Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Human T cell subpopulations defined by a monoclonal antibody. II. Evidence for cell cooperation in the response to alloantigens and generation of cytolytic cells

It has been shown previously that the 5/9 monoclonal antibody defines a small T cell subpopulation in human peripheral blood that includes all the cells responsible for proliferation to tetanus toxoid and to alloantigens as well as the helper cells for B cell differentiation. In the present study, human peripheral blood T cells were fractionated according to their reactivity with the 5/9 monoclonal antibody and stimulated in mixed lymphocyte culture (MLC). In spite of a strong proliferative response in MLC, 5/9+ cells generated no cytolytic activity against PHA-activated lymphocytes bearing the stimulating alloantigens (CTL activity) or against the K562 human cell line (NK activity). The precursors of these cytolytic effector cells were present in the 5/9- fraction. However, 5/9+ cells or soluble factors derived from 5/9+ cells were needed to induce 5/9- cells to respond in MLC and develop cytolytic activity. Both 5/9+ and 5/9- cell populations, upon MLC stimulation, were able to lyse L1210 mouse lymphoma cells in the presence of specific antibodies (ADCC).     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Immunomodulating activity of p-hydroxyphenyllactic and ascorbic acids

p-Hydroxyphenyl lactic acid (PHA) in a concentration of 5 . 10(-5) M produced a significant inhibition of cell proliferation in response to alloantigens in a one-way mixed lymphocyte culture (MLC) in colonic cancer patients and in blast transformation in response to suboptimal doses of Con A. Multiple administration of ascorbic acid in an optimal concentration to the culture increased the proliferative response of lymphocytes to alloantigens and Con A. PHA and ascorbic acid did not exhibit any immunomodulating action during the use of healthy donors' lymphocytes or lymphocytes from colonic cancer patients, transformed with optimal mitogen doses. PHA did not affect the production of cytotoxic T lymphocytes in the MLC of the spleens of allogeneic mice but inhibited lymphocyte proliferation in response to alloantigens in the MLC of the spleens obtained from B6 and vitamin A deficient animals.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

Interferon production and tumor cell killing by human lymphocytes stimulated in mixed-lymphocyte culture

The in vitro synthesis of interferon (IFN) by human lymphocytes stimulated in mixed-lymphocyte culture (MLC) was examined. The production of IFN in MLC was restricted to T lymphocytes and maximum levels of IFN were detected in supernatants from cells incubated for 5 to 7 days. The IFN produced was identified as IFN-gamma by antibody neutralization. To identify the T cell responsible for IFN production, purified T lymphocytes were separated into subpopulations after incubation in 5 mM theophylline. Theophylline-resistant (T-res) T cells retain the ability to form sheep erythrocyte (SRBC) rosettes and are depleted in IgG Fc receptor-positive T cells (T gamma cells). Theophylline-sensitive (T-sens) T cells fail to form rosettes after theophylline treatment and are enriched in T gamma cells. In addition, analyses using monoclonal antibodies showed that T-sens cells were enriched in OKM1-, HNK-1-, and 7.2-positive cells and T-res cells contained increased numbers of 9.6- and OKT4-positive cells. Following MLC stimulation, equivalent levels of IFN-gamma were produced by T-res and T-sens cells and both subpopulations maintained natural killer (NK)-like cytotoxicity against K562 target cells. Addition of partially purified IFN-gamma to unstimulated T-res and T-sens cells resulted in the maintenance of NK-like cytotoxicity in a manner analogous to that observed after MLC. Additional experiments indicated that peripheral blood lymphocytes depleted of 9.6- or OKM1-positive cells by complement-mediated lysis were devoid of cytotoxicity against K562 cells. Furthermore, MLC stimulation of 9.6- or OKM1-depleted cells failed to restore cytotoxic activity. In summary, these experiments demonstrate that the maintenance of NK-like cytotoxicity by MLC-stimulated T cells is associated with the synthesis of IFN-gamma, that MLC stimulated T-res and T-sens T-cell subsets produce equivalent amounts of IFN, and that 9.6- or OKM1-positive cells are required for the maintenance of NK-like cytotoxicity in MLC.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

Evidence for the presence of suppressor T lymphocytes in animals treated with cyclosporin A

Mice sensitized with alloantigens and treated with cyclosporin A (CsA) were incapable of generating antigen-specific cytolytic lymphocytes (CL). Lymphocytes from these CsA-treated animals could not be reactivated upon exposure to the same alloantigens in mixed lymphocyte culture (MLC), whereas their response to a third-party antigen remained intact, suggesting a long-lasting and specific effect of CsA. After being irradiated, these lymphocytes from CsA-treated animals were added to normal MLC and were shown to prevent normal lymphocytes from becoming cytolytic in a dose-dependent and antigen-nonspecific fashion. These suppressor cells were not detected in mice receiving CsA only, indicating that CsA did not induce but rather permitted the expression of suppressor cells possibly generated by allosensitization. The suppressor cells appeared to be T lymphocytes, because treatment with anti-Thy-1.2 antibody and C abrogated their suppressive activity. The present results suggest that activation and/or sparing of suppressor cells by CsA may account for the long-lasting unresponsiveness seen in CsA-treated animals.     

