Characterization of the blastogenic and cytotoxic responses of normal mice to ecotropic C-type viral gp71.

Spleen cells from normal (B6C3)F1 mice demonstrated natural cytotoxic reactivity mediated by a "null" cell population which, in part, had immunologic specificity for the major envelope glycoprotein (gp71) of the endogenous ecotropic murine leukemia virus. In contrast, the cytotoxic reactivity reflected in spleen cells of NIH Swiss nude mice apparently had no immunologic specificity. A significant level of blastogenic response could be generated in vitro by using normal (B6C3)F1 and AKR spleen cells in the presence of gp71. This reactivity was highly type specific. Moreover, using normal spleen cells from (B6C3)F1, both secondary blastogenic responses and cytotoxic T cell responses could be induced in vitro with purified soluble viral gp71. These findings extend further our previous studies on the existence of a natural immune response in normal mice to their endogenous MuLV by providing in vitro evidence for the expression of cell-mediated response in addition to the humoral response and that at least two effector cell types are operable in the cell-mediated phase.     

Studies of the immunological capacity of germ-free mouse radiation chimeras. IV. Cell-mediated immunity

The cell-mediated immune (CMI) response of germ-free mouse radiation chimeras was compared with that of conventional mice. Spleen or thymus cells from chimeric or normal mice were injected intravenously into lethally irradiated, allogeneic hosts. Spleens of the irradiated hosts were assayed for effector cells using the ⁵¹Cr release assay. Spleen cells from syngeneic and allogeneic chimeras and normal mice were equally active in giving rise to effector cells. However, thymus cells from allogeneic chimeras were completely inactive within 9 months post-bone marrow transplant while thymus cells from syngeneic chimeras and normal mice still remained functional. Although allogeneic chimeras contain cells potentially reactive toward host antigens, cells cytotoxic to host antigens were not detectable. In addition, these studies indicate the helper cell and effector cell, both associated with T-derived lymphocytes, represent two different populations.     

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.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

The ability of hapten-conjugated cells to induce cell-mediated cytotoxicity is affected by the mode of hapten linkage

Spleen cells from C57 $\text{B}\text{1}\text{6}$ mice were modified with varying concentrations of several haptens coupled in different ways, then assayed for their ability to induce a cytotoxic response against hapten-modified target cells when cocultured with syngeneic spleen cells. Haptens such as TNBS, DNBS, Fl-NCS, and AB-NCS which couple via the ?-amino groups of lysine were all capable of generating high levels of cytotoxicity when tested against hapten-coupled syngeneic tumor. One hapten, DNBM, which coupled via -COOH groups was effective in generating cell-mediated cytotoxicity. By contrast, similar haptens such as ABA and DNBA which couple as diazonium salts via tyrosine and histidine groups were not found to generate cytotoxic responses over wide ranges of concentrations. Possible explanations are discussed.     

Cell-mediated immunity to Friend virus-induced leukemia. III. Characteristics of secondary cell-mediated cytotoxic response.

By employing the 125IUdR release cytotoxicity assay, we have been able to measure the primary and secondary cell-mediated cytotoxic response of C57BL/6 mice to FBL-3 cells, a syngeneic Friend virus-induced leukemia. It was found that the secondary cell-mediated cytotoxic response occurred more rapidly after challenge (within 3 days) than the primary response, and the levels of reactivity were considerably higher. As in the primary response, the secondary cytotoxic reactivity of spleen cells was T cell dependent, being eliminated by pretreatment with anti-theta antibody plus complement. However, the secondary reactivity of pertioneal exudate (PE) cells was not entirely T-cell dependent. The specificity of the secondary cytotoxic response was analyzed by primary or secondary immunization with various tumor cells and by testing of cytotoxic lymphocytes against a variety of target cells. When spleen cells were used for testing, only tumor cells induced by Friend, Moloney, or Rauscher (FMR) leukemia viruses could produce secondary cell-mediated cytotoxic responses against FBL-3 cells. This correlated well with the specificity observed in the in vivo tumor transplantation protection studies. Similarly, spleen cells immune to FBL-3 had appreciable cytotoxicity against tumor cells induced by FMR viruses. The FBL-3 immune mice also gave significant protection against the challenge of FMR leukemias. When PE cells were used for testing, they gave higher levels of cytotoxicity against tumor cells induced by FMR viruses, but also gave less, but appreciable, cytotoxicity against non-FMR tumors. The latter reactivity might be related to the antigens induced by the murine endogenous type C viruses.     

Fractionation of lymphocytes involved in the generation of cell-mediated cytotoxicity over insolubilized conjugated histamine columns.

