The effects of neuraminidase and galactose oxidase on murine lymphocytes. I. Evidence for the differential delivery of signal(s) leading to cell proliferation and the differentiation of cytotoxic T cells.

The sequential treatment of normal C57BL/6 mouse spleen cell populations with neuraminidase (NA) and galactose oxidase (GO) resulted in cell proliferation, but not in the differentiation of cytotoxic T cells. In contrast, C57BL/6 spleen cells derived from animals primed 5 to 8 months earlier with alloantigen (P815 mastocytoma cells of the DBA/2 strain) both proliferated and demonstrated T cell-mediated cytotoxicity after NAGO stimulation. T cells differentiating into cytotoxic cells after NAGO treatment demonstrated properties similar to alloantigen-specific 'memory' T cells. These were: 1) cytotoxicity developed only from 'primed' cell populations, 2) cytotoxicity developed within 24 hr after NAGO treatment, 3) DNA synthesis was not required for the differentiation of cytotoxic cells during the first 24 hr of culture but both DNA synthesis and cell proliferation were required for the cytotoxicity developing after 24 hr, and 4) all cytotoxicity induced by NAGO showed specificity for the priming alloantigen. It was found, furthermore, that cytotoxicity could be induced at much lower GO concentrations than needed for increased DNA synthesis. We interpret this finding as an indication that NAGO can differentially deliver two 'signals' to T lymphocytes: one leading to cell proliferation, the other causing the differentiation of memory T cells into cytotoxic effectors.     

Induction of lymphocyte cytotoxicity by modification of the effector or target cells with periodate or with neuraminidase and galactose oxidase.

Treatment of mouse spleen cells with periodate (NaIO4) or with neuraminidase and galactose oxidase (NAGO) induces blastogenesis and renders the cells cytotoxic to mastocytoma (P815) target cells. Treatment of target cells (P815 cells and turkey erythrocytes) with NaIO4 or with NAGO renders them susceptible to cytolysis by untreated mouse spleen cells. The cytotoxicity induced by NaIO4 is reduced upon reacting the NaIO4-treated, effector or target cells with borohydride or hydroxylamine. Thus the formation of free surface aldehydes on either the effector or target cell induced a cytotoxic effect. It is postulated that cross-linkage via a Schiff base between effector and target cell initiates the cytotoxic effect. Cytotoxicity induced by NaIO4 or NAGO is immunologically nonspecific and is independent of major antigenic differences between effector and target cells. Phagocytic cells are not involved in NaIO4-or NAGO-induced cytotoxicity toward P815 target cells.     

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

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

Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of concanavalin A treatment on the allogeneic response of mice to challenge with P 815 mastocytoma: interleukin 2 treatment reverses concanavalin A suppression in vivo

Mice injected repeatedly with concanavalin A (Con A) prior to and following challenge with P 815 mastocytoma are suppressed in their cell-mediated cytotoxicity responses. Earlier studies showed that pretreatment of the animals with silica to affect macrophage (M phi) functions reversed the Con A suppression. In the present paper we have shown that peritoneal exudate cells (PEC) induced/activated by ip injection of Con A were able to transfer suppression to normal mice. Separation of the PEC populations into adherent and nonadherent cells abrogated their capacity to transfer suppression. It was further shown that Con A is not functioning in this in vivo system to block effector activity of cytotoxic cells on target cells, and PEC induced with Con A were not directly cytotoxic to target P 815 cells. Finally, we were able to show that the cytotoxicity response of Con A-suppressed mice could be enhanced by treatment with concentrated culture supernatants of normal mouse spleen cells, rich in interleukin 2 (IL 2) activity. Attempts to detect a recently described mouse serum inhibitor of IL 2 in normal or Con A-treated mice were unsuccessful and spleen cells from Con A-treated mice lost their capacity to generate IL 2 in vitro when cultured under appropriate conditions. Taken together, these results suggest that suppression of cell-mediated immune responses in Con A-treated mice results from interruption of the normal generation of IL 2 helper effects necessary for the activation of cytotoxic effector T cells in vivo.     

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

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

Continuously proliferating T killer cells specific for H-2b targets: selection and characterization.