In vitro stimulation of natural killer (NK) cells by soluble factor(s) generated in antigen-stimulated rhesus monkey peripheral blood lymphocyte cultures.

Peripheral blood lymphocytes from rhesus monkeys (Macaca mulatta) sensitized to keyhole limpet hemocyanin (KLH), when stimulated in vitro with KLH, developed natural killer (NK) cell activity that was assayed with Rous Sarcoma virus-transformed marmoset fibroblasts as targets in a 4-hr 51Cr-release assay. The supernatant fluids from 24- to 25-hr KLH-activated cultures were capable of stimulating NK development in nonsensitive lymphocyte cultures. The effector cells were neither macrophages nor B cells (plastic and nylon-wool nonadherent) and did not form E-rosettes with neuraminidase-treated sheep red blood cells. Cultures depleted of EA-rosetting cells, i.e., Fc-receptor-bearing lymphocytes, were incapable of generating NK activity when stimulated in vitro. Kinetic studies showed that peak DNA synthesis, as measured by 3H-T incorporation, preceded maximum cytotoxicity. Elimination of dividing cells by 5-bromo-2'deoxyuridine (BrdU) and light treatment during the interval from day 1 to day 4 inhibited the development of cytotoxicity on day 7. Cell replication was required for the induction of NK cells with KLH as well as with antigen-activated culture supernatant fluids. When cultures were left unstimulated for 4 days, NK activity could not be developed subsequently either by adding antigen, mitogen (PHA), or supernatant fluids from activated cultures.     

Cytotoxicity of human peripheral lymphocytes in cell-mediated lympholysis; antibody-dependent cell-mediated lympholysis and natural cytotoxicity assays after mixed lymphocyte culture.

Lymphocytes that have been purified by Ficoll-Hypaque centrifugation lose antibody-dependent and natural cytotoxic activities upon culture in tissue culture medium supplemented with human plasma. However, stimulation of peripheral lymphocytes in the mixed leukocyte culture (MLC) appears to enhance killer (K) and natural killer (NK) activities in addition to generating cytotoxic T ymphocytes. Enhancement of NK and antibody dependent activities appears to correlate with cell division as measured by 3H-thymidine uptake. However, elimination of dividing cells in the MLC by addition of 5-bromodeoxyuridine has no effect on NK and K cells activities. Since this treatment abolishes cell-mediated lympholysis mediated by cytotoxic T lymphocytes, it is a useful probe for determining the relative activities of NK, K, and cytotoxic T lymphocyte effector cells after lymphocyte stimulation.     

Suppression of the late stages of mitogen-induced human B cell differentiation by FC gamma receptors (FC gamma R) released from polymorphonuclear neutrophils

During short incubation in serum-free medium, polymorphonuclear neutrophils (PMN) release soluble material that can be characterized as receptors for Fc IgG (Fc gamma R) on the following evidence: it agglutinates erythrocyte-IgG antibody (EAG) complexes, it prevents the binding of EAG to EAG-rosette-forming cells, and it binds to EAG-rosette-forming cells after modulation of their Fc gamma R, allowing the formation of 'passive' rosettes. These Fc gamma R were isolated by affinity chromatography on sepharose 4B-IgG. This material was shown to interfere with the differentiation of peripheral blood B cells into Ig-secreting cells in cultures stimulated by pokeweed (PWM) or Nocardia opaca (NOC) extracts. The number of Ig-secreting cells determined by a reverse hemolytic plaque assay using protein A-coated sheep erythrocytes was decreased by addition of Fc gamma R over a wide range of dilutions. The number of Ig-containing cells was diminished in PWM-stimulated cultures, but not in cultures stimulated with NOC. Fc gamma R did not affect cell viability, nor did they interfere with plaque-forming cell assay. Fc gamma R was not suppressive when added before the 3rd day or after the 6th day of culture. The suppressor factor was shown to remain associated with Fc gamma R after elution at acidic pH; it was removed by absorption on Sepharose 4B-IgG, but not on pepsin-digested F(ab')2 fragments. The suppressive factor as well as the capacity to restore EAG rosette formation by modulated lymphocytes were destroyed by heating (56 degrees C, 30 min) or by freezing and thawing. Properties of Fc gamma R released from PMN in this system are similar to those of Fc gamma R released from unstimulated human peripheral blood lymphocytes (PBL) and to those of mouse Ig-binding factor produced by alloactivated T cells or T cell lines.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Ontogeny and postnatal persistence of a strong suppressor activity in man

