Flow cytometric analysis reveals the presence of asialo GM1 on the surface membrane of alloimmune cytotoxic T lymphocytes

Previous studies have demonstrated that natural killer (NK) cells express the glycolipid asialo GM1, as evidenced by the sensitivity of NK cells to treatment with anti-asialo GM1 serum and complement. Because alloimmune cytotoxic T lymphocytes (CTL) were found to be insensitive to treatment with anti-asialo GM1 serum and complement, it was concluded that asialo GM1 is expressed by NK but not by CTL. However, fluorescence studies indicated that a significant proportion of peripheral T cells did express asialo GM1. Flow cytometric studies were undertaken to determine the extent to which alloimmune CTL express asialo GM1. Affinity-purified, monospecific IgG anti-asialo GM1 antibodies were used to label cells from mixed lymphocyte cultures. Separation of asialo GM1-positive and -negative fractions by cell sorting revealed that the majority of CTL activity resides in the asialo GM1-positive population. When these studies are compared with similar studies of splenic NK activity, it is apparent that, despite the relative insensitivity of CTL to treatment with anti-asialo GM1 and complement, both CTL and NK activity are enriched in the asialo GM1-positive cell population obtained by cell sorting.     

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.     

Characterization of natural killer cells and their precursors in the murine bone marrow

We have fractionated murine bone marrow cells according to their density on bovine serum albumin (BSA) gradient and studied (a) the NK activity against YAC-1 targets, (b) the proportion of asialo GM1+ lymphocytes, (c) and the presence of large granular lymphocytes (LGL) in the different fractions (A, B, C, D). The NK activity was found mainly in the C fraction, but the proportion of asialo GM1+ cells was the same in every fraction. No LGLs were found in the bone marrow. Cells from the various fractions were also transplanted into irradiated recipients. Seven days later the highest NK activity was found in the spleens of mice injected with cells from the A + B fractions indicating that the immediate precursors for NK cells reside in the low density fractions of the BSA gradient. Mice transplanted with C or D fractions needed longer time to develop normal NK levels. The treatment of bone marrow cells before transplantation with anti-asialo GM1+ complement did not inhibit the development of NK activity, so it can be concluded that the precursor for NK is asialo GM1-.     

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.     

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.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Discrimination between macrophage-and NK-type tumoricidal activities via anti-asialo GM1 antibody

The usefulness of asialo GM1, a glycolipid surface marker, to define the effector cell types involved in tumor resistance in vitro and in vivo was assessed. Pretreatment of rat effector cells with anti-asialo GM1 antibody plus complement in vitro either abrogated or markedly diminished NK activity; in contrast, macrophage-type cytocidal activity was not diminished by such pretreatment. Similarly, systemic inoculation of anti-asialo GM1 antibody selectively eliminated NK activity, leaving macrophage-type tumoricidal reactivity intact. Finally, such pretreatment did not diminish host resistance in an in vivo tumor model in which the available evidence suggests a critical role for macrophages. The asialo GM1 marker may thus be useful in delimitating the tumoricidal capacity of cells exhibiting NK activity from that mediated by other cell types.     

Synergistic defense system by cooperative natural effectors against metastasis of B16 melanoma cells in H-2-associated control: different behavior of H-2+ and H-2- cells in metastatic processes

