers in the spleen 2 days postinfection. Protection was mediated by several classes of leukocytes, including those depleted of NK cell activity by antibody to asialo GM1 and those depleted of macrophages by size separation. Mice receiving these leukocytes produced significantly higher levels of IFN 6 hr postinfection (early IFN) than did HSV-1-infected mice not receiving donor leukocytes. Antibody to IFN, under conditions that blocked early but not late IFN synthesis, greatly enhanced HSV-1 synthesis in mice receiving leukocytes and completely removed the protective effect mediated by leukocytes. High doses of anti-asialo GM1 blocked both NK cell activity and early IFN production and resulted in high titers of HSV-1. This effect on virus synthesis was not seen if mice were given antibody 1 day postinfection. Lower doses of anti-asialo GM1, which still depleted NK cell activity but had no effect on early IFN production, did not enhance HSV-1 synthesis. Depletion of NK cell activity with a low dose of antibody had no effect on the reduced HSV-1 synthesis resulting from prophylactic IFN treatment or on the enhanced HSV-1 synthesis resulting from antibody to IFN treatment. Thus, resistance to acute HSV-1 infection in mice correlates with early IFN production but not with NK cell activity, suggesting that NK cells are not major mediators of natural resistance in this model and that the antiviral effect of IFN is not mediated by NK cells.     

Human natural cytotoxic lymphocytes: definition by a monoclonal antibody of a subset which kills an anchorage-dependent target cell line but not the K-562 cell line

A monoclonal mouse antibody (HNC-1A3) which defines a subset of human lymphocytes with natural cytotoxic activity was produced and studied. HNC-1A3+ cells represent 12 +/- 3% of peripheral blood mononuclear leukocytes. When sorted out using a fluorescence-activated cell sorter, they consist of 60% small lymphocytes, 35% large (predominantly agranular) lymphocytes, and 5% monocytes. They contain 30 +/- 6% E-rosette-forming cells, 6 +/- 1% OKT4+ cells, 17 +/- 6% OKT8+ cells and less than 2% OKT10+ or Leu-7 (HNK-1)+ cells. They are responsible for most of the natural cytotoxic activity against the MA-160 prostatic adenoma cell line but mediate an insignificant amount of cytotoxicity against the NK prototype target K562 cell line. Conversely, Leu-7+ cells which mediate most NK activity against K562 are weakly active against MA-160. Our data suggest a heterogeneity among leukocytes mediating natural cytotoxicity, with restricted specificities for the recognition sites on target cells.     

Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1)

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.     

Cells with natural killer activity in the cerebrospinal fluid of normal mice and athymic nude mice with acute Sindbis virus encephalitis

Sindbis virus causes an acute, nonfatal inflammatory encephalitis in weanling BALB/c mice. Mononuclear inflammatory cells are present in the cerebrospinal fluid (CSF) as well as in the parenchyma of the brain. Both aspects of this inflammatory response were eliminated by treatment with cyclophosphamide. Athymic nude (nu/nu) mice developed no inflammation in the brain, but did develop a CSF pleocytosis that peaked on day 2 after infection. The time course of the appearance of cells in the CSF was earlier in nu/nu mice than their heterozygote (nu/+) littermates. The pleocytosis in nu/nu mice reached a peak on day 2, whereas in nu/+ mice the peak was on day 4, as it is in normal BALB/c mice. To determine whether some of the CSF cells in nu/nu mice may be natural killer (NK) cells, NK activity was measured in a 4-hr assay by using a YAC-1 target cell. NK cell activity in the spleen and peripheral blood was induced by infection with Sindbis virus in nu/nu mice with a similar time course to that of nu/+ mice (peak 1 day after infection). CSF from nu/nu mice had NK activity present 2 days after infection that was greater than that present in either the peripheral blood or spleen. BALB/c and nu/+ mice had insufficient cells present for assay at day 2, but BALB/c mice had NK activity present in the CSF 3 and 5 days after infection that exceeded that in the peripheral blood or spleen. Brain interferon was detectable on day 1 in nu/nu mice, but not until day 2 in nu/+ mice even though the amounts of brain virus were the same in the two groups at all time points. It is concluded that cells with NK activity contribute to the CSF pleocytosis induced by acute Sindbis virus encephalitis.     