We report here on the ontogeny and postnatal persistence of an inhibited human immune response in which lymphocytes from human newborns strongly suppress the proliferation of adults' lymphocytes stimulated by phytohemagglutinin (PHA) or alloantigens in vitro. For this research we used a 2-way mixed lymphocyte culture (MLC) supplemented with PHA, with sex chromosomes acting as markers for dividing male and female cells, or alternatively a double chamber system. The proliferation of maternal lymphocytes was significantly suppressed by fetal lymphoid cells from the liver as early as the 8th week of gestation and by those from fetal blood at the 14th week or later during gestation. This strong suppressor activity persisted in 11-mo-old infants but usually disappeared after that time.     

Production and properties of immune interferon from spleen cell cultures of Toxoplasma-infected mice

In response to antigenic stimulation, spleen cells from Toxoplasma-infected mice produce a factor showing inhibitory activity against vesicular stomatitis virus infection in L cell cultures. When BALB/C and ICR mice were inoculated intraperitoneally with the low-virulent S-273 strain of T. gondii, such activity was first detected in 4 and 7 days and reached maximum levels at 10 and 14 days respectively, and retained these levels for at least three weeks. However, BALB/C mice, which are considerably more sensitive to Toxoplasma infection than ICR mice, produced significantly smaller amounts of interferon (IF) after challenge with the high virulent strain. The IF produced in this system possessed certain known properties of immune (type II) IF and was not neutralized by rabbit antiserum against mouse type I IF. The immune IF preparation also inhibited multiplication of Toxoplasma within nonphagocytic L cells in an IF-like fashion, whereas Newcastle disease virus-induced (type I) IF had no effect on this parasite. The antiviral and anti-Toxoplasma activity in immune IF preparations could not be distinguished solely on the bases of their molecular weight and isoelectric point. The experiments with anti-theta serum plus complement and with nylon wool column effluent cells strongly suggest that immune IF was produced by T lymphocytes and required the assistance of macrophages.     

Natural regulatory cells in murine bone marrow: inhibition of in vitro proliferative and cytotoxic responses to alloantigens

A lymphocyte-enriched fraction of murine bone marrow (BML), obtained by sucrose density fractionation, contains natural regulatory cells that can profoundly suppress the proliferative and cytotoxic response of syngeneic lymph node cells to irradiated alloantigens in a mixed lymphocyte culture (MLC). A close correlation exists between the inhibition of alloantigen-induced proliferation and the generation of cytotoxic effectors. The suppression of proliferation is dependent on the dose of BML added to the cultures but is not due to cell crowding, since red blood cells, thymocytes, and irradiated splenocytes, all syngeneic to the lymph node responders, do not inhibit proliferation to the degree observed with BML. The addition of BML to cultures does not cause the maximum proliferative response to change from the usual day 5 peak, indicating that there is no change in culture kinetics. The release of nonradioactive thymidine by BML cannot explain the suppression. The target of suppression is maximally affected during the first 24 hr of culture, since adding BML to MLC later than this resulted in negligible inhibition of proliferation. Thus, the natural regulatory cell-mediated suppression reflects inhibition of "early" events in the proliferative response to alloantigens.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. I. Activation of Listeria antigen-responsive T cells

Soon after rats are infected with Listeria monocytogenes (LM), Listeria antigen- (LMA) responsive lymphocytes are delivered to the animal's thoracic duct. These LM-responsive lymphocytes can be restimulated in vitro by LMA to generate cells that have a potent cytolytic capability. The activation of LMA responsive lymphocytes is immunologically specific and dependent upon the activity of histocompatible accessory cells. Neither cell-free LMA nor LMA-pulsed allogeneic accessory cells can promote a significant cytotoxic response by negatively selected responder lymphocytes. LM-dependent cytolytic lymphocytes differ from natural killer (NK) cells inasmuch as their activation is not facilitated by interferon (IF). Likewise, supernatants from cultures containing specifically sensitized thymus-derived (T) lymphocytes and histocompatible LMA-pulsed accessory cells fail to augment (day 2 and day 3 supernatants actually inhibit) the activation process. The results imply that the successful activation of LM dependent cytolytic lymphocytes requires the cooperative interplay of responder T cells and specific-antigen-pulsed accessory cells.