Normal Splenic Lymphocytes Research Articles

Sodium selenite inhibits deoxynivalenol-induced injury in GPX1-knockdown porcine splenic lymphocytes in culture

Deoxynivalenol (DON) is a cytotoxic mycotoxin that can cause cell damages. The main effect is to inhibit protein synthesis. Oxidative stress is one of the effects of DON. Selenium (Se) can ameliorate the cell damage caused by DON-induced oxidative stress, but it is unclear whether through selenoprotein glutathione peroxidase 1 (GPX1). We established GPX1-knockdown porcine spleen lymphocytes, and treated them with DON and Se. Untransfected porcine splenic lymphocytes (group P) and transfected cells (group M, GPX1 knockdown) were treated with or without DON (0.824, 0.412, 0.206, or 0.103 μg/mL, group D1-4), Se (Na2SeO3, 2 μM, group Se), or both (group SD1–4) for 6, 12, or 24 h. The cells were collected and the activities of SOD and CAT, levels of GSH, H2O2, malonaldehyde (MDA), total antioxidant capacity (T-AOC), and the inhibition of free hydroxyl radicals were determined. Levels of ROS were measured at 24 h. Compared with group P, the antioxidant capacity of group M was reduced. DON caused greater oxidative damage to the GPX1-knockdown porcine splenic lymphocytes than to the normal control cells. When Na2SeO3 was combined with DON, it reduced the damage in the GPX1-knockdown porcine splenic lymphocytes, but less effectively than in the normal porcine splenic lymphocytes.

High intensity focused ultrasound enhances anti-tumor immunity by inhibiting the negative regulatory effect of miR-134 on CD86 in a murine melanoma model

HIFU has been demonstrated to enhance anti-tumor immunity, however, the mechanism of which has not been well elucidated. Emerging evidence indicates that miRNAs play important roles in immune response. In this study, we used the B16F10 melanoma allograft mouse model to investigate the role of miRNAs in HIFU-enhanced anti-tumor immunity. We found that HIFU treatment decreased circulating B16F10 cells and pulmonary metastasis nodules while increased IFN-γ and TNF-α in the peripheral blood and cumulative mouse survival, which was associated with inhibition of miR-134 expression and activation of CD86 expression in tumor tissues. Further, we determined that miR-134 directly binds to the 3'UTR of CD86 mRNA to suppress its expression in B16F10 cells. When B16F10 cells transfected with miR-134 were co-cultured with normal splenic lymphocytes, the secretion of IFN-γ and TNF-α from lymphocytes was reduced and B16F10 cell survival was increased. HIFU exposure efficiently decreased miR-134 while increased CD86 expression in B16F10 cells in vitro. CD86 knockdown with siRNA markedly rescued the viability of HIFU-treated B16F10 cells that co-cultured with lymphocytes. Altogether, our results suggest that HIFU down-regulates miR-134 to release the inhibition of miR-134 on CD86 in melanoma cells, thereby enhancing anti-tumor immune response.

Highly oligomeric procyanidins from areca nut induce lymphocyte apoptosis via the depletion of intracellular thiols

Procyanidins are plant-derived polyphenolic compounds possessing a variety of biological activities, such as immunomodulation, and induction of tumor cell apoptosis. We previously reported that total extract of areca nut exhibited a suppressive effect on the metabolic activity and cytokine expression in normal splenic lymphocytes. As areca nut contains a rich amount of polyphenols, the objective of the present study was to investigate the pro-apoptotic effect of polyphenol-enriched areca nut extract (PANE) and its fractionated oligomeric procyanidins in splenic lymphocytes. Our data showed that PANE markedly induced lymphocyte apoptosis in a concentration- and time-dependent manner. Notably, the fractionated oligomeric procyanidins from pentamers to decamers were active in inducing the apoptosis, whereas monomers to tetramers were inactive. In addition, a marked diminishment in the level of intracellular thiols was revealed in lymphocytes treated with pentamers to decamers. Pretreatment with N-acetyl- L-cysteine, a precursor of glutathione, resulted in significant attenuation of both apoptosis and thiol diminishment induced by areca procyanidins. Taken together, our results indicated that highly oligomeric procyanidins derived from areca nut exhibited a chain length-dependent pro-apoptotic effect in primary lymphocytes, which is mediated, at least in part, by the diminishment of intracellular thiols.