H-2+ and H-2- cells of B16 melanoma were established by repeated fluorescence-activated cell sorting. The H-2- line formed no metastasis in untreated C57BL/6 mice, whereas the H-2+ cells showed evidence of metastatic development. This difference was ascribed mainly to the increased susceptibility of H-2- cells to attack by natural effector mechanisms, particularly asialo GM1+ NK cells. After treatment with both anti-asialo GM1 serum and whole body irradiation (400 rad), numerous colonies of H-2- cells formed in the lung, whereas the metastasis was only marginally enhanced by irradiation and moderately by treatment with anti-asialo GM1 serum. With the H-2+ cells, treatment with each modality significantly increased the number of metastatic colonies. Therefore collaboration of asialo GM1+ NK cells and radiosensitive natural effectors seems to be the main mechanism involved in the synergistic effects on defense against H-2- cell metastasis, and to a lesser extent against H-2+ cell metastasis. Irradiation (1000 rad) to the right lung to abrogate the organ-associated defense increased the colonies, particularly in the H-2+ cells. On the other hand, treatment with anti-asialo GM1 serum increased colonization in the early phase of metastasis with H-2- cells and may have abolished asialo GM1+ NK cells capable of recognizing the reduced expression of H-2 antigens and eliminating H-2- cells in the blood-born phase. Natural defense mechanisms probably exert suppressive effects on the metastasis of H-2+ cells, mainly in the organ-associated phase after extravasation.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

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.     

Role of lymphokine-activated killer cells as mediators of veto and natural suppression

Fresh bone marrow cells have veto activity but little if any NK activity. By contrast, lymphokine-activated bone marrow cells have potent natural suppressor as well as veto activity, and also have cytolytic activity characteristic of lymphokine-activated killer cells. Veto activity of fresh bone marrow cells is eliminated by 9 Gy irradiation and by depletion of cells expressing Qa-2, but is unaffected by removal of cells expressing Thy-1, Qa-5, Ly-5, or asialo GM1. By contrast, the veto and NS activities of lymphokine-activated bone marrow cells are both abrogated by C lysis depletion of cells expressing Qa-2, Qa-5, Thy-1, asialo GM1, NK1, and Ly-11, but are unaffected by depletion of cells expressing Ly-2. Bone marrow cells depleted of Qa2+ cells fail to generate veto or natural suppressor activity when cultured in Con A-conditioned medium, unlike bone marrow cells depleted of mature NK1.1+ NK cells. Cloned NK cell line F8 is able to mediate both natural suppression and veto. These findings indicate that bone marrow veto and natural suppression are not mediated by T or NK cells present de novo in the bone marrow, but are dependent on proliferating cells that phenotypically resemble pre-NK cells. The progeny of these cells have the phenotype and functional activity of lymphokine-activated killer cells, and are capable of directly mediating both veto and natural suppression.     

Preparation of monoclonal antibodies against a glycolipid asialo GM1

Five IgM monoclonal antibodies (MAbs), MW-1, MW-2, MW-3, MW-4, and MW-5, against a glycolipid asialo GM1 were prepared from hybridoma clones obtained by the fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with asialo GM1 adsorbed to naked Salmonella. All the MAbs reacted only with asialo GM1 when their reactivities were examined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC)-immunostaining using structurally related glycolipids. The MAbs showed a complement-dependent lysis of mouse natural killer (NK) cells, but the lytic activities were weaker than that of a rabbit polyclonal anti-asialo GM1 antibody. When they were mixed, the anti-NK activity was increased to a level almost comparable to that of the polyclonal antibody. These results suggest that all the MAbs obtained are specific for asialo GM1 and that they may be different in fine specificity for the glycolipid. Significance of the MAbs in immunological and neurochemical studies is discussed.     

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.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Anti-glycoprotein D monoclonal antibody protects against herpes simplex virus type 1-induced diseases in mice functionally depleted of selected T-cell subsets or asialo GM1+ cells.

