Fresh Spleen Cells Research Articles

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Endothelial precursor cell cross-match using Tie-2-enriched spleen cells.

Non-HLA antibodies against human endothelial progenitor cells (EPC) in pre-transplant recipient serum can have a deleterious influence on the graft. EPC enriched from peripheral blood have been commonly used for EPC cross-matching. In the present study, we describe cross-matches using EPC enriched from fresh or frozen-thawed spleen cell preparations, thereby widening the sample source for deceased-donor cross-matching and retrospective studies. EPC cross-matches were performed retrospectively using spleen cells and the flow cytometric XM-ONE cross-match test kit. Healthy controls (n=28) showed no IgG antibodies against EPC. When sera of 11 random dialysis patients were studied, 2 patients (18%) exhibited IgG EPC antibodies. When pre-transplant sera of 20 kidney graft recipients with good long-term graft outcome (serum creatinine 1.0±0.2mg/dL measured 2463±324days post-transplant) were investigated using frozen-thawed and then separated Tie-2-enriched spleen cells of the original transplant donor, 3 patients (15%) had pre-transplant IgG EPC antibodies. When pre-transplant sera of 5 patients with intra-operative graft loss were studied employing the original donor spleen cells, 4 (80%) patients showed IgG EPC antibodies. Cross-matches with spleen cell-derived EPC using the XM-ONE assay are technically possible. Our very preliminary experience suggests clinical relevance.

Effects of 3D nanocomposite bioceramic scaffolds on the immune response.

The interaction of new nanocomposite mesoporous glass/hydroxyapatite (MGHA) scaffolds with immune cells involved in both innate and acquired immunity has been studied in vitro as an essential aspect of their biocompatibility assessment. Since the immune response can be affected by the degradation products of bioresorbable scaffolds and scaffold surface changes, both processes have been evaluated. No alterations in proliferation and viability of RAW-264.7 macrophage-like cells were detected after culture on MGHA scaffolds which did not induce cell apoptosis. However, a slight cell size decrease and an intracellular calcium content increase were observed after contact of this cell line with MGHA scaffolds or their extracts. Although no changes in the percentages of RAW cells with low and high contents of reactive oxygen species (ROS) are observed by the treatment with 7 day extracts, this study has revealed modifications of these percentages after direct contact with scaffolds and by the treatment with 24 h extracts, related to the high reactivity/bioactivity of this MGHA nanocomposite at initial times. Furthermore, when normal fresh murine spleen cells were used as an experimental model closer to physiological conditions, no significant alterations in the activation of different immune cell subpopulations were detected in the presence of 24 h MGHA extract. MGHA scaffolds did not affect either the spontaneous apoptosis or intracellular cytokine expression (IL-2, IL-10, IFN-γ, and TNF-α) after 24 h treatment. The results obtained in the present study with murine immune cell subpopulations (macrophages, lymphocytes B, lymphocytes T and natural killer cells) support the biocompatibility of the MGHA material and suggest an adequate host tissue response to their scaffolds upon their implantation.

In vivo augmentation of NK cell activity by methanol extract of cultured cambial meristematic cells of mountain wild ginseng (P4393)

Abstract Although mountain wild ginseng (MWG, Panax ginseng C.A.Meyer) has been regarded to be more effective than cultivated ginseng, very few data have been accumulated until now because of its limited availability and expensiveness. Some of us established a technique of isolation and culture of innately undifferentiated cambial meristematic cells from MWG as an attractive alternative. Using methanol extract of cultured MWG cambium-derived cells (MEGC), we here investigated in vivo immunomodulatory effect in a mouse model. Mice were intraperitoneally given MEGC (200 mg/Kg) and adaptive immunity was unaffected by MEGC treatment. Without Ag immunization, splenic NK cell activity in MEGC-treated mice was significantly higher when compared to controls. However, number of NK1.1+ cells and surface expression of activation/ inhibitory receptors and activation markers were unaltered by MEGC. Instead, RT-PCR data clearly demonstrated that MEGC treatment increases constitutive expression of Granzyme B and perforin in fresh spleen cells. When spleen cells were cocultured with Yac-1 in the presence of IL-2, Granzyme B and IFNγ levels were also higher in MEGC-treated group. However, these effects were achieved solely at early phase of culture (< day 1) and lower concentration of IL-2. Collectively, MEGC augments in vivo NK cell activity through up-regulated constitutive expression of cytolytic mediators and data suggest that ginseng may act at early phase of NK cell response.

