Normal Mouse Lymphocytes Research Articles

The bovine lymphoid system: Binding and stimulation of peripheral blood lymphocytes by lectins

We tested the ability of 11 different lectins to stimulate DNA synthesis in bovine peripheral blood lymphocytes, using a wide range of experimental conditions. Five of the 6 lectins which induced DNA synthesis (concanavalin A, succinyl-concanavalin A, phytohemagglutinin M, pokeweed mitogen and lipopolysaccharide) did so under conditions similar to those optimal for stimulation of murine lymphocytes. The other lectin (peanut agglutinin) stimulated normal bovine lymphocytes whereas it does not stimulate normal mouse, rat, guinea pig or human lymphocytes. The binding of 5 different lectins to bovine peripheral blood lymphocytes was measured by fluorescene microscopy. Four of the lectins bound to various proportions of cells. Double labeling experiments using a rhodamine-labeled goat anti-bovine immunoglobulin reagent and fluorescein-labeled lectins showed that both peanut agglutinin and soybean agglutinin bound to lymphocyte populations which were negative for surface immunoglobulin. The majority of lymphocytes negative for surface immunoglobulin bound peanut agglutinin, indicating that this lectin may bind specifically to bovine ‘T’ lymphocytes.

Inverse correlation between cell‐surface adhesiveness and malignancy in mouse fibroblastoid cell lines

The correlation between tumorigenicity and cell-surface adhesiveness was investigated in eight mouse fibroblastoid cell lines. Four of the lines (Mc 11-Mc 15) were derived from mouse MC-induced fibrosarcomas, two were derivatives of L cells (A9, A9HT), and two (clone 3 and clone 7 H7F4) were obtained by fusion of A9HT cells with normal diploid mouse lymphocytes. Quantitative comparison of cellular adhesive properties by the Latex particle adherence assay indicated that higher malignancy of some lines (Mc 11, 13, 14, A9HT cl 7 H7F4) was regularly associated with lower cell-surface adhesiveness and conversely, lower malignancy of the other lines (Mc 15, A9, cl 3) with high cell-surface adhesiveness. The differences in adhesiveness of cell lines of high and low malignancy were determined by cell subsets not attaching and Latex particles (24.6-61.9% of cells in highly and 4.6-12.0% in slightly tumorigenic lines) and cell subsets attaching more than 25 Latex particles per cell (14.6-17.2% in highly and 46.1-71.4% in less tumorigenic lines).

Release of soluble “blocking” and “suppressor” factors from normal lymphocytes treated with RNA from spleens of tumour-bearing mice

RNA extracted from the spleens of tumour-bearing (TLRNA) and tumour-immune (ILRNA) mice was shown to transfer to normal lymphocytes (NL) the ability to produce factors that blocked specific tumour-cell cytotoxicity and mediated specific antibody-dependent cell cytotoxicity (ADCC). Aliquots of normal C3H mouse lymphocytes were treated with TLRNA or ILRNA and cultured in vitro in the absence of tumour antigen. Supernatants were collected at 24h intervals and tested in a microcytotoxicity assay for blocking and ADCC activities. Factors that inhibited tumour destruction by specifically sensitized lymphocytes at the level of both the tumour cells and effector cells were demonstrable in culture supernatants of NL pretreated with TLRNA (50 or 100 microgram/4 X 10(6) cells) but not ILRNA. However, treatment of NL with either RNA resulted in the production factors that mediated tumour-specific ADCC. Cytotoxicity testing and absorption studies of the tumour cell and a control cell (LM) indicated that factors mediating ADCC and blocking at the target-cell level were specific for the tumour. Suppressor activity at the effector-cell level was not absorbed by tumour cells and represents a separate and distinct mechanism of immunosuppression. These data indicate that RNA faithfully transfers "suppressive" as well as "positive" types of immune responses that have been reported previously for lymphocytes obtained directly from tumour-bearing and tumour-immune animals.

