In vivo treatment of mice and hamsters with antibodies to asialo GM1 increases morbidity and mortality to pulmonary influenza infection

The role of natural killer (NK) cells in host defenses against influenza virus infections in the lung was investigated by using rabbit antiserum to asialo GM1 (RAGM1), a neutral glycosphingolipid expressed on the plasma membrane of NK cells and some mouse pulmonary macrophages. Intravenous or intratracheal (i.t.) administration of RAGM1 resulted in depletion of the (in vitro) NK activity in lung and spleen or lung alone, respectively. The NK activity was depleted as early as 12 hr post-inoculation of antiserum, but returned to the normal range of activity by 4 days after antibody administration. RAGM1 serum treatment had no effect on the cytotoxic macrophage activity expressed by the plastic-adherent mononuclear cell populations isolated from mouse or hamster lung. Treatment of mice or hamsters with an i.t. or i.v. inoculation of RAGM1 rendered both species of laboratory animals susceptible to increased morbidity and mortality during a pulmonary influenza infection. These data support the hypothesis that a population of NK cells exist in an extravascular compartment within the lung, and that this local population of NK cells in the lung is crucial to the early natural pulmonary defenses during influenza infection.     

Alloantigen-activated lymphocytes from mice bearing a spontaneous "nonimmunogenic" adenocarcinoma inhibit its growth in vivo by recruiting host immunoreactivity

Nylon wool columns eluting lymphocytes from the spleen of mice bearing a clinically evident spontaneous, nonimmunogenic adenocarcinoma of recent origin (TS/A) do not display cytotoxic response, release of lymphokines, and proliferation in vitro against TS/A cells, nor do they inhibit TS/A tumor growth in a Winn-type neutralization assay in vivo. After 5-day co-culture with allogeneic spleen cells from mice differing at multiple minor histocompatibility antigens only, these lymphocytes are still noncytolytic against TS/A cells, whereas they release interferon-gamma, mediate delayed-type hypersensitivity (DTH) reactions, and inhibit TS/A tumor growth in the Winn assay. In the Winn test, alloactivated lymphocytes from TS/A tumor-bearing mice are more effective than those from normal mice on a per cell basis. The induction of this TS/A tumor inhibition ability depends on the presence in the cultures of Thy-1+ lymphocytes. The presence of Lyt-2+ lymphocytes is also important, whereas that of asialo GM1+ is not. The TS/A inhibition in vivo by alloactivated lymphocytes mostly depends on Thy-1+, Lyt-2- and asialo GM- lymphocytes, even though a few Thy- cells are also very efficient tumor inhibitors. The alloactivated lymphocytes inhibit TS/A tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor rejection. TS/A tumor rejection leaves a specific DTH and an immunologic memory resulting in rejection of a second lethal TS/A challenge in a significant number of mice.     

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.     

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.     

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.     

Description of phenotypically distinct T-lymphocyte subsets which mediate helper/DTH and cytotoxic functions in the Syrian hamster

Recent studies describe aberrations in the functions of T lymphocytes from Syrian hamsters. A current proposal links the apparent functional deficiencies of cytotoxic and suppressor T cells with anomalies found in class I molecules of this species (no polymorphism is detected) and speculates that hamsters possess limited heterogeneity of T cell subpopulations, particularly a class I-restricted subset. The present work tests this hypothesis by examining the extent of T cell heterogeneity defined by differential cell surface antigen expression. A panel of mouse monoclonal antibodies against hamster lymphocyte antigens was generated. MAb #20 and #110 bound to most, if not all, peripheral T cells; a third antibody, #38, divided T cells into two subpopulations which were functionally distinct. Cells within the #38-negative subset produced easily detectable IL 2 and mediated delayed-type hypersensitivity to influenza virus. In contrast, isolated 38+ cells produced little IL 2 and required the addition of exogenous T cell growth factor for proliferation to Con A. Treatment of immune cells with mAb #38 and complement abrogated cytolysis to TNP-haptenated or influenza-infected targets. Thus, Syrian hamsters possess at least two T cell subpopulations of discrete functional ability and unique cell surface antigen expression. Although the data suggest that T cells analogous to those of the class I-restricted, Lyt 2+ subset are present in the hamster, it is predicted that the scope of their composite antigen receptor repertoire may be limited by the monomorphism of class I molecules in this species.     

A murine cytotoxic T lymphocyte clone from the intestinal mucosa that is antigen specific for proliferation and displays broadly reactive inducible cytotoxic activity

Thy-1+, Lyt-1-,2+, asialo GM1+ cytotoxic T lymphocyte (CTL) clones have been isolated from the intestinal mucosa of mice primed with alloantigens. Two different types of cytotoxic clones have been obtained. The first type is functionally similar to most splenic and lymph node-derived CTL clones in that they are strictly antigen specific with respect to proliferation and cytolytic activity. The second type of CTL clone has several unique characteristics. Although these clones are also antigen specific with regard to proliferation, they are not cytolytic under standard growth conditions in medium containing 4% rat concanavalin A-induced spleen cell supernatant. After culture for 4 days in the presence of high concentrations of interleukin 2, cells become activated and exhibit broad lytic potential. Moreover, during the activation process, these CTL begin to express a murine T cell surface antigen, CT-1, which is associated with activated cytotoxic cells. The findings reported in this report should now allow us to precisely define, both phenotypically and functionally, specific lymphocyte populations that make up the gut-associated lymphoid tissues. These data also describe a new type of effector CTL that differs from other cytotoxic cells reported to date, because it is antigen dependent for proliferation, but requires signals mediated by lymphokines for lytic activation.     