Lysis of uninfected and virus-infected cells in vivo: a rejection mechanism in addition to that mediated by natural killer cells

To examine the lysis of virus-infected cells in vivo, uninfected and lymphocytic choriomeningitis virus (LCMV)-infected L-929 cells were labeled in vitro with [125I]-iododeoxyuridine and implanted intravenously into mice. Natural cytotoxicity against both uninfected and virus-infected cells was demonstrated in normal uninfected mice, but LCMV-infected cells were cleared from the lungs and whole bodies more rapidly than uninfected cells. Treatment of L-929 cells with defective interfering LCMV inhibited standard virus synthesis and protected the target cells from enhanced in vivo rejection. The in vivo rejection was apparently mediated by a cellular constituent of the host immune response and not simply a result of virus-induced cytopathic effects on the target cell, as hydrocortisone acetate and cyclophosphamide each reduced rejection of both target cell types and eliminated the enhanced rejection of LCMV-infected cells. The enhanced rejection of LCMV-infected cells was not restricted by histocompatibility antigens, indicating that classic T-cell recognition was not involved in the lysis, and since the enhanced rejection of LCMV-infected cells was mediated by mice treated with cobra venom factor, complement was also not involved in the lysis. Although moderate levels of interferon (102 U/ml) were present in the sera and although there was a modest activation of natural killer (NK) cells in the lungs of LCMV-infected cell recipients but not uninfected cell recipients, the enhanced rejection of virus-infected cells did not appear to be NK cell mediated. Normal mice and mice depleted of NK cell activity by in vivo treatment with antibody to asialo ganglio-n-tetraosylceramide ( AGM1 ) rejected uninfected and LCMV-infected L-929 cells similarly. This antibody markedly inhibited the rejection of NK-sensitive YAC-1 cells. In addition to the natural cytotoxicity directed against virus-infected cells, a second nonspecific rejection mechanism appeared in response to treatment protocols which induced interferon. Polyinosinic-polycytidylic acid and infection with LCMV augmented in vivo rejection of both uninfected and LCMV-infected L-929 cells but eliminated the differential rejection of the virus-infected cells. Infection with LCMV also augmented the in vivo rejection of the NK-sensitive target cell, YAC-1. In vivo treatments with anti- AGM1 sera only moderately inhibited the elevated rejection of uninfected and LCMV-infected L-929 cells, indicating that the enhanced rejection of these target cells was predominantly mediated by a mechanism other than that mediated by NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Natural killer cells vs cytotoxic T cells in the peripheral blood of virus-infected mice

The cell-mediated immune response of mice against various enveloped RNA and DNA viruses expressed by immune lymphocytes from the spleen and the peripheral blood (PBL) were compared. PBL from mice of various strains infected with vaccinia virus, vesicular stomatitis virus (VSV), or lymphocytic choriomeningitis virus (LCMV) were tested on histocompatible or incompatible target cells infected with the homologous virus. PBL from immune mice showed clear H-2 restriction, but additionally, they expressed high natural killing (NK) activity on YAC-1 cells. The high NK-cytolytic activity of PBL on YAC-1 differed significantly from that expressed by splenic lymphocytes. In both lymphocyte populations lysis was detected as early as 1 day after infection; NK activity decreased in the spleen after day 4 post infection, whereas that of PBL persisted at high levels for up to 10 days after infection. Treatment of mice with anti-asialo GM1 in vivo abrogated NK activity in PBL effector cells tested in vitro. These results may explain some of the difficulties to observe MHC-restricted cytotoxic T cells in PBL from humans or primates during primary infections with virus.     

Induction of alloreactive cytotoxic T cells by acute virus infection of mice

Alloreactive cytotoxic T lymphocytes (CTL) distinct from virus-specific CTL and activated natural killer (NK) cells were generated during acute lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice. The alloreactive CTL shared similar antigenic markers (Thy-1.2+, Lyt-2.2+, and asialo GM1-) with the virus-specific CTL that appeared at the same time 7 days postinfection, but had different target specificities. These alloreactive CTL lysed allogeneic but not syngeneic or xenogeneic targets. These were distinct from activated NK cells, which lysed all target cell types, peaked 3 days postinfection, and had a phenotype of asialo GM1+, Thy-1 +/-, Lyt-2.2-. Cold target competition studies indicated that there were several subsets of alloreactive T cells with distinct specificities, and that these alloreactive T cells were not subsets of the virus-specific T cells. Similar types of alloreactive CTL were induced at much lower levels in C3H/St mice. This may indicate that the generation of this "aberrant" T cell activity is under genetic control. Hence, the LCMV infection of C57BL/6J mice induces several cytotoxic effector populations including alloreactive CTL, activated NK cells, and virus-specific CTL. Virus infections therefore have the ability not only to polyclonally stimulate B cells, as previously described, but also to stimulate CTL.     

