The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Lymphocyte-mediated cytolysis of allogeneic tumor cells in vitro. III. Enzyme sensitivity of target cell antigens.

Target tumor cells pretreated with high concentrations of papain or Pronase were resistant to lysis by cytotoxic T lymphocytes (CTL), whereas treatment with trypsin or neuraminidase had no protective effect. Parallel determinations of the H-2 content of target cells following enzyme treatment showed that approximately 80% of surface H-2 was removed by papain or Pronase, 40% by trypsin, and virtually none by neuraminidase treatment. Both susceptibility to lysis by CTL and content of surface H-2 after papain treatment were fully restored by 6 hr at 37 °C in nutrient medium. These findings suggest that lymphocyte-mediated cytolysis (LMC) determinants (target cell antigens bound by CTL) are sensitive to degradation by papain and Pronase but are resistant to the enzymatic action of trypsin and neuraminidase. That a similar pattern of enzyme sensitivity is shown by serologically defined H-2 antigens indicates that both functional classes, LMC and H-2, may have a structural association.     

Effects of heparin and benzyl alcohol on lymphocyte-mediated cytotoxicity in vitro

The effect of heparin on the in vitro lysis of EL4 tumor cells by immune BALB/C lymphocytes was investigated by using a ⁵¹Cr-release cytotoxicity. assay. Powdered heparin did not inhibit lymphocyte-mediated cytotoxicity (LMC) at concentrations up to 500 units/ml. Heparin containing 9 mg/ml of benzyl alcohol (BA), as preservative, significantly reduced the LMC. BA alone at 1 mg/ml inhibited LMC without any apparent toxic effect on the target cells or on the immune lymphocytes. The inhibitory effect of three different heparin preparations was found to be related to the BA concentration rather than the amount of heparin. However, low concentrations of heparin (0.5 or 1 unit/ml) significantly enhanced the LMC. Our findings are in contrast to previous reports suggesting a depressive effect of heparin on LMC.     

Lymphocyte cytotoxicity in x-irradiation-induced rat small bowel adenocarcinoma. III. Blocking by 3 M KCL extract

Hypertonic salt extracts (3 M KCl) of x-irradiation-induced Holtzman rat small bowel adenocarcinomas blocked the in vitro destruction of allogeneic cultured cells of this malignancy by sensitized lymphoid cells obtained from tumor-bearing animals. The protective effect were mediated by a blocking action at both the effector and the target cell level. The extracts were separated into 50% ammonium sulfate soluble and insoluble fractions with the soluble fraction being more effective in blocking the cytotoxic responses through interaction with the lymphoid cells whereas the insoluble one had a greater effect upon tumor target cells. Associated with both fractions was the oncofetal glycoprotein previously identified with the cellular membrane of this x-ray-induced malignancy. Immunoglobulins were identified with insoluble fraction; some were able to bind the oncofetal protein, thus clasifying it as a fetal antigen. The protective effects of the soluble fraction and this neoantigen were found to be citric acid labile, whereas the effects due to the insoluble fraction were unchanged.     

T-rosettes in hemodialysis patients and renal allograft recipients

Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by ³H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release ⁵¹Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.     

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO₄ for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the ⁵¹Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.     

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing ¹²⁵I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the ¹²⁵I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.     

Immune cytolysis of human tumor cells mediated by xenogeneic "immune" RNA

Normal, nonimmune, human peripheral blood lymphocytes, when incubated with RNA extracted from lymphoid organs of guinea pigs or sheep immunized with human tumor cells, mediated the immune cytolysis of those tumor cells in vitro. Lymphocytes incubated without RNA, or with control RNA preparations, failed to evidence cytotoxic activity. Treatment of the active RNA preparations with ribonuclease abrogated the cytotoxic activity, but treatment with deoxyribonuclease or pronase did not effect activity.     

