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BALC/c female mice were exposed to 300 R of total body X-irradiation following which erythropoiesis was studied for the next 8 weeks. The data indicate that the initial effects of radiation injury on erythropoiesis is marked depression of both splenic and marrow erynthropoiesis and the development of ineffective release of maturing RBCs from these organs, as demonstrated by marked decreases in organ radioiron uptake and in radioiron reappearance in RBCs, respectively. The data suggest a complex interrelationship between splenic and marrow erythropoiesis following erythropoietic perturbation. We conclude that the mouse spleen may function in a booster capacity to marrow erythropoiesis in the attainment and maintenance of a new steady-state condition.

The non-antigen specific, late acting T helper cell replacing factor TRF can be purified to greater than 99.9% by a combination of molecular sieving and hydrophobic chromatography. Ion exchange chromatography on QAE columns provides an additional means of purification. In the presence of 4M urea, the biological activity of TRF focuses as a single peak, with a Pi of 5.2 ± 0.1. A goat antiserum raised against partially purified TRF specifically inhibits its biological activity and also inhibits the generation of plaque forming cells in normal mouse spleen cultures. Preliminary results obtained with hybridomas derived from peripheral blood lymphocytes of the immunized goat fused with mouse myeloma P3X63Ag8 are described.

Lipopolysaccharide (LPS) has been purified from Bacteroides gingivalis. The purity of this LPS is evidenced on ultracentrifugation, immunoelectrophoresis, and chemical analyses. On comparison with LPS from aerobic enteric organisms, LPS from Bacteroides gingivalis exhibits minimal potency when tested for pyrogenicity in rabbits and mitogenicity in mouse spleen cells. On the other hand, purified LPS from Bacteroides gingivalis exhibits high potency in its ability to stimulate 45Ca release from prelabeled fetal rat bones and to inhibit Ca influx into osteoclast-like cells.

Several investigators have reported a reduction in the number of colony-forming units (CFU) in the spleens of mice exposed to hypoxia and have postulated that this is secondary to a direct toxic effect of hypoxemia on the CFU. To test the hypothesis that at least part of this effect could be secondary to the stress of the hypoxic exposure, mice were given 12 consecutive daily injections of corticotropin (ACTH), and the number of spleen and marrow CFU were assayed by the spleen colony technique. There was a marked decrease in spleen CFU in these mice when compared with control animals, but marrow CFU were not reduced. These tesults indicate that part of the effect of hypoxemia on CFU may be secondary to the stress of the exposure and suggest that spleen CFU are sensitive to steroid hormones. Further investigation of this and other differences between spleen and marrow CFU could lead to a better understanding of stem cell differentiation.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against chick red blood cells (CRBC) can be detected by flow cytometric (FCM) analysis of cellular DNA content. When compared to a standard chromium release assay FCM analysis shows several advantages: (1) equivalent cytotoxicity can be detected after 1 h compared to 4 h for 51Cr; (2) equivalent cytotoxicity can be seen at a 5-fold lower effector-to-target ratio; and (3) no radiolabeling is needed. When mouse spleen cells were fractionated based on adherence to the plastic, adherent cells showed the highest ADCC by both FCM and 51Cr release.

Mouse IgG1, IgG2a and IgG2b antibodies against sheep red blood cells are equally effective, on a weight basis, in suppressing the primary in vitro immune response of normal mouse spleen cells against sheep erythrocytes. Significant suppression was obtained with as little as 10 ng of antibody per culture.

Substantial increases in both 3β-OH sterol and fatty acid synthesis were observed after concanavalin A addition to mouse spleen lymphocytes cultured in serum-free media. The rate of sterol synthesis increased linearly up to 60 h. The rate of fatty acid synthesis increased up to 20 h, reaching a plateau in synthetic activity which was the maintained. CO 2 production from acetate was slightly stimulated by concanavalin A. In contrast to sterol and fatty acid synthesis, the rate of CO 2 production in both mitogen-stimulated and resting cultures declined with time. Dibutyryl cyclic AMP had a strong inhibitory effect on concanavalin A-stimulated sterol and fatty acid synthesis from acetate, but only a slight effect on CO 2 production. Delayed addition of dibutyryl cyclic AMP resulted in reduced inhibition. The data suggest a sequence of initiation for fatty acid and sterol synthesis prior to DNA synthesis and a possible regulatory role of cyclic AMP in this initiation. The results support the hypothesis that lymphocyte activation is sequential within the spleen cell population and is accompanied by fatty acid and sterol synthesis.