XenCSA: Cell Surface Antigens Related to the Major Glycoproteins (gp70) of Xenotropic Murine Leukemia Viruses

Abstract Antisera from rabbits immunized with rabbit corneal (SIRC) cells infected with xenotropic murine leukemia viruses (MuLV) were tested for their reactivity with MuLV and the surface of cells exogenously or endogenously infected with MuLV. The sera demonstrated class specificity for gp 70 determinants of intact xenotropic MuLV. Only molecules with characteristics of gp70 were precipitated out of radiolabeled antigens from the surface of cell lines exogenously infected with MuLV or from lymphocytes of normal mice. Cross-reactivity between virion envelope and cell surface antigens was demonstrated by absorption testing. The set of crossreactive antigens present on the envelopes of xenotropic MuLV and murine lymphocytes was termed XenCSA for xenotropic MuLV envelope-related cell surface antigen. XenCSA expression on thymocytes and spleen cells from 63 strains of inbred mice was assessed by immunofluorescence using flow microfluorometry. Lymphocytes from both tissues of all strains expressed XenCSA but to a highly variable extent. Based on mean fluorescence values, thymocytes and spleen cells from each strain were classified as being high or low for XenCSA. There was a high association for expression of XenCSA and GIX on thymocytes but no correlation with Fv-1, T1a, H-2, Pca-1, or GCSA. The level of XenCSA expressed on the cell surface was also independent of the amount of infectious xenotropic MuLV produced by those cells.

Fatty acid positional specificity in phospholipids of L1210 leukemia and normal mouse lymphocytes

The positional distribution of fatty acids in the choline and ethanolamine phosphoglycerides of the L1210 murine leukemia cells was determined and compared to that of normal mouse lymphocytes. The major phospholipids of both cell types had appreciable degrees of positional specificity as evident from the higher percentage of saturated fatty acids in position 1 and of polyunsaturated fatty acids in position 2. The L1210 cells had less arachidonate and more linoleate in position 2 of choline and ethanolamine phosphoglycerides as compared to the normal lymphocytes. However, there were similar proportions of saturated, monoenoic and polyenoic fatty acids in positions 1 and 2 of the phospholipids of the L1210 leukemia cells and the lymphocytes, These data demonstrate that fatty acid positional specificity is retained in the major phospholipids of this rapidly growing tumor.

Anti-tumour cytotoxicity of poly(A)-containing messenger RNA isolated from tumour-specific immunogenic RNA.

The transfer of tumour-specific cytotoxicity against a murine fibrosar-coma has been demonstrated in vitro using xenogeneic RNA extracted from tumour-cell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA. Treated cells were tested in vitro using an adaptation of the Takasugi and Klein microcytotoxicity assay. Percent cytotoxicity was calculted using cells treated with fractions of normal RNA as control. An increase in tumour cytotoxicity was found with poly(A)-containing immune RNA. The optimum dose of poly(A)-tailed immune RNA was estimated as 6.5 microgram of RNA per 4 x 10(6) lymphocytes. Populations of lymphocytes were separated using glass and nylon wool. T- and B-enriched populations were treated with various RNA components. The adherent cell population showed no significant cytotoxicity, whilst treatment of the nonadherent population with poly(A)-tailed immune RNA produced high levels of cytotoxicity.

B lymphocyte development, heterogeneity, and function

Thymus-independent (B) lymphocytes are a heterogeneous group of cells which, it is increasingly clear, undergo an ordered sequence of differentiation steps from stem cells to antibody producing cells. Through the study of the immunologic potential of B lymphocytes from neonatal mice and from mice with an X-linked immune defect, it has become possible to assign certain functions to cells of distinct differentiated states. Furthermore, a differentiation antigen, Lyb 5, has been identified which acts as a marker of a particular subset of B lymphocytes in normal mice. Initial studies of the functional properties of Lyb 5+ and Lyb 5- B lymphocytes are described.

Fractionation of immune RNA capable of transferring tumor-specific cellular cytotoxicity

Xenogeneic immune RNA (I-RNA) extracted from the spleens and lymph nodes of guinea pigs previously immunized with a murine fibrosarcoma was able to convert normal mouse lymphocytes to effector cells specifically cytolytic to the same murine tumor in vitro. This effect of I-RNA was dose-dependent, and destroyed by treatment with RNase, but not with DNase or pronase. I-RNA was fractionated by ultracentrifugation on a 5–20% sucrose density gradient and the fraction capable of transferring cell-mediated cytotoxicity (CMC) was shown to have a sedimentation coefficient of 8–16 S. I-RNA was also fractionated by oligo(dT)-cellulose affinity chromatography and the active fraction was found to possess polyadenylic acid (poly(A)) sequences thus resembling messenger RNA. The immunological activity of the poly (A)-containing RNA fraction was tumor-specific and RNase-sensitive. In further experiments, I-RNA fractionated by sucrose density gradient ultracentrifugation was subsequently chromatographed. on an oligo(dT)-cellulose column. CMC was transferred only by the fraction which sedimented at 8–16 S and also contained poly (A).

Mycoplasma-dependent activation of normal lymphocytes: induction of a lymphocyte-mediated cytotoxicity for allogeneic and syngeneic mouse target cells.