Secretory processes in lymphocyte function

The secretion of immunoglobulin by plasma cells has been considered a classical example of the non-regulated pathway of protein secretion, in which newly synthesized protein is processed by the Golgi, packaged into small vesicles, and immediately secreted without intracellular storage. In the case of lymphokine secretion by T lymphocytes, it is generally not clear whether this non-regulated pathway is also being used, as opposed to the regulated pathway which has been proposed to operate in the cytotoxic lymphocyte mechanism. In this case, as in mast cells and endocrine cells, proteins are synthesized and then stored in cytoplasmic granules. The secretion is triggered (regulated) by a membrane receptor-ligand interaction, which for the cytotoxic lymphocytes is part of the target cell binding process. In cytotoxic T lymphocytes, this secretion process can be measured by following the appearance of a granule serine protease in the medium, and it has been shown to be triggered by target cells or by immobilized antibodies which bind the T celt receptor complex. In addition to cytotoxic lymphocytes, cloned T helper cells contain this serine protease in cytoplasmic granules with a low internal pH. Helper lymphocytes secrete this enzyme in response to (1) soluble antigen which has been processed by cells bearing the appropriate MHC antigens; (2) immobilized antibodies against the T cell receptor complex; (3) a combination of phorbol ester and calcium ionophore. Thus in both helper and cytotoxic lymphocytes, the regulated pathway of protein secretion clearly operates after triggering by the T cell antigen receptor.     

Soluble precursor of membrane-associated protein kinase in uterine smooth muscle cells

Incubation of smooth muscle strips from rat uterus with isoproterenol resulted in redistribution of protein kinase activity between the cytosol and a 20,000 to 50,000g membrane fraction. Similarities in the elution properties of the cytosolic and membrane-associated forms of the enzyme on DEAE-cellulose ion exchange chromatography further suggested the two forms were the same. The nature of membrane binding of the soluble enzyme was investigated using smooth muscle microsomal and cytosol fractions. Membranes readily bound the soluble enzyme when the two subcellular compartments were reconstituted and incubated at 30 °C for 10 min. The extent of binding was proportional to the ratio of membranes to cytosol and was characterized by the inhibition of soluble enzyme activity toward exogenous substrates in a Triton X-100 reversible manner. In marked contrast to the binding of soluble protein kinase to heart particulate fractions, binding of the cytosol enzyme to smooth muscle cell membranes was unaffected by ionic strength or cAMP. The latter property indicated holoenzyme was bound in a manner similar to the free catalytic subunit of cAMP-dependent protein kinase and suggested the enzyme was bound by association between the membrane and the catalytic subunit. Binding of cytosol protein kinase to the membranes rendered the enzyme insensitive to trypsin digestion and the capacity of the smooth muscle cell membranes to bind the soluble enzyme exceeded that of other rat tissue fractions. Resistance to salt extraction and proteolysis, as well as its detergent dependence, suggested the soluble enzyme became an integral or intrinsic membrane protein following association with the membrane. The ability of membranes to incorporate [γ-³²P]ATP into phosphoprotein was lost on detergent extraction of protein kinase and restored in an apparently specific manner when extracted and washed membranes were reconstituted with soluble enzyme. The intrinsic nature of membrane protein kinase and the apparent specificity with which the soluble enzyme was hound by membranes further indicated that, in myometrium. hormone-induced translocation of protein kinase is an important mechanism by which enzyme activity is increased in the vicinity of its in situ substrates.     

Functional characteristics of T cell receptors during sensitization against histocompatibility antigens in vitro

Using the mixed leukocyte culture (MLC) reaction as a model system for the generation of cytotoxic effector lymphocytes, we have examined changes which occur in recognition-binding function during T cell sensitization. Properties of recognition-binding units on normal and immune T lymphocytes were assessed through the ability of T cells to bind to specific cellular immunoadsorbants at various stages of in vitro sensitization. While antigen-specific recognition-binding function was readily detected on fully cytotoxic effector cells, we were unable to detect functionally specific binding of unsensitized lymphocytes to cellular immunoadsorbants. The ability of cells undergoing sensitization in MLC to bind specifically to target cell monolayers appeared congruent in time with cytotoxic function. These results suggest that a fundamental membrane-associated change occurring during T cell sensitization may be the development of a strong and specific target-cell binding function.     

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.     