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.     

IgG antibodies to asialoGM1 are more sensitive than IgM antibodies to kill in vivo natural killer cells and prematured cytotoxic T lymphocytes of mouse spleen

IgG and IgM antibodies, which were isolated from the anti-asialoGM1 (GA1) serum, had different effects against natural killer (NK) and prematured cytotoxic T cells (CTLs) in in vivo administration and in in vitro treatment. In in vitro treatments, the IgM antibody killed NK cells of nude mouse spleen in the presence of complement (C') 12 times more potently than the IgG antibody did, and either antibody with C' killed pre-CTL. In in vivo administrations, only the IgG antibody was effective in diminishing NK activity of the nude mouse spleen cells and in suppressing antigen-specific CTL induction from primed spleen cells by in vitro stimulation with X-irradiated tumor cells. The IgM antibody was not effective at all in either system. The in vivo effect of the IgG on NK activity was blocked by preadministration with silica or carrageenan but not by that with cobra venom factor (CVF). These results indicate that in vivo administration of anti-GA1 antiserum leads to macrophage-mediated depletion of CTL precursors as well as NK cells.     

Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities

The susceptibility of cytotoxic effector lymphocytes and their induction to in vivo or in vitro treatment with rabbit anti-neutral glycolipid ganglio-N-tetraosylceramide (anti-ASGM1) antiserum was investigated. Intravenous injection of anti-ASGM1 antiserum eliminated measurable natural killer (NK) cell activity in spleen cells of mice infected for 5 days with Vaccinia virus, or for 8 days with lymphocytic choriomeningitis virus (LCMV) if injected 24 hr prior to testing. In addition, this treatment lowered measurable virus-specific cytotoxic T cell activity by 60 to 95%. Virus-specific cytotoxic T cell and NK cell activity generated during a primary infection in vivo was also sensitive to treatment in vitro with anti-ASGM1 antiserum (1/300 to 1/600 dilution) plus rabbit complement at a dilution of 1/15 (20 to 50% cell death, more than 30-fold decrease of cytotoxic activity); in vitro treatment with rabbit complement alone often enhanced NK and cytotoxic T cell activity slightly. In vivo treatment with anti-ASGM1 before primary immunization decreased generation of primary CTL only if high doses of anti-ASGM1 antiserum were injected twice. Antiviral T cells generated during secondary stimulation in vitro and alloreactive cytotoxic T cells from a mixed lymphocyte culture were resistant to treatment in vitro with anti-ASGM1 plus complement at the end of the culture period. Treatment in vitro of in vivo-primed responder spleen cells with anti-ASGM1 plus complement before their addition to a secondary restimulation culture resulted in complete inhibition of a secondary antiviral cytotoxic T cell response. In vivo treatment with anti-ASGM1 24 hr before their spleen cells were harvested and restimulated in vitro significantly reduced the virus-specific T cell activity of mice that had been immunized with virus several weeks previously. A cloned T cell line exclusively exerting NK-like activity was resistant, and two cloned virus-specific cytotoxic T cell lines were susceptible to treatment with anti-ASGM1 plus complement in vitro. These results caution the general use of rabbit anti-ASGM1 as a marker to distinguish NK from CTL cells; they indicate a possible relationship between NK and CTL cells and suggest that in vitro culture of lymphocytes may alter or select the cell surface expression or availability of the ASGM1 marker(s).     

Lymphokine-activated killer (LAK) cells. II. Delineation of distinct murine LAK-precursor subpopulations