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.     

Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Adoptive transfer assays performed in this laboratory have shown that peritoneal exudate cells from 10-day primiparous hamsters are cytotoxic to SV40-transformed sarcoma cells (WF5-1) carrying fetal antigen, whereas peritoneal exudate cells from multiparous hamsters are less cytotoxic. This suggests a suppressor activity might be present during subsequent pregnancies that reduces the responsiveness of lymphocytes from pregnant hamsters to stimulation by fetal antigens on tumor cells. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi- and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.     

Marker and function analysis of natural killer and alloreactive T cells during early stages of dimethylbenzanthracene carcinogenesis. The immunity system during the precancer period

The effect of the chemical carcinogen dimethylbenzanthracene (DMBA) on cellular immunity was studied at a 6-mg dose which induces adenocarcinomas and adenoacanthomas in more than 70% of BalB/c mice within 1 year after administration. DMBA caused a significant reduction of splenic natural killer (NK) activity and responsiveness to alloantigens in mixed lymphocyte reactions (MLR). These activities decreased soon after the carcinogen treatment and remained suppressed during the entire tumor induction period. There was a linear correlation between the reduction in NK activity and a selective decrease in the number of asialo GM1 positive cells in the spleen. However, cell sorting experiments using the flow cytometer have shown that the lytic activity per cell of asialo GM-1 positive cells in untreated mice and in DMBA-treated ones was similar. There was no correlation between the suppressed response of the T cells in MLR and the percentage of T cell subpopulations residing in the spleen of the DMBA-treated mice. The decrease in the number of NK cells and the reduced MLR activity in the spleen occurred simultaneously with a decrease in the potential of bone marrow precursor cells to reconstitute NK and MLR activity in the spleen of lethally irradiated mice. These results indicate that the carcinogen DMBA effects the immune system at various levels and either eliminates or inactivates precursor cells as well as mature lymphoid 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.     

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.     

The generation of effector T cells in influenza A-infected, cyclosporine A-treated mice

Specific effector T cells that mediate DTH to influenza virus were found to be formed in vivo in CsA-treated mice. The activity of these cells could only be measured when they were transferred into untreated naive mice. The cells mediating DTH were H-2 restricted in the I region of the MHC. When effector T cells that mediated DTH were transferred into CsA-treated recipients, no DTH activity could be detected. Influenza-specific cytotoxic T cells could not be detected in the spleens of CsA-treated mice given virus intravenously, even when drug treatment was started 3 days after virus administration. There was only a partial restoration of cytotoxic activity when spleen cells from CsA-treated infected mice were cultured in the presence of virus-infected stimulators. This seemed to indicate that Class I-restricted responses were more susceptible to CsA than the generation of Class II (or I-region-restricted) responses.     

Lymphokine-activated killer cells are generated before classical cytotoxic T lymphocytes after bone marrow transplantation in mice

Lymphokine-activated killer (LAK) cells are demonstrable within 2 wk after syngeneic or allogeneic (H-2-compatible) bone marrow transplantation in mice. Classical cytotoxic T lymphocytes (CTL) are not active until at least 4 wk after transplant. Both LAK cells and CTL bear the Thy-1 marker and do not possess the murine natural killer cell marker asialo GM.     

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.     

Immunity to herpes simplex virus type 2. Suppression of virus-induced immune responses in ultraviolet B-irradiated mice

Ultraviolet B irradiation (280 to 320 nm) of mice at the site of intradermal infection with herpes simplex virus type 2 increased the severity of the herpes simplex virus type 2 disease and decreased delayed-type hypersensitivity (DTH) responses to viral antigen. Decrease in DTH resulted from the induction of suppressor T cells, as evidenced by the ability of spleen cells from UV-irradiated mice to inhibit DTH and proliferative responses after adoptive transfer. Lymph node cells from UV-irradiated animals did not transfer suppression. DTH was suppressed at the induction but not the expression phase. Suppressor T cells were Lyt-1+, L3T4+, and their activity was antigen-specific. However, after in vitro culture of spleen cells from UV-irradiated mice with herpes simplex virus type 2 antigen, suppressor activity was mediated by Lyt-2+ cells. Culture supernatants contained soluble nonantigen-specific suppressive factors.     

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.     

Modeling of influenza-specific CD8+ T cells during the primary response indicates that the spleen is a major source of effectors

The biological parameters that determine the distribution of virus-specific CD8(+) T cells during influenza infection are not all directly measurable by experimental techniques but can be inferred through mathematical modeling. Mechanistic and semimechanistic ordinary differential equations were developed to describe the expansion, trafficking, and disappearance of activated virus-specific CD8(+) T cells in lymph nodes, spleens, and lungs of mice during primary influenza A infection. An intensive sampling of virus-specific CD8(+) T cells from these three compartments was used to inform the models. Rigorous statistical fitting of the models to the experimental data allowed estimation of important biological parameters. Although the draining lymph node is the first tissue in which Ag-specific CD8(+) T cells are detected, it was found that the spleen contributes the greatest number of effector CD8(+) T cells to the lung, with rates of expansion and migration that exceeded those of the draining lymph node. In addition, models that were based on the number and kinetics of professional APCs fit the data better than those based on viral load, suggesting that the immune response is limited by Ag presentation rather than the amount of virus. Modeling also suggests that loss of effector T cells from the lung is significant and time dependent, increasing toward the end of the acute response. Together, these efforts provide a better understanding of the primary CD8(+) T cell response to influenza infection, changing the view that the spleen plays a minor role in the primary immune response.     

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.