BALB/c (H-2d) thymus-derived lymphocytes sensitized to C57BL/6 (H-2b) alloantigens have been propagated in vitro for over 9 months. These T lymphocytes are specifically cytotoxic to H-2b target cells but are stimulated to proliferate by both H-2b and H-2k spleen cells. This indicates that for these selected cells the antigen requirements for cell proliferation are different from those for cell-mediated cytotoxicity. If not continuously stimulated with allogeneic spleen cells, the cytotoxic cultures fail to divide and rapidly lose their cytotoxic activity. Allogeneic erythrocytes do not stimulate cell proliferation in "quiescent" cell cultures and allogeneic tumor cells do so only in the presence of spleen cells. However, "quiescent" cell cultures display cytotoxicity in the presence of phytohemagglutinin A as do cell cultures which have lost their cytotoxic activity although they proliferate upon allogeneic stimulation. The significance of these findings is discussed.     

Cytotoxic T-lymphocyte reactivity with individual Sendai virus glycoproteins.

Liposomes were constituted with affinity-purified Sendai virus glycoproteins HN and F and phosphatidylcholine (PC) or phosphatidylethanolamine: phosphatidylserine (PEPS). The glycoprotein-bearing recombinant vesicles (RV) were used to modify the surface of P815 mastocytoma cells (H-2d) or EL4 lymphoma cells (H-2b). The cells treated with HN-F-PCRV, HN-PEPSRV, or F-PEPSRV were shown by surface immunofluorescence to retain antigen for at least 2 h at 37 degrees C after treatment. The modified cells were used in cytotoxicity assays with effector spleen cells from either DBA/2 (H-2d) or C57BL/6 (H-2b) immunized by inoculation of active Sendai virus. Cells modified by treatment with HN/F-PCRV showed susceptibility to cytolysis similar to that in actively infected cells. Cells modified with HN-PEPSRV or with F-PEPSRV were also susceptible. The sum of reactivities of the anti-HN component and the anti-F components was close to that seen with HN- and F-bearing targets. Syngeneic but not allogeneic target cells expressing Sendai virus glycoproteins were bound and lysed by the effector cells, which was expected if the interactions were major histocompatibility complex restricted. The activity was attributed to cytotoxic T lymphocytes, since it was depleted by treatment with anti-Thy 1.2 antibody and complement.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Lectin-dependent cell-mediated cytotoxicity: induction of a unique effector cell population.

Spleen cells from C57BL/6 (H-2^b) mice were assayed for their ability to mediate lectin-dependent (Con A, PHA) cell-mediated cytotoxicity (LDCC), following immunization with erythrocytes, bovine serum albumin, Bacillus Calmette Guerin, and allogeneic (H-2^d) P815 cells. Sensitization with viable, but not formaldehyde-fixed, P815 cells resulted in lectin-dependent lysis of syngeneic EL-4 cells. All other sensitization procedures failed to produce LDCC. Spleen cells from mice challenged with high (10⁸) doses of P815 cells were capable of mediating both direct (anti-P815) cytotoxicity and LDCC, while challenge with low (10⁴) doses of P815 cells produced strong LDCC reactivity in the apparent absence of direct cytotoxicity (DCMC). Characterization of the effector cells indicated that LDCC reactivity was mediated by an activated, non-adherent T cell population. The effector cells appear to be unique in that LDCC could be induced in the absence of DCMC, LDCC activity appeared prior to DCMC, and DCMC could be removed by adsorption on P815 monolayers without depleting LDCC reactivity.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.     

Lack of reconstitution of nude mice alloreactivity by purified interleukin 2 and induction of non-H-2-specific effector cells by crude supernatants