Monoclonal antibodies reactive with chicken interleukin-17

Chicken interleukin-17 (chIL-17) gene was previously characterized through cloning from a chicken intestinal expressed sequence tag (EST) cDNA library. To further investigate the biological properties of chIL-17, six monoclonal antibodies (mAbs) against a bacterially expressed chIL-17 recombinant protein were produced and their binding specificities characterized. Antibodies which were initially selected on the basis of their specific binding reactivity with recombinant chIL-17 in ELISA were further characterized by Western blot analysis. Monoclonal antibodies specific for chIL-17 identified 20 and 21 kDa protein bands in the culture supernatant and cell lysate of CU205 cells. These mAbs also recognized specific bands for chIL-17 in the cell lysate from conconavalin A (Con A)-activated, but not from normal splenic lymphocytes. Furthermore, these mAbs detected a 16 kDa protein in the lysate of CU205 cells treated with tunicamycin and stained an intracellular protein in CU205 cells in flow cytometric analysis. Together, these results indicate that these new mAbs are specific for chIL-17 and will be a useful tool for structural and immunological studies of IL-17 in poultry.

The role of NO in macrophage dysfunction at early stage after burn injury

Aim: To explore the role of nitric oxide (NO) in macrophage dysfunction at early stage after burn injury. Method: Peritoneal macrophages were isolated and cultured from early stage burnt mice. NO production and inducible NO synthase (iNOS) expression in the macrophages were checked by the Greiss method and real-time PCR (TaqMan), respectively. l-Arginine, the substrate of NO producing, or N-monomethyl- l-arginine ( l-NMMA), a competing blocker of NOS was administered to the culture, the changes of NO, TNF-α and PGE2 productions were measured, additionally the changes of the iNOS, TNF-α and COX-2 expression were assayed by real-time PCR. After that, the effects of l-arginine and l-NMMA were determined on burnt macrophage influencing the proliferation of normal splenic lymphocytes. Result: A large amount of NO was produced by macrophages from post burn hour 6 (6PBH) with a high level of iNOS expression. l-Arginine could increase NO production in a dosage-dependent manner, while l-NMMA attenuated NO production, but neither could affect iNOS expression. Moreover, l-arginine enhanced productions of both the latter produced TNF-α and PGE2 from burnt macrophages, and the expressions of TNF-α and COX-2 were improved significantly, while l-NMMA did reverse ways. It was found that macrophages from post burn hour 24 mice could inhibit Con A-stimulated normal splenic lymphocytes dramatically, l-NMMA could decrease this function significantly, but l-arginine could not influence the suppression. Conclusion: Our experiment indicated NO derived from burnt macrophage played a vital role in macrophage producing excessive TNF-α and PGE2, and suppressing lymphocyte function at early stage after burn injury.

Cell Surface Expression of the Endoplasmic Reticular Heat Shock Protein gp96 Is Phylogenetically Conserved

In mammals, the heat shock protein gp96 complexed to antigenic peptides elicits T cell adaptive immunity. By itself, however, gp96 can evoke responses that are characteristic of innate immunity. Interestingly, this protein, which resides in the endoplasmic reticulum, is expressed on the surface of certain mouse tumor cells. Given that heat shock proteins are highly conserved, we investigated whether the cell surface expression of gp96 is also evolutionarily conserved. Our data reveal that gp96, most likely containing the endoplasmic reticulum retention motif (KDEL), is expressed on the surface of three different Xenopus lymphoid tumor cell lines, each derived from a different spontaneously arising thymic tumor. Levels of expression differ among the tumor lines tested, with more immunogenic tumors expressing greater amounts of surface gp96. Moreover, a high level of gp96 surface expression is detectable on a subset of Xenopus normal nontransformed splenic lymphocytes (mainly surface IgM+ B cells) but not on other normal cells, including macrophages and nucleated erythrocytes. Surface expression of a gp96 protein homologue occurs also on some, but not all, T and B cell clones derived from peripheral blood cells of the channel catfish, as well as on lymphocyte-like cells, but not on erythrocytes from the hagfish, a primitive agnathan vertebrate lacking markers of an adaptive immune system. gp96 is actively directed to and retained on the plasma membrane of Xenopus lymphocytes and tumor cells and hagfish lymphocyte-like cells by a process that requires vesicular transport. This selective surface expression of gp96 on some immune cells from different vertebrate classes is consistent with an ancestral immunological role of gp96 as danger-signaling molecule.