Characterization of antibody-mediated inhibition of natural killer (NK) cytotoxicity: Evidence for blocking of both recognition and lethal hit stages of cytolysis

Rat antisera prepared against murine, periodate-activated alloimmune cytotoxic lymphocytes (termed RAT¹) have previously been shown to effectively block T-cell-mediated cytotoxicity (CMC) at the “lethal hit” stage of cytolysis (J. C. Hiserodt and B. Bonavida, J. Immunol.126, 256, 1981). Both natural killer (NK) and cytotoxic T lymphocytes (CTL) have been shown to mediate lysis by the same pathway, namely binding of effector to target cells, programming for lysis, and killer cell-independent target cell lysis. This result suggested that the molecular mechanism of NKCMC and CTLCMC may also be similar. In this context, RAT¹-mediated blocking of CTL was examined for its ability to block NKCMC. The results show that (1) addition of RAT¹ serum or IgG fractions blocked NKCMC in the absence of complement in a 4-hr ⁵¹Cr-release assay, and blocking was directed at the effector cell; (2) at the single-cell level, RAT¹ serum blocked the formation of conjugates between effector and target cells; (3) in a Ca²⁺-pulse experiment, in which the effectors and targets were first allowed to bind in the absence of Ca²⁺ for 1 hr at 37 °C, followed by the addition of Ca²⁺ to initiate the lytic event, RAT¹ was capable of blocking cytotoxicity after conjugate formation at the Ca²⁺-dependent lethal hit stage of cytolysis. The similarity of results in RAT¹ blocking experiments of both the CTL and NK systems suggests a common molecular mechanism of cytolysis.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.     

Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2

Large granular lymphocytes (LGL) have been characterized phenotypically and functionally as cytotoxic T lymphocytes, NK cells or lymphokine-activated killer cells. The most prominent morphologic feature of LGL is large cytoplasmic granules that are thought to contain the molecules responsible for cell lysis. In this study, we describe the morphologic and functional characteristics of IL-2-dependent cytotoxic lymphocytes derived from feline PBL. Stimulation of feline PBL with Con A followed by culturing in 50 U of gibbon monkey IL-2 human rIL-2 induced long term lymphocyte cultures. These lymphocytes are cytotoxic for the feline leukemia virus-induced T cell lymphoma (FL74), in a 4-h 51Cr release assay. All cell lines are either constitutively cytotoxic for FL74 cells, or cytotoxic in a lectin-dependent cell cytotoxic assay, the latter being a characteristic of low passage cultures. In contrast, no cell lines express self lysis or lysis for other lines. [3H]TdR uptake showed that 1 U of human rIL-2 produces a 50% maximal proliferative response by feline lymphocytes suggesting a high degree of homology between the ligand binding sites of feline and human IL-2R. Feline cytotoxic lymphocytes possess abundant cytoplasm containing large azurophilic granules characteristic of LGL. These granules are bound by a bilipid membrane and contain numerous smaller membrane-bound vesicles 50 to 60 nm in diameter. A model is proposed, whereby subsequent to binding of LGL to target cell the large granules fuse to the LGL plasma membrane and release the small vesicles into the binding pocket. The vesicles then transport the lytic molecules directly and selectively to the target cell membrane.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Resistance of cytolytic lymphocytes to perforin-mediated killing. Murine cytotoxic T lymphocytes and human natural killer cells do not contain functional soluble homologous restriction factor or other specific soluble protective factors

CTL and NK cells produce a cytolytic pore-forming protein (perforin, cytolysin) localized in their cytoplasmic granules. These cytotoxic cells are resistant to killing mediated by other lymphocytes and by purified perforin. A membrane factor, known as homologous restriction factor (HRF), has been suggested to confer protection to different cell types against both C- and perforin-mediated lysis. The granules of human large granular lymphocytes have been reported to contain, in addition to perforin, a soluble HRF activity that can be eluted from anion-exchange columns at 115 mM NaCl. Here, we report that a soluble HRF activity is absent in the granules or the cytosol of murine CTL and human NK cells. Our data indicate that the inhibition attributed to HRF could be explained by exogenous EDTA added during granule fractionation. EDTA was shown to bind to Mono Q and to elute at 90 to 120 mM NaCl. A second perforin-inhibitory activity was also eluted from such a column. However, it was present in preparations obtained not only from CTL and NK cells, but also from some perforin-susceptible tumor cell lines, indicating that it has nonrestricted distribution and suggesting that it is probably irrelevant to the perforin-protection mechanism. Our results argue against a role for soluble granule HRF or other soluble factors in mediating resistance of cytotoxic lymphocytes against perforin-mediated lysis.     

In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.

Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA did not influence the results of the chromium release assay. Attempts to block the cell-mediated cytotoxicity in vitro by coating tumor cells with an anti-MATSA serum failed. It was concluded that cell-mediated immune responses against Marek's disease tumor cells are directed against an as yet undefined antigen(s).     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.