To verify or to challenge the reports indicating that IL-2 was the only molecule involved in the reconstitution of nu/nu mice alloreactivity in vitro, Balb/c (H-2d) nu/nu spleen cells were primed in culture against C57/B16 (H-2b) in the presence of crude IL-2-containing supernatants or purified IL-2. The generation of cytotoxic effectors was evaluated against a panel of 51Cr-labeled target cells. Although crude IL-2-containing supernatants sustained the generation of cytotoxic effectors, purified "natural" IL-2 (from different origins) and recombinant IL-2 were not able to do so. Con A or PHA were identified as cofactors synergizing with IL-2 to induce effectors from nu/nu spleen cells. These effectors efficiently lysed EL4 (H-2b, tumor line), but not mitogen-induced blast cells from the same strain. They also lysed targets bearing irrelevant allogenic H-2 specificities. Cold competition experiments confirmed the lack of H-2 specificity of such effectors: lysis of EL4 cells (H-2b) was inhibited strongly by YAC-1 cells (H-2a, very sensitive to NK lysis) or P815 cells (H-2d, autologous to the nu/nu effectors). Our results clearly challenge earlier conclusions and indicate that IL-2 alone does not reconstitute nude mice alloreactivity. Crude supernatants containing IL-2 and mitogen induce nonspecific effectors with patterns of reactivity similar to those of activated natural killers. We think that the cytotoxicity observed in these conditions in nude mice results from the mitogenic triggering of some kind of prethymic killer cells which subsequently are expanded by IL-2.     

A reappraisal of the effector cells mediating mitogen induced cellular cytotoxicity.

The ability of mitogens to induce cytotoxic effector reactions in vitro has been studied to investigate basic mechanisms of cell mediated cytotoxicity. The type of mitogen, the source of effector cells, and the nature of the target cell are all critical variables in determining the characteristics of the cytotoxic event in this system. Spleen cells and bone marrow cells from congenitally athymic nude mice as well as from their heterozygous control littermates were capable of mediating lysis of RBC targets in the presence of either PHA or Con A. Removal of macrophages from these effector populations by adherence columns, density gradient centrifugation, and carrageenan treatment failed to abrogate this cytotoxic capacity. However, purified macrophages themselves also were capable of mediating mitogen induced killing of RBC targets, although the kinetics of this cytotoxicity were substantially different from that induced by lymphocytes. In contrast to these observations, the capacity of mitogen stimulated cells to kill metabolically active complex targets like the P815 mastocytoma or cultured L cells appears to be exclusively a T lymphocyte dependent function. In addition, blastogenic transformation of the effector cells with the T cell mitogens PHA and Con A, but not with the B cell mitogen LPS, leads to enhanced killing of these complex targets. These data suggest that mitogen or lectin induced cellular cytotoxicity can detect at least three different active effector cell types (B cells, T cells, and macrophages) acting via at least four different mechanisms.     

The role of HLA-DR determinants in monocyte-macrophage presentation of herpes simplex virus antigen to human T cells

Spleen cell killing of target cells can manifest through spleen cell-target cell interaction in the presence of mitogenic lectin, lectin-dependent cell-mediated cytotoxicity (LDCC). Spleen cells from C57B1/6 mice immunized with C3H mouse cells were found to be capable of cytotoxicity against autologous and other C57B1/6 spleen cells in the presence of Con A. Thus, alloimmune spleen cells are capable of an anti-self cytotoxic response in the presence of mitogenic lectin, antiautologous LDCC. Antiautologous LDCC is blocked by preincubation of cytotoxic cells with colchicine, an inhibitor of the cytotoxic effector mechanism. Analysis of alloimmune spleen cell subpopulations suggests that the antiautologous LDCC cell is an immature alloimmune cytotoxic cell (prekiller cell). Potent LDCC was found in alloimmune spleen cell preparations depleted of alloimmune cytotoxic T cells (killer-depleted) by three passes on allogeneic cell monolayers genetically identical with the immunizing cell. However, some LDCC effectors were also found to adhere to the adsorbing target, suggesting that there is some maturational diversity among LDCC effectors.     

Studies on T cell clonal expansion. II. The in vitro differentiation of pre-killer and memory T cells.