Effect of cell fusion on metastatic ability of mouse hepatocarcinoma cell lines.

AIM:To study the effect of cell fusion on metastatic ability of mouse hepatocarcinoma cells and the factors involved in the process of metastasis.METHODS: By the method of successively increasing the concentrations, cell fusion and limit dilution, 8-Ag resistant cells were selected, and HGPRT-Hca-P cells and eight cloned hybridoma cells were obtained. To observe their metastatic ability, they were inoculated into mice foodtaps and the drainage lymph nodes were examined under microscope.RESULTS:The end concentration of 8-Ag which was used to select HGPRT deficient Hca-P cells was 30mg/L.All the cells selected died in HAT culture medium in one week.Fused cells appeared approximately 9 days later. They were round, transparent and a little larger than their parental cells. Eight clones of hybridoma cells were obtained and named as PSH1-PSH8. The metastatic rate of HGPRT-Hca-P cells and PSH7 cells was 28.6% and 71.4% respectively, the difference being significant (P < 0.05). The metastatic rate of other clones was no more than 20% and there was no significant difference from HGPRT-Hca-P cells (P >0.05).CONCLUSION:In normal mice splenic lymphocytes, there are some factors that could inhibit tumor metastasis, however, there are some other factors accelerating tumor cells to metastasize. The establishment of PSH7 provides an experimental model which could be used to study the factors involved in metastasis.

Role of cytokines in GVL (ESb lymphoma) and GVHD after adoptive transfer of allogeneic T lymphocytes in mice.

ESb lymphoma cells injected i.v. into DBA/2 (H-2d) mice multiply rapidly in the liver and kill all mice in a few days. Adoptive transfer of allogeneic C57B1/6 (H-2b) tumor-immune or normal splenic lymphocytes to sublethally irradiated DBA/2 mice induced a marked antitumor state, graft-versus-leukemia (GVL), increasing the mean survival time 2-3-fold, but also induced an acute and lethal graft-versus host disease (GVHD). We have undertaken experiments to try to dissociate GVL from GVHD. Transfer of immune spleen cells induced a greater GVL than transfer of normal spleen cells with an equivalent to GVHD. Three to five million immune or normal CD8+ T lymphocytes were sufficient to induce both GVL and GVHD. Individual DBA/2 mice were labeled and followed. In mice undergoing GVHD, the spleens were repopulated by donor (H-2b) lymphocytes, and tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were present in the sera of 26 of 27 and 18 of 20 mice, respectively, together with increased amounts of TNF-alpha and IL-6 mRNA in their spleens. This was in contrast to DBA/2 mice receiving allogeneic cells but not developing GVHD. Both interferon-alpha/beta (IFN-alpha/beta) and IL-12, which had proven very effective in association with adoptive transfer of syngeneic immune T lymphocytes in inhibiting ESb metastases, enhanced GVHD when administered with allogeneic immune or normal spleen cells, and >90% of mice died. Intensive IL-2 treatment inhibited GVHD while maintaining GVL.

Studies on the tissue-related phenotypic heterogeneity of murine B cells.