Mycoplasma arthritidis, M. hominis, and M. arginini were tested for their ability to induce a cytotoxic response from normal CBA mouse lymphocytes against 51Cr-labeled allogeneic target cells. In most cases, the mycoplasmas alone were not toxic for the target cells. Furthermore, the mycoplasmas did not result in decreased lymphocyte viability but, in fact, contributed to enhanced lymphocyte survival. In the absence of normal CBA lymphocytes, mycoplasmas alone did not induce a significant amount of cell damage in either the allogeneic or the syngeneic target cells. Strains of M. arthritidis and M. hominis, when added to the lymphocyte-target cell mixtures, induced statistically significant increases in 51Cr release from both target cell types at each assay period after 6 h. The release of 51Cr was taken as a measure of cell death. M. arginini induced only low levels of cytotoxicity or none at all. Both arthritogenic and non-arthritogenic strains of M. arthritidis induced the cytotoxic response. The degree of cytotoxicity produced was directly related to the size of the initial inoculum. The presence or absence of serum in the culture medium did not contribute significantly to the cytotoxicity response.

Mycoplasma-dependent activation of normal lymphocytes: role of arginine and nonviable mycoplasma antigen in the induction of lymphocyte-mediated cytotoxicity for syngeneic mouse target cells.

The ability of arginine supplementation to abrogate or potentiate Mycoplasma arthritidis-mediated cytotoxicity induction by normal CBA mouse lymphocytes was investigated. Also, nonviable antigens of M. arthritidis were assayed for their ability to induce a cytotoxic response. The presence of additional arginine in the culture medium had no significant effect on the amount of cytotoxicity produced. In some cases, an increase in cytotoxicity was seen in the presence of additional arginine. Amino acid analysis of the culture supernatants showed that the arginine in the nonsupplemented medium was exhausted during the assay, whereas ample amounts of arginine were present in the supplemented medium. However in both cases the amount of cytotoxicity was comparable. Nonviable antigens of M. arthritidis were unable to induce a significant cytotoxic response in the presence of normal lymphocytes. The same antigens exhibited a suppressive response when tested for blastogenesis in the presence of normal lymphocytes. A discussion of lymphocyte involvement is presented.

Kinetics of small lymphocytes in normal and nude mice after splenectomy.

Autoradiography and various quantitations on lymphoid tissues have been used to evaluate the kinetics of small lymphocytes in normal (+/nu or +/+) and congenitally athymic nude (nu/nu) NMRI mice 1 month after splenectomy or sham-splenectomy. The results indicate that splenectomy causes depressed thymic activity and diminished numbers of T lymphocytes in peripheral lymphoid tissues. The total number of cells in these tissues as well as the blast cell activity, were within normal limits. Bone marrow lymphocyte numbers and kinetics as well as blood lymphocyte levels in splenectomized and sham-splenectomized normal animals were comparable. Blood lymphocyte numbers were at normal levels in splenectomized nude mice, in spite of reduced numbers of bone marrow and thoracic duct lymphocytes. It is suggested that increased number of newly-formed lymphocytes, found in lymph nodes and blood of splenectomized mice, are released from the lympho-myeloid organs in compensation for the loss of long-lived, thymus-derived cells.

Separation of cells with different histocompatibility (H‐2) antigens by passage through cellular immunoadsorbent columns

The anti-Ig column technique of Wigzell et al. (Scand. J. Immunol. 1972. 1:75) was adapted to the separation of mouse cells with different H-2 antigens. Fractionation was achieved by a two-step procedure: first the cells were treated with anti-H-2 sera, and thereafter passed over anti-mouse Ig columns. Antibody-coated cells were selectively retained in the anti-Ig column. The method could be applied to the separation of normal mouse lymphocytes or lymphoma cells with different H-2 antigen complexes and for the selective isolation of H-2 loss variants from a mouse carcinoma/fibroblast hybrid.

Synthesis of a liver enzyme in hybrid cells.

Rat hepatoma cells were fused with cells of an established mouse lymphoma line, with normal diploid mouse macrophages, lymphocytes and fibroblasts and with normal diploid rat macrophages and lymphocytes. The liver-specific enzyme tyrosine aminotransferase was produced by almost all the hybrid cells, but usually at a lower level than in the parental hepatoma cells. Most of the hybrids also showed increased levels of this enzyme after exposure to dexamethasone. In the rat x mouse hybrids, the electrophoretic mobility of the enzyme indicated that only the rat hepatoma enzyme was produced. The findings are difficult to explain in terms of simple models involving a single diffusible repressor or activator of tyrosine aminotransferase synthesis.