Characterization of an interaction between fetal hemoglobin and lipid A of LPS resulting in augmented induction of cytokine production in vivo and in vitro

A previously described extract of sheep fetal liver was reported to reverse many of the cytokine changes associated with aging in mice, including an augmented spleen cell ConA-stimulated production of IL-4 and decreased production of IL-2. Similar effects were not seen with adult liver preparations. These changes were observed in various strains of mice, including BALB/c, DBA/2 and C57BL/6, using mice with ages ranging from 8 to 110 weeks. Preliminary characterization of this crude extract showed evidence for the presence of Hb gamma chain, as well as of lipid A of LPS. We show below that purified preparations of sheep fetal Hb, but not adult Hb, in concert with suboptimally stimulating doses of LPS (lipid A), cooperate in the regulation of production of a number of cytokines, including TNFα and IL-6, in vitro. Furthermore, isolated fresh spleen or peritoneal cells from animals treated in vivo with the same combination of Hb and LPS, showed an augmented capacity to produce these cytokines on further culture in vitro. Evidence was also obtained for a further interaction between CLP, LPS and fetal Hb itself in this augmented cytokine production. These data suggest that some of the functional activities in the fetal liver extract reported earlier can be explained in terms of a novel immunomodulatory role of a mixture of LPS (lipid A) and fetal Hb.

Immune and non-immune factors in cryopreserved tissues

Background Although cryopreserved tissues are used clinically, the effects of cryopreservation on antigenicity leading to immune and non-immune responses are not well known. Methods We investigated the change of inflammatory effects of cryopreserved tissue by using spleen and aortic allografts from Class I antigen–disparate B6.C-H-2 bm1 (bm1; K bm1 , IA b , D b ), Class II antigen–disparate B6.C-H-2 bm12 (bm12; K b , IA bm12 , D b ) and Class I and Class II antigen–disparate (bm1 × bm12)F1 ( K bm1 × b , IA b × bm12 , D b ) mice against C57BL/6 Cr Slc (B6; H-2 b ) mice. Cryopreservation was done in a programmed freezer and cryopreserved tissues were kept in the vapor phase of liquid nitrogen for 2 weeks and thawed at room temperature. Results Cryopreserved B6 spleen cells expressed almost the same levels of Class I ( K b and D b ) and Class II ( IA b ) antigens as observed in fresh B6 spleen cells. Cryopreserved bm1 and bm12 spleen cells had the same stimulator activities in mixed-lymphocyte reaction (MLR) and cytotoxic T-lymphocyte (CTL) assays compared with fresh bm1 and bm12 spleen cells, respectively. To elucidate the effects of cryopreserved tissues on immune response of recipients, descending aortas of (bm1 × bm12)F1 mice were implanted into the right common carotid artery of B6 ( H-2 b ) mice with the cuff technique and the reactivities of recipient B6 mice against Class I antigen–disparate bm1 antigens and Class II antigen–disparate bm12 antigens were examined 4 weeks after implantation. In both MLR and CTL assays against bm1 or bm12 antigens, anti-donor reactivities were augmented and there was no significant difference between B6 mice grafted with fresh aortic allografts and those grafted with cryopreserved ones. Histologic analysis showed that mild infiltration of mononuclear cells into the adventitia was observed in both fresh and cryopreserved aortic allografts. The fibrous change was observed more strongly in cryopreserved aortic allografts compared with fresh aortic allografts. Conclusions Cryopreservation has no effect on eliciting immune responses to Class I or Class II alloantigens, but has some effect on promoting fibrous change.

DNA immunization with hepatitis C virus (HCV) polycistronic genes or immunization by HCV DNA priming-recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1-transgenic mice.

We studied immune responses to hepatitis C virus (HCV) genes delivered as DNA encoding the entire HCV protein coding genome in two polycistronic plasmids encoding HCV capsid-E1-E2-NS2-NS3 and HCV NS3-NS4-NS5 in HLA-A2.1-transgenic mice. Immune responses to HCV DNA prime and recombinant canarypox virus boost were also studied with the above constructs. At 8 weeks after a canarypox virus boost, the DNA prime/canarypox virus boosting regimen induced potent cellular immune responses to HCV structural and nonstructural proteins on target cells expressing the HLA-A2.1 allele. High frequencies of gamma interferon-secreting cells, as detected by enzyme-linked immunospot assay, were obtained in response to several endogenously expressed HCV proteins. We also observed cytotoxic-T-lymphocyte reactivity in response to endogenously expressed HCV proteins in fresh spleen cells without in vitro expansion. Upon challenge with a recombinant vaccinia virus expressing HCV proteins at 2 months postimmunization, the HCV DNA prime/canarypox virus-immunized mice showed a complete reduction in vaccinia virus titers compared to HCV DNA prime/boost- and mock-immunized controls. Immune responses were still detectable 4 months after canarypox virus boost in immunized mice. Interestingly, at 10 months postimmunization (8 months after canarypox virus boost), the protection in HCV DNA prime/boost-immunized mice against recombinant HCV-vaccinia virus challenge was higher than that observed in HCV DNA prime/canarypox virus boost-immunized mice.