Spleen cells from normal BALB/c mice were cultured in vitro with irradiated C57BL/6 stimulating cells. Five days later the T cell-mediated cytotoxic activity of the effector cells was assessed with a 51Cr-release assay that used H-2bEL-4 tumor cells as targets. Before the BALB/c responding lymphocytes were sensitized they were fractionated by passing the spleen cells over insolubilized histamine rabbit serum albumin Sepharose columns (H-RSA-S) or over rabbit serum albumin Sepharose (RSA-S) control columns. Fractionation of cells over the H-RSA-S columns depleted or significantly reduced the cytotoxic potential of the unretained cells. All cytotoxic potential was recovered when the cells that adhered to the H-RSA-S were eluted from the columns. In contrast, no effect on responsiveness was detected after the cells had been fractionated over the control column. The loss of response potential by the cells that did not adhere to H-RSA-S could not be accounted for by removal of macrophages nor by the concentration of cells with suppressor activity in the effluent. These cell fractionation studies raise the possiblity but do not prove that cytotoxic precursor cells may express amine receptors that could be responsible for their retention by insolubilized histamine columns.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(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.     

Lymphokine-activated cell-associated antigen involved in broad-reactive killer cell-mediated cytotoxicity

Spleen cells from rats which had been hyperimmunized with mouse lymphokine-activated killer (LAK) cells, were fused with the mouse myeloma cell line, P3 X 63 Ag8.653. Antibodies secreted by 1500 cultures were selected by their blocking effect on LAK cell-mediated cytotoxicity in the absence of complement. Two monoclonal antibodies (KBA4 and KBA6) greatly inhibited the cytotoxic activity of LAK cells, which were induced from mouse spleen cells by culture with recombinant human interleukin 2 (r-IL-2). These antibodies also blocked the cytotoxic activity of natural killer (NK) cells, but activated macrophages (A-M phi) were only slightly sensitive to them. However, no effect of the antibodies on the cytotoxic activity of cytotoxic T lymphocytes (CTL) was detected. These data suggest that the specific antigen, lymphokine-activated cell-associated (LAA) antigen, defined by these monoclonal antibodies may be associated with the recognition mechanisms of broad-reactive killer (BRK) cell-mediated cytotoxicity. The observation that low levels of LAA antigen are distributed in all lymphoid cells and that it was significantly enhanced by treatment of the cells with r-IL-2 suggests that the antigen may be involved in lymphocyte-activation mechanisms. We also found that the LAA antigen consists of two distinct polypeptides with Mr of 180,000 and 95,000 Da, which are similar to that of LFA 1 antigen. However, the biological characteristics of LAA antigen did not coincide with those of LFA 1. Therefore, KBA MAb may recognize a carbohydrate epitope distinct from that of LFA 1.     

Immunogenicity of subcellular fractions and molecular species of MuLV-induced tumors

Summary YBA, a Moloney virus-induced leukemia in CBA mice, and a relatively weak immunogenic tumor, was screened for the presence of immunogenic antigens. The tumor was subjected to homogenization and subcellular fractionation on sucrose gradients; the immunogenic subcellular fractions underwent further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the subcellular fractions and the SDS-PAGE-isolated molecular species were tested by (their) subcutaneous injection into syngeneic mice and examination of their splenocytes examined against tumor cell and normal cell targets by the chromium release cell-mediated lympholysis assay. Tumor cell homogenates were also separated by SDS-PAGE and tested for immunogenicity without prior fractionation.Splenocytes from mice that had received injections of certain SDS-PAGE-isolated epitopes derived from YBA tumor homogenate or its light and heavy subcellular fractions generated effective cytotoxic responses against YBA target cells after 6 days in vitro cultivation. In contrast, intact YBA tumor cells or non-separated tumor homogenates failed to induce an efficient cytotoxic response. The effector cells induced with the immunogenic SDS-PAGE-isolated epitopes of YBA tumor were specific, since they cytolysed the homologous target cells more efficiently than unrelated target cells or syngeneic normal cells. The activity of these effector cells was affected by varying the effector : target ratio. Augmentation of the cytotoxic responses was obtained when the splenocytes of mice immunized with SDS-PAGE-isolated epitopes of YBA tumor were restimulated in vitro, with the homologous neoplastic cells.Immunogenic SDS-PAGE epitopes were isolated from YAC tumor also (YAC is a Moloney-induced tumor of A mice). The effector cells induced with these separated epitopes were characterized as thymus-derived cells and not as natural killer cells.The results suggest that (1) the molecular repertoire of YBA and YBA tumors contain immunogens that can induce a specific antitumor cell-mediated response; (2) the isolated molecular species injected are more efficient immunogens than the entire, unseparated homogenate sample or a dose of 10⁸ intact inactivated tumor cells; and (3) the gel matrix may be responsible for the enhanced cell-mediated response induced against the weakly immunogenic tumor.     