Mode of action of immunosuppressive substances in sera of tumor-bearing mice.

Sera from mice with transplanted 3-methylcholanthrene (MCA)-induced tumors inhibited the proliferative response of normal mouse lymphocytes to mitogens and allogeneic cells. The sera were not toxic to these cells and did not inhibit mitogen responses by an increased binding of mitogen to serum components. The sera could have prevented the initiation of the proliferative response or could have inhibited cells already proliferating. The uptake of 3H-uridine, an event preceding DNA synthesis, was also suppressed. The sera had no effect on proliferation of a normal tissue culture line or an allogeneic tumor cell line induced by MCA. However, sera from tumor-bearing mice slowed growth of the autochthonous tumor cells and allogeneic lymphoma cells but did not completely block their proliferation. Exposure of lymphoid cells to the sera for a period as brief as 1 hour markedly decreased the ability of cells to respond subsequently to mitogens, and washing of the cells did not restore that ability. The inhibitory activity of the sera was only partially removed by absorption with lymphoid cells or cell lines.

Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the theta-antigen by non-ionic detergents.

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid-urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the theta-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid-urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and mu-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3-4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid-urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The theta-alloantigen was isolated from CBA T-cells both by acid-urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the theta-positive thymoma WEHI 105. The theta-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.

Morphological Differences between Thymus- and Bone Marrow-Derived Lymphocytes: 1. A light microscopic and experimental study in unstimulated mice

Simple morphological criteria are described, enabling one to distinguish two types of lymphocytes in normal unstimulated mice: ‘Th’, predominant in the thymus (90-92%) and ‘Bm’, predominant in the bone-marrow (85-93%). Their distribution in the peripheral lymphoid organs agreed significantly with the observed average distribution of T and B lymphocytes. Thy-mectomized and irradiated bone-marrow-restored CBA mice, as well as nude mice, showed a striking lack of Th cells. In vitro depletion of θ-bearing or Ig-bearing lymphocytes lead to a correlated depletion of Th or Bm lymphocytes, respectively: blood lymphocytes treated with anti-θ serum + complement showed a decrease in Th lymphocytes corresponding to the percentage of cells killed by the anti-θ serum. The selective retention of Ig-bearing spleen cells on Wigzell's anti-lg columns caused a depletion of Bm lymphocytes. It is concluded that the Th type is the morphological expression of the T lymphocytes, and the Bm type that of the B lymphocytes. The implications of this way of distinguishing T and B lymphocytes by simple morphology, and of the actual relationships between T and B cell differentiation, are discussed.

In vitro conversion of normal mouse lymphocytes by plasmacytoma RNA to express idiotypic specificities on their surface characteristic of the plasmacytoma immunoglobulin

Using a “blind” experimental protocol, we have reaffirmed that RNA-rich extracts from plasmacytoma can convert normal mouse lymphocytes in vitro so that normal surface immunoglobulins (Ig) are replaced by specific plasmacytoma Ig as judged by an immunocytoadhesion assay that detects cells bearing the idiotypic specificity of the plasmacytoma Ig. No loss of biological activity could be detected when extensive degradation of DNA in the extracts by DNase was explicitly demonstrated or when de novo RNA synthesis by the cells was inhibited by actinomycin D, thus eliminating a role for DNA in the cell conversion. Loss of biological activity also failed to occur after the RNA-rich extracts were treated with pronase or when the amount of contaminating protein was markedly reduced; however, loss of activity did occur when de novo protein synthesis by the cells was inhibited by puromycin or cycloheximide, thus eliminating the possibility that the specific plasmacytoma Ig or a fragment thereof was passively transferred onto the cell surface with the RNA molecule as a carrier. However, mild RNase treatment that causes a relatively small increase in the amount of 4S RNA and yet retaining relatively large amounts of 18S and 28S RNA, as might result from only a few breaks in the intact RNA molecules, effectively destroyed the biological activity of the RNA-rich extracts, reaffirming the requirement for intact RNA. When the RNA-rich extracts were fractionated by sucrose density gradient centrifugation, the activity was limited to the 12S to 23S fractions.