Lymphokine-activated killer (LAK) cells can lyse a number of tumor target cells regardless of whether the tumors are natural killer (NK) sensitive or resistant. LAK can also lyse autologous lymphoblasts that have been modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS). In this study, we examined the surface markers of murine LAK precursors. It was found that depletion of Thy 1- or Lyt 2-bearing precursor cells abolished the ability of spleen cells to generate LAK against TNBS-self, but had no effect on the generation of LAK against tumor cells. Depletion of asialo-GM1 (AGM1)-bearing precursors abolished the generation of LAK against all target cells tested. Normal spleen cells were fractionated on a Percoll density gradient and two fractions were examined: fraction (Fxn) 3, which is enriched for NK activity but depleted of the ability to generate cytotoxic T lymphocytes (CTL), and Fxn 5, which had no NK activity but was enriched for the ability to generate CTL. Both fractions were capable of generating LAK, although Fxn 5 required a relatively larger amount of interleukin 2 (IL 2). Upon examination of the surface markers of LAK precursors in these fractions it was found that the precursors in Fxn 3 giving rise to LAK against tumors were Thy-1-, Lyt-2-, AGM1+, whereas the precursors in Fxn 5 were Thy-1+, Lyt-2+, AGM1+. The precursors generating LAK against TNBS-self were Thy-1+, Lyt-2+, AGM1+ in both fractions. The time kinetics of LAK generation in both fractions were different, with Fxn 3 showing much earlier kinetics. These data delineate at least two different LAK precursors defined by their buoyant density, by their surface markers, and by their susceptible target cells. These data also may resolve the confusion in the literature regarding the phenotype of LAK precursors.     

Murine anti-CD3 monoclonal antibody induces potent cytolytic activity in both T and NK cell populations

Antibodies specific for the CD3 complex have the capacity to both stimulate and inhibit a variety of T cell functions. We show here that a monoclonal antibody to the epsilon chain of CD3 can induce efficient non-MHC-restricted cytolytic activity in murine lymphocytes with peak activity occurring after 48 hr of incubation. In a panel of targets, the anti-CD3-activated effectors lysed tumor cells but not normal lymphoblasts. Cytolysis was not dependent on the presence of the antibody in the cytolytic assay. Moderate to high cytolytic activity was elicited from lymph nodes, spleen, and thymus by anti-CD3 treatment in vitro, whereas only low activity was apparent in bone marrow. The precursors of anti-CD3-activated cells consisted largely of mature T cells, although a smaller component of immature T cells was also involved. Thus, separation of thymocytes based on adhesion to peanut agglutinin revealed that both positive (immature) and negative (mature) fractions could be activated, while cytotoxic pretreatment of spleen cells with an antibody (J11d) to immature T cells before anti-CD3 activation significantly decreased the resulting cytotoxicity. The majority of precursors in spleen were Thy 1+ and CD8+ and/or AGM1+. Antibody depletion studies showed that the effector cells have both a T and a NK component consisting of Thy 1+, CD5+, CD8+, CD4-, and AGM1- cells and Thy 1-, CD5-, CD8-, CD4-, and AGM1+ cells, respectively. The production of significant amounts of IL-2 and TNF in culture following anti-CD3 treatment, along with the synergistic effect of exogenously added IL-2, suggests that one or both of the effector cell types could be induced by lymphokines. The intraperitoneal administration of the anti-CD3 antibody induces cytolytic activity in vivo. Therefore, the direct activation of cytolysis by anti-CD3 antibody and the additional effects, both direct and synergistic, of lymphokines produced by the activated lymphocytes could conceivably provide a potent anti-tumor therapy.     

Phenotypic and functional analysis of the cellular response in regional lymphoid tissue during an acute virus infection

A phenotypic and functional analysis has been made of the cellular response in regional lymphoid tissue of C57BL/6J mice infected with lymphocytic choriomeningitis virus. Massive recruitment of nondividing cells occurred from 3 days after infection, with total numbers of CD8+ T lymphocytes, B220+ B cells, and Thy-1- B220- null cells being high from day 4 to day 6. In contrast, the peak counts for CD4+ T cells were recorded on day 4 and declined dramatically thereafter. Enhanced expression of IL-2R and Ly-24, both of which can be regarded as T cell activation markers, was found for both the CD4+ and the CD8+ subsets, being most prominent for the CD8+ T cells on day 6. Evidence of T cell proliferation was not recognized until days 5 and 6, coincident with enhanced responsiveness of the lymphocytes to rIL-2 and the development of virus-specific cytotoxic activity. Elimination of the CD4+ T cells by treatment of mice with mAb did not modify either the pathogenesis of lymphocytic choriomeningitis, or the expression of activation markers on the CD8+ T cells which are known to be the key effectors in this disease. Thus, the pattern of responsiveness for the CD8+ population is of recruitment to the lymph node, progressive increase in the expression of activation markers and enhanced sensitivity to rIL-2, with late proliferation and generation of cytotoxic activity. This model provides a system for the rigorous in vivo analysis of parameters influencing lymphocyte differentiation and activation in a virus infection.