Abrogation of tumor induced Ly49 expression on mouse spleen cells by Mitomycin C

Mouse spleen cells were activated with IL2 for 4 days in the presence or absence of paraformaldehyde fixed YAC (PFY) tumor cells (spleen cell: PFY ratio 100:1). Fresh spleen cells had poor expression of Ly49A but the expression was significantly upregulated by IL2 activation. Addition of PFYs resulted in a further boosting of Ly49A expression. Besides Ly49A, the Ly49C receptors were also upregulated by PFYs. Upregulated Ly49 expression was not restricted to NK cells (NK1.1 positive cells) but was also seen on T cells (TCRβ positive cells). Time kinetics studies indicated that maximum upregulation of Ly49 in response to PFY cells occurred on day 3 and 4 and the expression declined thereafter. Ly49 expression in response to PFYs was completely blocked if spleen cells were pre-treated with Mitomycin C, an inhibitor of DNA synthesis. These results show that the addition of a small number of paraformaldehyde fixed YAC cells to spleen cell cultures undergoing IL2 activation, resulted in a significant upregulation of Ly49 receptors and this process was dependent upon cell proliferative activity.

Induction of specific transplantation immunity by oral immunization with allogeneic cells.

Oral administration of antigen has been shown to be effective for both positive and negative modulation of immune responses. In the present study we characterized changes in the reactivity of the immune system after oral immunization with allogeneic spleen cells. Mice were orally immunized for 10 consecutive days with fresh allogeneic spleen cells, and the phenotype, proliferative response, cytotoxic activity and cytokine production profile of recipient spleen cells were assessed 1 or 7 days after the last immunization dose. Although no significant changes in the proportion of CD4+, CD8+ or CD25+ cells were observed in the spleen of orally immunized mice, significant activation of alloreactivity in spleen cells was found. Cells from orally immunized mice exhibited enhanced proliferation and cytotoxic activity after stimulation with specific allogeneic cells in vitro, and produced considerably higher concentrations of interferon-gamma (IFN-gamma) and significantly less interleukin (IL)-4 than did cells from control mice. The production of IL-2 was essentially unchanged and that of IL-10 was only slightly increased. The systemic allosensitization induced by oral immunization was demonstrated in vivo by increased resistance to the growth of allogeneic tumours induced by subcutaneous inoculation of high doses of tumour cells. In addition, orthotopic corneal allografts in orally immunized recipients were rejected more rapidly (in a second-set manner) than in control, untreated recipients. These data show that oral immunization with allogeneic cells modulates individual components of the immune response and that specific transplantation immunity, rather than tolerance, is induced in the treated recipients.

Induction of proliferation in resting B-cells by a factor released by activated mouse spleen cells.

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.

LeIF: A Recombinant Leishmania Protein That Induces an IL-12-Mediated Th1 Cytokine Profile

Abstract We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major-infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-γ (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-γ. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-γ by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1–226) maintained the ability to stimulate IFN-γ from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags.

Cyclophosphamide given after active specific immunization augments antitumor immunity by modulation of Th1 commitment of CD4+ T cells.