Nucleolar changes during the differentiation of erythroblasts in the mouse spleen were examined quantitatively by electron microscopy. On the basis of a three-dimensional nuclear analysis, as reported previously, the erythroblasts could be classified into four types: small (S), medium (M), large (L) and extra-large (EL). Extra-large and large erythroblasts have large prominent nucleoli which are usually attached to the nuclear membrane. Medium and small erythroblasts have small nucleoli which are generally separated from the nuclear margin. The volumetric ratio of nucleoli to nucleus obtained by a point-counting method is 0.157 +/- 0.016 in EL; 0.135 +/- 0.011 in L; 0.035 +/- 0.004 in M and 0.015 +/- 0.004 in S, and the volume of nucleoli is 37.8 microns3 in EL; 17.3 microns3 in L; 2.3 microns3 in M; 0.5 microns3 in S, respectively. The number of nucleoli per nucleus is largest (3.9) in EL and smallest (0.6) in S. The nucleolar changes are discussed in relation to the developmental sequence of the erythroid series.

SummaryAntibody production by normal mouse spleen cells in vitro requires the presence of both adherent and nonadherent cell populations. The data presented here demonstrate that by the addition of alpha-tocopherol (vitamin E) to the medium nonadherent spleen cells can be stimulated to respond to sheep red blood cells in the relative absence of adherent cells. In addition, alpha-tocopherol will enhance in vitro immune responses by populations of spleen cells containing both adherent and nonadherent cells.

Culture conditions are reported for serum-free media supporting an antibody forming cell (AFC) response to sheep erythrocytes as great as the response in conventional serum-supplemented media. Serum-free RPMI 1640 medium facilitated reproducible AFC responses in CBA, C57BL and BALB/c spleen cell cultures, providing that bicarbonate was used as buffer. Satisfactory responses could not be obtained if bicarbonate was replaced by N -2-hydroxy-ethyl piperazine- n -2-ethane sulphonic acid (HEPES). A low AFC response to sheep erythrocytes of T lymphocyte-depleted cell populations in serum-free media indicated the advantage of this type of media over serum-supplemented media in studies of T-B cell collaboration. Serum-free media may also find general application when nonspecific stimulatory or inhibitory effects of serum must be avoided.

New Zealand Black (NZB) and NZB by New Zealand White (NZW) F 1 hybrid ( B W ) mice develop clinical signs of autoimmune disease between 6 and 10 months of age but spleen cells from these strains have a greatly reduced in vitro response to sheep erythrocytes (SRBC) as early as 5–6 weeks of age. This hyporesponsiveness can be only partially restored with 2-mercaptoethanol, allogeneic macrophages or spleen cells, or allogeneic factor. The response of NZB and B W spleen cells to the thymic independent antigen DNP-Ficoll is nearly normal. The reduced in vitro SRBC response was found to be attributable to splenic T and B cells rather than macrophages. Macrophages from NZB mice were found to function normally. The in vitro behavior of NZB lymphocytes is very similar to non-autoimmune mice infected with common murine viral pathogens. NZB and B W mice may be making an active immune response as early as 5 weeks of age.

This report describes a miniaturized version of the mouse mixed leukocyte culture utilizing serum-free and mouse serum supplemented media. Parameters that optimize cellular responses of mouse spleen cells against alloantigens are defined.

A bacterial antitumor and immunopotentiating agent, OK-432, induced Interferon in the spleen cell cultures but not in the thymus cell cultures of various inbred strains of mice. When 1 × 10 7 spleen cells were cultured in the presence of 5 μg/ml of OK-432, interferon activity was detected as early as 4 hr later and reached a maximum level of about 160 to 500 units/ ml 24 hr later. OK-432-induced interferon was mainly an IFN-γ of molecular weight approximately 40,000, but also contained IFN-α and IFN-β.