Evidence for the presence in unimmunized mice of two populations of bone marrow derived B lymphocytes, defined by differences in adherence properties

The presence of two populations of bone-marrow-derived lymphocytes in normal unimmunized mice was inferred from data showing that cells forming antibody to either the dinitrophenyl (DNP) determinant or sheep erythrocytes (SRC) arose spontaneously in antigen-free tissue culture medium in contrast to cells forming antibody against FγG, which arose spontaneously in much smaller numbers, and which in cultures of spleen cells from the athymic (nude) mouse were undetectable. Measures amplifying the degree of spontaneous activation of antibody forming cells (AFC) to DNP and SRC, such as the inclusion in cultures of large doses of polymerized flagellin (POL) or flagella (FLA), lipopolysaccharide (LPS) or the presence of allogeneic cells had no effect on the absence of nonantigen dependent activation of FγG-reactive B cells. However when appropriately activated, by FγG plus a “second signal” provided by the presence of POL, FγG-reactive B cells became fully susceptible to the effect of the presence of allogeneic cells in amplifying the numbers of AFC produced in cultures. An ancillary observation suggested that a proportion of B cells reactive to DNP and SRC from CBA mice passed through macrophage adherence columns, while only a small percentage of B cells reactive to FγG could be demonstrated in the filtrate. An hypothesis accounting for these observations is that the population of B cells reactive to FγG, the activation of which is absolutely antigen dependent, differs from a proportion of the population of B cells reactive to DNP and SRC in that this proportion of the latter populations has been altered by previous activation by cross-reacting antigens. The spontaneous appearance of non antigen dependent AFC to DNP and SRC in tissue culture, and the increase in this number induced by a factor produced by allogeneic recognition, are a functional reflection of such an alteration by previous contact with antigen. It seems possible that a loss of a tendency of the virgin B cell to adhere to glass or plastic is another consequence of prior interaction with antigen.

Failure to produce cell-free lymphotoxin.

NORMAL human, mouse and guinea-pig lymphocytes have been reported to secrete a cytotoxic factor, lymphotoxin (LT), on stimulation with lectins or staphylococcal filtrates; sensitized lymphocytes behave similarly on stimulation with specific antigen. Lymphotoxin is suggested to be present in the supernatant of lymphocyte cultures and to be capable of killing non-specifically a large range of target cells1. Purification and physico-chemical characterization of human and murine lymphotoxin have been carried out1. This work has been confirmed by several workers2–5, whereas others observed a cytotoxic activity only, if lymphocytes, stimulant and target cells were present in the same culture6–10,16. The implication that mouse and guinea-pig lymphocytes secrete LT was not upheld by my experiments. Lymphoid cells were obtained from normal mouse spleens (BDF! hybrids (C57B1/6/J × DBA/2/J) ; C3H, Balb/c). Cells were released into Hanks Balanced Salt Solution (Hanks BSS, GIBCO) by teasing with broad forceps. Small clumps were removed by sedimentation. The resulting suspension was spun, the cells washed three times (Hanks BSS) and finally resuspended in medium RPMI 1640 (GIBCO) containing 10% inactivated foetal calf serum (FCS; Flow). Total cell counts (Model A Coulter Counter) and viability tests (Trypan blue exclusion) were performed. Viability was in the range of 90%. Guinea-pig spleen cell suspensions (individual outbred guinea-pigs) were prepared in the same way except that TC 199/10% FCS (Flow) was used throughout. Viability ranged from 77–94%. All steps were performed at 4° C using pre-cooled materials; centrifugation was carried out at room temperature using a Sorvall GLC-1 centrifuge.

DEPLETION OF SPECIFIC GRAFT-VERSUS-HOST REACTIVITY FOLLOWING ADSORPTION OF NONSENSITIZED LYMPHOCYTES ON ALLOGENEIC FIBROBLASTS

SUMMARY Antigen recognition in transplantation reactions induced in vitro by mouse fibroblasts manifests itself in specific binding of T lymphocytes to membrane antigens of fibroblast monolayers. To deplete graft-versus-host (GVH) reactivity we interacted normal mouse lymphocytes with allogeneic fibroblasts; 4-6% of H-2d lymphocytes adhered to H-2k or H-2b monolayers. The nonadhering cells showed a reduced capacity to bind to cells of the original adsorbing H-2 phenotype. However, adherence to fibroblasts of a different H-2 was not impaired. The capacity of the nonadherent cells to evoke GVH reactivity in vivo was measured by assaying (1) splenomegaly of X-rayed animals injected with the nonadherent lymph node cells; (2) cytotoxicity to target cells of the repopulated spleen cells; and (3) enlargement of the draining lymph nodes in a local GVH reaction following injection of nonadherent spleen cells. All of these test systems showed that GVH reactivity was markedly decreased or eliminated in an immunospecific manner by adsorption. Specific adherence demonstrated the diversity of T lymphocytes with regard to cell receptors for alloantigens. This approach may provide a method for the specific elimination of antihost-reactive cells in the transplantation of bone marrow or lymphoid cells.