In order to evaluate the regulatory effect of cyclophosphamide (CPA) on active specific immunization (ASI)-induced antitumor immunity, we examined the timing of CPA (100 mg/kg) with ASI, and focused on whether CPA given after ASI augments antitumor immunity by modulation of Th1 commitment of CD4+ T cells. We examined the effect of CPA combined with ASI using sonicated tumor supernatant (SS) and recombinant interleukin-1 beta (rIL-1 beta). Survival of i.p. tumor inoculated mice after ASI (days -12, -9, and -6) followed by 100 mg/kg CPA (day -3) (ASI-CPA) was significantly prolonged compared with that of mice treated with ASI alone, whereas CPA (day -15) treatment before ASI (CPA-ASI) completely abrogated the survival prolongation by ASI alone. In early stage (day 0) after ASI-CPA treatment, the CD4+ T cells were determined to play an important role in the protective immunity for the following reasons: 1) the CD4+/CD8+ ratio of spleen cells from immunized mice was higher than that of the control or CPA alone treated group; and 2) the tumor neutralizing activity of fresh spleen cells was abrogated by CD4+ T-cell depletion in vitro. CD4+ T cells of mice treated with ASI-CPA produced more interferon (IFN)-gamma and IL-2 and less IL-4 than those of the ASI alone group. These results suggest that the protective immunity induced by ASI was augmented through the modification of the Th1 and Th2 balance by CPA injection after ASI.

Priming of cytotoxic T lymphocytes by five heat-aggregated antigens in vivo: conditions, efficiency, and relation to antibody responses.

Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, beta-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100 degrees C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K(b) hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D(b)-restricted CTL specific for the peptide 49-57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100 degrees C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100 degrees C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.

Enhancement of lymphokine-activated killer cell activity by fibronectin.

In this study, we demonstrated that immobilized fibronectin (FN) enhanced lymphokine-activated killer cell (LAK) activity, and that the enhanced LAK activity was completely abrogated by an anti-VLA-5 monoclonal antibody and RGD peptide. Fresh spleen cells expressed VLA-4, VLA-6, and vitronectin receptor, whereas VLA-5 was expressed only on the spleen cells activated with interleukin-2. LAK cells showed increased adhesion to immobilized FN compared with that to control bovine serum albumin, and the increased adhesion of LAK cells to immobilized FN was inhibited by anti-VLA-5 monoclonal antibody. Conjugate-formation assay showed that the LAK cells cultured on immobilized FN bound to target cells more efficiently than the control LAK cells, and that anti-LFA-1 monoclonal antibody inhibited the LAK-target cell binding. Immobilized type IV collagen and laminin, as well as FN, enhanced LAK activity. All of these results suggest that the interaction of integrins expressed on LAK cells with extracellular matrix proteins acts in a costimulatory manner for the enhancement of LAK activity, and that anchorage is necessary for full activation of LAK cells.

A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes

Murine lymphocytes readily undergo spontaneous and glucocortocoid-induced apoptosis in vitro. It has been previously demonstrated that during apoptosis, many cell types including lymphocytes, enzymatically cleave their DNA, thus demonstrating a sub-G 0 DNA peak when stained with propidium iodide and analyzed by flow cytometry. In a mixed population, it is often desirable to phenotypically identify distinct populations or subsets undergoing apoptosis, thus requiring multiparameter analysis of surface phenotype and DNA content. Paraformaldehyde fixation procedures, although common for surface evaluation, have not been extensively used in methods quantifying apoptosis. To measure apoptosis in a mixed lymphocyte population, we evaluated a gentle detergent permeabilization and paraformaldehyde fixation procedure combined with propidium iodide (PI) DNA staining, adapted from existing methods for cell cycle studies. With this method and rigorous gating techniques which we defined, we detected both apoptotic and debris fractions within the sub-G 0 cell cycle region of a glucocortocoid-treated murine lymphocyte cell line. Using this cell line, WEHI 231.7, as a lymphocyte model, we developed a logical gating strategy to exclude debris from analysis. We further demonstrated that apoptosis in freshly isolated murine lymphocytes detected with paraformaldehyde fixation and PI staining was quantitatively comparable to PI staining with ethanol fixation, or nick translation labeling of DNA strand breaks (TUNEL). Finally, using fresh murine spleen cells, we demonstrated that paraformaldehyde fixation preserves surface protein staining, allowing multiparameter analysis of immunophenotype and apoptotic or cell cycle stats in a mixed lymphocyte population. Thus, this method offers an inexpensive and technically simple alternative for assessing apoptosis and surface phenotype.

Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors.

We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.

CD4+ T cells recovered from a mixed immune lymphocyte-tumor cell culture induce thymidine incorporation by naive rat lymphocytes in response to tumor cells.

Spleen cells from tumor-immune rats incorporate thymidine when co-cultured for 4 days with syngeneic cancer cells. Non-adherent cells, recovered from a 7-day mixed culture with cancer cells, had lost their capacity for incorporating thymidine when exposed again to the same tumor cells; however, in these conditions, the non-adherent cells induce thymidine incorporation by fresh naive spleen cells. This response is restricted to tumor lines which originate from the same tumor as the cells used for immunization and is due to memory and/or activated CD45RC- CD4+ T cells. Our results indicate that tumor-specific T cells maintain their capacity to respond to tumor antigens, even after an extended culture time with tumor cells. In another study, in the same conditions, spleen cells from normal or tumor-bearing rats did not evoke significant responses.