Passive transfer of a monoclonal antibody (MAb) specific for glycoprotein D (gD) is highly effective in preventing the development of herpes simplex virus type 1-induced stromal keratitis. In the present study, we investigated whether animals which had been functionally depleted of T-cell subsets or asialo GM1+ cells would continue to be responsive to MAb therapy. BALB/c mice were depleted of CD4+, CD8+, or asialo GM1+ cells by treatment with anti-L3T4, anti-Lyt 2.2, or anti-asialo GM1 antibodies, respectively. Functional depletion of CD4+ cells was documented by the loss of delayed-type hypersensitivity responsiveness, while CD8+ cell depletion was accompanied by abrogation of cytotoxic lymphocyte activity. Anti-asialo GM1 treatment led to the loss of natural killer cell lytic activity. Mice depleted of the desired cell population and infected on the scarified cornea with herpes simplex virus type 1 uniformly developed necrotizing stromal keratitis by 3 weeks postinfection. A single inoculation of anti-gD MAb (55 micrograms) given intraperitoneally 24 h postinfection strongly protected hosts depleted of CD4+ cells against stromal keratitis. Likewise, antibody treatment in CD8+ or asialo GM1+ cell-depleted hosts was as therapeutically effective as that seen in non-cell-depleted mice. We also observed that in cell-depleted mice, the virus spread into the central nervous system and caused encephalitis. The CD4+ cell-depleted mice were the most severely affected, as 100% developed fatal disease. Anti-gD MAb treatment successfully protected all (32 of 32) CD4+-, CD8+-, or asialo GM1(+)-depleted hosts against encephalitis. We therefore conclude that antibody-mediated prevention of stromal keratitis and encephalitis does not require the obligatory participation of CD4+, CD8+, or asialo GM1+ cells. However, when mice were simultaneously depleted of both CD4+ and CD8+ T-cell subsets, antibody treatment could not prevent fatal encephalitis. Thus, antibody can compensate for the functional loss of one but not two T-lymphocyte subpopulations.     

Origin and differentiation of natural killer cells. I. Characteristics of a transplantable NK cell precursor

To study the origin and differentiation of natural killer (NK) cells, we developed an assay for the transplantable precursor of NK(YAC-1) cells present in the bone marrow. Mice were depleted of endogenous NK(YAC-1) cells by injection of anti-asialo GM1 antibody, followed by lethal whole body irradiation. Normal syngeneic bone marrow cells were transplanted into such pretreated mice. Regeneration of NK(YAC-1) activity in the recipient mice was monitored by two different assays: the ability of spleen cells to lyse YAC-1 cells in vitro and the ability to clear i.v. injected, 125IUdR-labeled YAC-1 cells from the lungs. With both assays, a dose-response relationship between the number of bone marrow cells injected and the degree of NK(YAC-1) activity generated could be demonstrated. However, the lung clearance assay appeared superior because the NK regeneration could be detected earlier and with lower numbers of injected marrow cells. With this assay, several characteristics of the NK precursors and their differentiation could be defined. 1) The generation of mature, lytic NK cells from their transplantable precursor requires an intact "marrow microenvironment" in the recipient mice, because differentiation failed to occur in mice rendered osteopetrotic by estradiol treatment. 2) The NK(YAC-1) precursors lack the surface antigens (NK-2.1, asialo GM1, Qa-5, Thy-1) that are characteristically seen on mature NK cells. 3) The NK-precursors could be eliminated from the bone marrow with anti-Qa-2 or anti-H-2 antisera + complement, indicating that these two antigens are expressed on the precursors. The relationship between NK(YAC-1) precursors and multipotent myeloid stem cells (CFU-S) was investigated by utilizing W/Wv and Sl/Sld mutant mice. Bone marrow cells of W/Wv anemic mice, although markedly deficient in CFU-S, have a normal frequency of NK(YAC-1) precursors. Sl/Sld mice that lack a suitable microenvironment for the development of CFU-S allowed normal differentiation of NK(YAC-1) precursors when transplanted with normal bone marrow cells. Together, these data suggest that multipotent myeloid progenitor cells, as defined by the CFU-S assay, and the NK(YAC-1) precursors are not closely related.     