Specific unresponsiveness to skin allografts in anti-lymphocyte serum-treated, marrow-injected mice: participation of donor marrow-derived suppressor T cells

Sequential changes of cell-mediated immune reactivities were examined in anti-lymphocyte serum-(ALS) treated, C3H/He (C3H; H-2k) bone marrow-injected (C57BL/6 X A)F1 (B6AF1; H-2b/k.d) mice bearing enhanced C3H skin grafts. Spleen cells of these mice exhibited marked suppression of the proliferative response to phytohemagglutinin and concanavalin A. When the spleen cells were assayed for the direct lymphocyte-mediated cytotoxicity against H-2k targets, their lytic activity remained low until the time of graft rejection, in contrast to the increasingly high cytotoxic activity exhibited by spleen cells of control B6AF1 mice given only ALS and C3H skin grafts. When spleen cells of marrow-injected B6AF1 mice were cultured with mitomycin-C treated C3H spleen cells, the proliferative response was significantly suppressed the throughout the course, despite the early appearance of high "secondary-type" cytotoxic activity. Co-culture experiments demonstrated the presence of C3H antigen-specific suppressor cells in the ALS-treated, marrow-injected mice bearing intact allografts. Treatment of spleen cells with anti-H-2, anti-Thy 1 and anti-I-J sera and C revealed that the suppressor cells present late in the marrow-injected mice were T cells of donor C3H bone marrow cell origin.     

Susceptibility of Friend virus antigen-modulated erythroleukemic cells to lysis by T lymphocytes from mice with dormant Friend virus infections.

Spleen cells from DBA/2 mice with dormant Friend leukemia virus (FLV) infections or from mice immunized with x-irradiated FLC-745 erythroleukemic cells are not cytolytic for FLC-745 cells when tested directly, but acquire cytolytic activity in vitro by cultivation with x-irradiated FLC-745 cells. Spleen cells cultured from normal mice do not acquire such cytolytic activity. Cytolytic activity resides in the T lymphocyte population. Alloantiserum, but not antisera against FLV-virion polypeptides, inhibited the lysis of FLC-745 cells by cytolytic T lymphocytes. To understand further the role of cellular and humoral immune anti-FLV responses in mice with dormant FLV-infections, in vitro experiments were conducted that mimicked the in vivo FLV-specific immune environment of these mice. We found that FLV-immune serum from mice with dormant FLV-infections modulated FLV-antigen expression on the surfaces of FLC-745 cells without affecting their susceptibility to lysis by cytolytic T lymphocytes. These results suggest that cytolytic T lymphocyte restrain the outgrowth of FLV-antigen-modulated erythroleukemic cells in mice with dormant FLV infections and that the targets for cell-mediated lysis may be H-2d-associated antigens.     

K cell cytotoxicity against antibody-coated chicken erythrocytes in tumor-bearing mice: its development with progressively growing tumor and the effect of immunization against the tumor.

Antibody-dependent (K cell) cytotoxic activity of spleen cells from mice bearing a chemically induced fibrosarcoma has been studied by using antibody-coated chicken erythrocytes as target cells. Spleen cells from tumor-bearing animals caused a significantly greater degree of target cells destruction than did those from control animals. The elevated cytotoxic activity in tumor-bearing animals increased with time after the tumor inoculation and correlated directly with the size of the tumor. The development of increased cytotoxic activity could be circumvented by surgical removal of the tumor. Mice that received x-irradiated tumor cells of x-irradiated tumor cells followed by a live challenge did not show a tumor growth and also failed to show increased K cell cytotoxic activity. It has been concluded that the increased K cell activity results directly from the active growth of tumor. The role of K cells in immunosurveillance has been discussed.     

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.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

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.     

Immune regulation of the L5178Y murine tumor-dormant state. II. Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice

L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state. Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation. L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination. Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth. The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures. MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity. Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice. These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.     

In vitro murine lymphocyte blastogenic responses to Cryptosporidium parvum.

Spleen and mesenteric lymph node lymphocytes from both Cryptosporidium parvum-exposed and unexposed mice were cultured with antigen (Ag) prepared from C. parvum oocysts. Spleen lymphocytes from oral-, intraperitoneal-, or oral + intraperitoneal-exposed mice did not respond significantly (P greater than 0.05) to Ag stimulation. Spleen lymphocytes from multioral-exposed mice, however, demonstrated significant (P less than or equal to 0.01) Ag-specific blastogenesis. Mesenteric lymph node lymphocytes did not respond to in vitro Ag stimulation regardless of the route of in vivo priming. These results demonstrate an in vitro cell-mediated immune response against C. parvum by lymphocytes in murine spleen.     

Cytotoxicity of lymphoid cells induced by Maclura pomifera (MP) lectin.

The cytotoxic activity of lymphoid cells stimulated with Maclura pomifera (MP) lectin was investigated. Spleen cells of Lewis (LEW) or Brown Norway (BN) rats induced a cell-dependent release of ⁵¹Cr from syngeneic, allogeneic, and xenogeneic erythrocytes when incubated with MP for 4–16 hr. The activity of MP differed from that of concanavalin A (Con A). MP exhibited a greater activity with spleen cells while Con A was more active when bone marrow cells were tested. Activity induced by MP required the presence of the lectin for at least 4 hr and was inhibited by melibiose, an inhibitor of MP binding. MP also stimulated phagocytosis by peritoneal macrophages of LEW rats, but phagocytosis was not responsible for the cytotoxic effect measured by ⁵¹Cr release. The ability of aggressor cells to bind MP did not correlate with their cytotoxic activity. The cytotoxic activity of spleen cells from athymic nude mice was equivalent to that of cells from euthymic littermates when stimulated with MP.