The development of B cells is accompanied by their ability to specifically enter the peripheral lymphoid tissues. Recently, we described a novel rat monoclonal antibody (IBL-2; IgG2b/k reacting with a 26/29-kD heterodimeric structure of the cell surface. This mAb has been found to recognize differentially the peripheral B cells of mice depending on their tissue origin. The majority of splenic B cells as well as the mature B cells in the bone marrow were stained with this mAb, whereas the B lymphocytes isolated from LN or Peyer's patches displayed only negligible reactivity. We extended these observations by analyzing the relationship between the expression of IBL-2 antigen and L-selection on the surface of B-cell precursors in the bone marrow by multiparameter flow cytometry. Within the B220 positive compartment, a significant difference of L-selectin expression could be observed between the various IBL-2-reactive subsets. Furthermore, we investigated whether evidences for the establishment of tissue-associated phenotypic heterogeneity similar to that found in normal mice could be found upon the adoptive transfer of normal unselected splenic lymphocytes into SCID recipients (Spl-SCID). It has been found that a large part of the splenic B cells preserved their IBL-2 reactivity, whereas the LN B cells had lost the IBL-2 antigen in Spl-SCID. These data indicate that the phenotypic difference within the SCID mice may be the result of the migration of B lymphocytes from the spleen toward the lymph nodes, and the altered expression of the IBL-2 antigen correlates with this process.

Activation of benzoporphyrin derivative in the circulation of mice without skin photosensitivity.

In vitro experiments with benzoporphyrin derivative monoacid ring A (BPD) confirmed earlier studies that it was taken up rapidly (within 30 min) to maximum concentrations by all cells tested. It was also confirmed that rapidly dividing tumor cell lines and mitogen-activated murine T lymphocytes took up significantly more (5-10-fold) BPD than did normal splenic lymphocytes. Further experiments were undertaken to determine whether BPD could be activated by whole-body irradiation with red light in the blood of animals, shortly after intravenous (i.v.) administration, in the absence of skin photosensitivity. It was found that shaved and depilated mice injected i.v. 60 min earlier with BPD at between 0.5 and 1.0 mg/kg could tolerate 160 J/cm2 of broad-band red light (560-900 nm) delivered, at a relatively low rate, over a 90 min time interval without developing skin photosensitivity or general phototoxicity. During the treatment time, plasma levels of BPD were between 0.7 and 1.0 micrograms/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating BPD. When L1210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total-body light treatment (160 J/cm2 of 590-900 nm light delivered over 90 min), significant reductions in circulating clonogenic tumor cells were observed in blood samples taken immediately following treatment. This indicated that sufficient light was being delivered to BPD in the blood flowing in the peripheral vasculature to effect cytotoxicity to cells containing the photosensitizer without causing either vascular or skin photosensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Blastogenesis of splenic lymphocytes to specific antigens and PHA in Paragonimus westermani infected mice

Paragonimus westermani is a common fluke in Korea. The present study aimed to observe the cell mediated immune response in experimental paragonimiasis of mice. The mouse (BALB/c) was orally inoculated with 40 metacercariae of P. westermani from Cambaroides similis. During the infection (1, 2, 4, 6 weeks) of mouse, blastogenic response of splenic lymphocytes to P. westermani adult antigen, metacercaria antigen, and PHA were observed. Sera from infected and noninfected mice added to normal mouse splenic lymphocytes with or without PHA. The blastogenic response of splenic lymphocytes to PHA was reduced after 1 week of infection. However after 6 weeks of infection, the response was restored to the control level. The blastogenic response of splenic lymphocytes to P. westermani adult or metacercaria antigen increased significantly on 1 week after infection, and maintained up to 6 weeks after infection. The response of non-infected mice was suppressed by addition of the infected mouse serum. The present results suggested that cellular immunity was involved in P. westermani infected mice and that P. westermani anti-serum inhibited proliferation of T lymphocytes.