Spleen cells from C57BL/6 mice immunized with a DBA/2 mastocytoma (P815) were harvested at various stages of the immune response and cultured in vitro in the presence and absence of antigen. Killer T cell activity in immune spleens could not be demonstrated until 6 or 7 days after antigen, but spleen cells harvested as early as 3 or 4 days and cultured for 24 hr at 37 degrees C showed significant cytotoxicity. This increased activity was not augmented further by culturing with antigen. "Memory" T cells, whose in vitro differentiation into killer cells required the presence of antigen, could not be demonstrated until 9 or 10 days after alloantigenic stimulation. Once produced, however, these cells persisted for at least 6 months. Memory cells, like killer T cells bound avidly to homologous allogeneic monolayers. There were indications that the memory T cell pool was heterogeneous. On one hand, when cells harvested 10 days after stimulation were exposed to antigen in vitro, their lytic activity increased within 24 hr but showed no further increases when the culture period was extended. In contrast, 45-day-old immune cells showed increasing lytic activity throughout a 4-day exposure to antigen. Augmentation of lytic activity in both cell populations was independent of DNA synthesis through the first 24 hr of culture. Subsequent increases in the activity of 45-day cells was dependent upon cell proliferation. Both the antigen-independent augmentation of lytic activity which followed culturing of immune cells, and the antigen-induced differentiation of memory cells were reversibly inhibited by a series of drugs which raised lymphocyte cAMP levels.     

Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice

Proliferation of islet-associated leukocytes occurred when isolated islets from 20-wk-old female nonobese diabetic (NOD) mice were cultured with 10 U/ml rIL-2 for 7 days. Co-culture of these leukocytes with freshly isolated islets from 6- to 8-wk-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that these leukocytes were composed mainly of CTL specific for islet cells. First, morphologically, these proliferating cells adhered to NOD islets at 6 h and killed islets within 48 h of culture, but these phenomena could not be observed in the other tissues from NOD mice. These islet-derived cells were cytotoxic to NOD islet cells in a 51Cr-release assay, whereas no appreciable cytotoxicity was observed when NOD Con A-induced splenic blasts or fibroblasts were used as targets. Second, a flow cytometric analysis showed that these cells consisted of 97% Thy-1.2, 69% Lyt-2, 8% L3T4, and 4% asialo-GM1-positive cells, whereas Mac-1-positive cells could not be seen in these assays. After treatment with anti-Thy-1.2 or Lyt-2 mAb and C, these cells lost their activity to lyse NOD islet cells. However, these cells still had a full killing activity after the depletion of L3T4 or asialo GM1-positive cells. Third, islet cells from BALB/c, DBA/2, and B10.GD mice which share the same H-2K Ag with NOD mice were susceptible to cytolytic activity of these cells, whereas islet cells from NON, C57BL/6, C57BL/10, and C3H mice remained intact. Furthermore, anti-Kd antibody was capable of blocking this cytolysis. These results suggest that CTL expressing Thy-1.2 and Lyt-2 phenotypes appear to recognize the islet cell Ag with the restriction of MHC class I Kd, and then destroy NOD islet cells.     

The role of Ia antigens in the activation of T cells by concanavalin A: an evidence for the species restriction between T cells and accessory cells

Comparisons were made between the characteristics of cytotoxicity activation in mouse spleen cells, induced by specific anti-H-2 antiserum, interferon (IF), interferon inducer (poly (I:C)), and mitogens (concanavalin A (Con A), pokeweed mitogen (PWM)). Important differences were found in the cytotoxicity activation associated with these agents: (a) The cytotoxic enhancing effect of anti-H-2 antiserum was comparatively rapid and was more pronounced in the first 4 hr of the assay, whereas, IF, poly (I:C), and the mitogens were most effective at 20 hr. (b) Anti-IF antiserum could neutrilize the stimulatory effects of IF and poly (I:C) on the cytotoxic activity of mouse spleen cells but had no influence on the stimulatory effects of anti-H-2 antiserum, PWM, and succinyl Con A. (c) The stimulatory effect of anti-H-2 antiserum was target specific and was observed when the K562 cell line was used as the target but not when YAC, P815, or WFu/G1 target cell lines were used. Stimulatory effects of all the other agents were nonspecific and did not depend on the target cells employed. (c) Cellular fractionation studies suggested that the alloantisera and interferon directly activated the natural killer (NK) cells without any participation of T-cells, B-cells, or macrophages. Both T-cells and NK cells, however, contributed to the mitogen-enhanced cytotoxicity of mouse spleen cells. These results indicated that the mechanism by which anti-H-2 antisera induce NK augmentation in mouse spleen cells is distinct from that of interferon and mitogens.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.