An oligosaccharide biosynthetic defect in concanavalin A-resistant Chinese hamster ovary (CHO) cells that enhances NK reactivity in vitro and in vivo

We have examined a concanavalin A-resistant (Con AR) Chinese hamster ovary cell (CR-7) that has a defect in the synthesis of asparagine-linked oligosaccharides and consequently an altered expression of membrane carbohydrate. The CR-7 mutant, which has a decreased ability to incorporate mannose into oligolipid and membrane glycoprotein and an increased membrane fucose, was more sensitive to natural killer (NK) cell lysis than the parental wild type (CHO-WT). Splenocytes mediating the lysis of the CR-7 line were asialo GM1+, nonadherent, IFN stimulatable, absent in the bg/bg mutant, and co-fractionated on Percoll density gradients with cells mediating lysis of the YAC-1 murine lymphoma. The increase in NK lysis correlated with enhanced binding of NK cells to the mutant determined by adsorption on tumor monolayers, cold target inhibition, and target binding analysis. A revertant of CR-7 (RCR-7), which showed wild-type levels of NK lysis, was intermediate in its ability to bind or cold target inhibit NK cells. The CR-7, CHO-WT, and RCR-7 lines were equally sensitive to hypotonic lysis and cytotoxicity by human lymphokine-activated killer (LAK) cells suggesting that the mutation did not nonspecifically alter membrane fragility. The NK-sensitive CR-7 line was less tumorigenic after subcutaneous injection in nude mice when compared with the parental CHO-WT or RCR-7 lines. This decreased tumorigenicity could be reversed by the i.v. injection of antiserum directed at the NK cell determinant asialo GM1. In conclusion, a ConAR tumor cell with a demonstrable oligosaccharide biosynthetic defect, exhibited enhanced NK lytic sensitivity and was poorly tumorigenic in vivo, a feature which may also be a consequence of its altered NK reactivity.     

Induction of interleukin 3 but not interleukin 2 or interferon production in the syngeneic mixed lymphocyte reaction

The syngeneic mixed lymphocyte reaction (SMLR) was assayed in the medium containing syngeneic normal mouse serum (NMS), by using nylon-adherent stimulator cells and nonadherent responder T cells, which were prepared from murine spleens in the absence of fetal calf serum (FCS) to avoid any sensitization to xenogeneic protein antigens. The responder cells in this SMLR, without definite background proliferation, generated specific proliferative response to the syngeneic stimulator cells in a dose-related fashion. The SMLR was accompanied by production of interleukin 3 (IL 3) but not interleukin 2 (IL 2) or interferon (IFN). No cytotoxicity against the syngeneic or allogeneic target cells was induced. Correlating with no production of IL 2 or IFN, no natural killer (NK) activity was detected. The proliferation was not inhibited by addition of specific antiserum for IFN-gamma. In contrast, proliferation in the responder cells when incubated with allogeneic stimulator cells was inhibited by anti-IFN-gamma serum and accompanied by production of IL 2 and IFN as well as IL 3, and by augmentation of NK activity and generation of cytotoxic T cells. Cell surface analysis revealed that the cells producing IL 3 in this SMLR system were Thy-1+ Lyt-1+2- helper T cells. Cells responding to the SMLR culture fluids with DNA replication were Thy-1-Lyt-1-2- asialo GM1- no-marker cells, which were the same as a population responsible for partially purified IL 3. On the other hand, when the responder cells were exposed to FCS before culture and assayed for SMLR in the FCS-free NMS medium, variable levels of IL 2 production were induced in response to the stimulator cells. The responder cells generated a high background DNA replication in the absence of syngeneic stimulators, suggesting that this IL 2 production may result from the stimulation of T cells by FCS as a foreign antigen. Overall, these results suggest that the SMLR may be a cellular interaction, in which non-T cells stimulate Lyt-1+2- helper T cells to produce IL 3 but not IL 2 or IFN. This IL 3 can, in turn, induce proliferation of IL 3 responding cells, which appear to be early precursors in lymphocyte differentiation, but no proliferative response or activation of IL 2- and IFN-dependent mature T cells or NK cells.