The spontaneous apoptotic cell death of normal human lymphocytes in vitro: The release of, and immunoproliferative response to, nucleosomes in vitro

Nucleosomes released spontaneously in short-term culture from murine spleen cells have a significant immunoproliferative effect in vitro, including the stimulation of anti-DNA antibody responses. The present studies show that during short-term cultures, tonsil lymphoid cells also undergo spontaneous apoptosis revealed morphologically (electron microscopy) by the appearance of changes in nuclear chromatin, typical of apoptosis and similar to morphologic changes of apoptosis in cultured normal splenic lymphocytes. This process is followed by the release, in the >30-kDa cell free supernatant fraction, of core histones (H2A, H2B, H3, H4) and low molecular weight DNA (approx 160 bp) constituents of nucleosomes. The >30-kDa tonsil lymphocyte cell-free supernatant material containing the constituents of core nucleosomes, as well as the >30-kDa supernatant fractions of tonsil cell lysates harvested at the same time, had a significant immunoproliferative effect on human or murine lymphocytes, increasing both DNA and immunoglobulin synthesis (protein A plaque-forming cells). Thus the release of immunoproliferative nucleosomes form dying human lymphoid cells provides an autocrine lymphocyte stimulatory network which may be important in immunoproliferative disorders and in normal cell turnover. Apoptosis in vivo may also provide a potential source for the circulating nucleosomal DNA identified in plasma in some systemic lupus erythematosus patients as well as contributing to increased polyclonal B lymphocyte stimulation and autoantibody responses in this disorder.

Immunosuppressive effects of four trichothecene mycotoxins

The trichothecenes are a family of mycotoxins contaminating some food and feeds. We have studied the inhibitory effect of four of them including less toxic compounds: T-2 toxin, DAS, DON and Trichodermin, on normal human peripheral blood lymphocytes and murine splenic lymphocytes. The results show that even for the least toxic compound, inhibiting concentrations can be realised in the blood by common alimentary contamination.

B-Lymphocyte-Derived Erythroid Burst-Promoting Activity Is Distinct From Other Known Lymphokines

Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte-derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum-free human bone marrow culture, BPA was localized entirely to the B-lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte-derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

IgD-binding factors from mouse T lymphocytes.

Incubation of normal mouse splenic lymphocytes with dimeric mouse IgD results in the formation of T-cell factors that selectively inhibit rosette formation of Fc delta receptor-positive lymphocytes with IgD-coated erythrocytes. The rosette-inhibiting factors bound to IgD-Sepharose and were recovered by elution at acid pH. The results indicate that the rosette-inhibiting factors are IgD-binding factors. Neither IgE-binding factors nor IgG-binding factors inhibited IgD rosettes. The IgD-binding factors induced by IgD consist of two species of molecular weight 78,000 and 37,000, respectively. The source of IgD-binding factors is Lyt-1+ T cells bearing Fc delta receptors. It appears that the binding of IgD to the receptors on the T cells induces the formation of IgD-binding factors.

Composition of Extracts of Airborne Grain Dusts: Lectins and Lymphocyte Mitogens

Airborne grain dusts are heterogeneous materials that can elicit acute and chronic respiratory pathophysiology in exposed workers. Previous characterizations of the dusts include the identification of viable microbial contaminants, mycotoxins, and endotoxins. We provide information on the lectin-like activity of grain dust extracts and its possible biological relationship. Hemagglutination of erythrocytes and immunochemical modulation by antibody to specific lectins showed the presence of these substances in extracts of airborne dusts from barley, corn, and rye. Proliferation of normal rat splenic lymphocytes in vitro provided evidence for direct biological effects on the cells of the immune system. These data expand the knowledge of the composition of grain dusts (extracts), and suggest possible mechanisms that may contribute to respiratory disease in grain workers.

Composition of extracts of airborne grain dusts: lectins and lymphocyte mitogens.

Airborne grain dusts are heterogeneous materials that can elicit acute and chronic respiratory pathophysiology in exposed workers. Previous characterizations of the dusts include the identification of viable microbial contaminants, mycotoxins, and endotoxins. We provide information on the lectin-like activity of grain dust extracts and its possible biological relationship. Hemagglutination of erythrocytes and immunochemical modulation by antibody to specific lectins showed the presence of these substances in extracts of airborne dusts from barley, corn, and rye. Proliferation of normal rat splenic lymphocytes in vitro provided evidence for direct biological effects on the cells of the immune system. These data expand the knowledge of the composition of grain dusts (extracts), and suggest possible mechanisms that may contribute to respiratory disease in grain workers.

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells.

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells.

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.