Interleukin-3 inhibits the generation of nonspecific killers by interleukin-2.

Interleukin (IL)-3 has effects on a wide variety of cell types, including immature cells of the immune system, as well as mature cells such as granulocytes. We have investigated the effects of IL-3 on generation of cytolytic cells that can kill tumor cells. Previously, we have shown that IL-3 can enhance the development of cytolytic T cells (CTL) reactive with the line 1 tumor in a CD4-dependent manner. It is of interest that we found the development of CTL in response to IL-3 was not accompanied by increased development of nonspecific killer cells. This was in contrast to IL-2, which enhanced development of both CTL and nonspecific cells. To determine if IL-3 could inhibit the development of nonspecific killers, we added IL-3 to cultures of fresh spleen cells stimulated with high levels of IL-2. In this assay, IL-3 showed very potent inhibitory activity against the generation of nonspecific killers. To determine if IL-3 could inhibit the IL-2-driven generation of non-specific killers in vivo, we injected a mixture of IL-2- and IL-3-producing line 1 cells. Each cytokine-producing transfectant was also injected alone. The cytotoxicity of tumor-infiltrating lymphocytes (TIL) from these tumors confirmed that the presence of IL-3 inhibits the generation of nonspecific killer cells in vivo.

The murine NK2.1 antigen: a 130 kD glycoprotein dimer expressed by a natural killer cell subset of the spleen, thymus and lymph nodes

Murine natural killer (NK) cells express a few antigens not found on other leukocyte subsets. The NK1.1 antigen, that is present in only a few mouse strains, has been extensively characterized whereas our knowledge of the NK2.1 antigen, which is more commonly expressed, remains, as yet, limited. Our laboratory has previously reported the production of a mAb (4LO3311) recognizing a murine NK cell-specific molecule with a similar strain distribution as the NK2.1 antigen formerly defined with an NZB anti-BALB/c antiserum. In this study, we demonstrate by sequential immunoprecipitation that 4LO3311 represents the first NK2.1 antigen-specific mAb. This reagent was used to immunoprecipitate the NK2.1 antigen from 125I-labeled lysates of fresh NK-enriched spleen cells. SDS-PAGE analyses revealed that the NK2.1 antigen is expressed at the cell surface as a N-glycosylated disulfide-linked protein dimer with ~65 kD subunits. The NK2.1 antigen is likely to be anchored in the plasma membrane by a peptide moiety since its expression on NK cells was not affected by treatment with phosphatidylinositol-specific phospholipase C. In addition to be present on a splenic NK cell subset, the NK2.1 antigen is shown to be expressed by a small number of CD4 −CD8 −thymocytes and by a subset of CD4 − CD8 −IgG − lymph node cells. Finally, it is shown here that unlike the NKR-P1, the rat homologue of the murine NK1.1 antigen, neither the NK2.1 nor the NK1.1 antigen is expressed by polymorphonuclear leukocytes.

Suppression of T-cell proliferation by CD8+ T cells induced in the presence of protoscolices of Echinococcus multilocularis in vitro.

Immunoregulatory influences of protoscolices (PSC) of Echinococcus multilocularis on murine T-lymphocyte functions have been examined in an in vitro system. Proliferative responses of spleen cells stimulated with concanavalin A (ConA) or anti-CD3 monoclonal antibodies were depressed by the addition of PSC. In the presence of PSC, both interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression by lymphocytes stimulated with ConA were significantly reduced. However, exogenous IL-2 reconstituted both the ConA-stimulated proliferative responses and IL-2R expression. These findings suggest that PSC of E. multilocularis can suppress lymphoid cell responses via influences on IL-2 production. Indeed, addition of CD(8+)-enriched cells from cultures stimulated with ConA plus PSC to fresh spleen cells showed marked suppression of the ConA responses. IL-2 production as well as IL-2R expression on the spleen cells so treated were suppressed. These findings reveal a suppressive immunologic function induced by E. multilocularis PSC that involves inhibition of IL-2 production and reduction of IL-2R expression. The PSC-induced CD8+ cells appear to play a key role in the suppressive regulation of host immune responses against E. multilocularis.