Two murine lymphomas, L5MF-22 of B10.129(5M) (H-2b) origin and P388 of DBA/2 (H-2d) origin, were inoculated into lethally irradiated hybrid (C57BL/6 x DBA/2)F1 (B6D2F1) mice (H-2b/H-2d). Marked localized graft resistance was found in the spleen and occasionally in the liver of recipient mice as a result of a non-T-dependent hybrid resistance (HR). Significant reduction of HR of B6D2F1 mice could be obtained when viable lymphoma cells were inoculated along with inactivated cells of the same tumor or of genetically related leukemias. These results suggest that in vivo competition for HR effectors can take place in mouse spleen and liver leading to a depression of the localized resistance.

A high-affinity, low-capacity receptor activity for 17 β-estradiol has been identified in cytosols of mouse spleen, normal or erythropoietic. It appears to be a protein which sediments at 5S in high-salt gradients and with a peak at 4S and a shoulder at 8-10S in low-salt gradients. In addition to 17 β-estradiol, only 17 α-estradiol and 5(10)-estren-3 α, 17 β-diol competed successfully with the steroid ligand. A K d of 1.5 × 10 −9 M and 4.1 fmoles/mg protein for the number of binding sites were calculated for the receptor activity of early erythropoietic spleen cytosols.

Spleen cells from ageing (24–30-month-old) mice, manifesting low response to concanavalin A (Con A) and phytohemagglutinin (PHA), were separated by peanut agglutinin (PNA) into agglutinable (PNA+) and unagglutinable (PNA−) fractions. The PNA+ cells were found to suppress the response of young (2–3-month-old) mouse spleen cells to the mitogens Con A and PHA. On the other hand, PNA− cells of the ageing mice expressed a response to these mitogens, at levels higher than those of the unseparated cells. Re-mixing of the PNA− and PNA+ cells of the ageing mouse spleens at various proportions indicated that the PNA− cells are indeed suppressible by the PNA+ cells. Anti Thy-1.2 treatment to the PNA+ fraction suggested that this suppression was not exerted by T cells. It thus appears that at least part of the reduced lymphoid cell function in ageing is related to an effect of suppressor cells, interfering with the expression of available reactive cells.

Partially purified interleukin 2 (IL-2) promotes proliferation of mouse spleen T, but not B cells, and of peanut-agglutinin-negative (PNA-), and cortisone-resistant "mature" thymocytes, but not of PNA+ "immature" thymocytes. Within the cortisone-resistant thymocyte population, IL-2-responsive cells were found in the blast cell fraction. Proliferation was measured by [3H] thymidine incorporation and subsequent increase in viable cells. The mitogenic effect of IL-2 could not be inhibited by 50 mM methyl-alpha-D-mannoside which excludes contaminating concanavalin A (Con A) as a cause of mitogenicity. The relative increase in viable cells in IL-2 vs. control cultures was abrogated by 1.5 mM hydroxyurea. A possible effect of IL-2 on cell survival is thus ruled out. IL-2, when acting as comitogen with Con A, affected only PNA- and cortisone-resistant thymocytes. These cells also showed high intrinsic IL-2 release when stimulated with Con A such that a comitogenic effect of externally added IL-2 was only seen at low cell concentrations. PNA+ thymocytes could neither be induced to release IL-2 nor did these cells become Con A-responsive under the influence of IL-2, thereby excluding an IL-2-mediated maturation.

In previous studies we have observed the production of a cytotoxic factor by the T-lymphocytes in the spleen of dengue type-2 virus-(DV) infected mice which killed normal mouse spleen cells in vitro. In the present study types of spleen cells affected in vitro by the cytotoxic factor (CF) have been investigated. It was observed that it kills most of the macrophages, one-third of T-lymphocytes and a few of the granulocytes, normoblasts and megakaryocytes. It had no effect on B-lymphocytes. The proportion of killed cells could not be significantly enhanced by retreatment of cells by additional CF. The cytotoxic activity of CF is not affected by adsorption with susceptible or non-susceptible cells. CF also kills lymphoid cells of animals of other species, viz. albino rats, rabbits, guinea pigs but had no effect on those of rooster. It had no effect on various cell cultures.

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of 3H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominantly in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.

The effect of three zirconium (Zr) compounds, zirconium oxychloride (ZrOCl2), zirconium oxide (ZrO2) and zirconium silicate (ZrSiO4) on the viability and DNA synthesis of cultured spleen cells from C57BL mice was studied in vitro. The viability of spleen cells was determined by the trypan blue stain method using cells cultured in a CO2 incubator at 37°C for 1-4 days.The degree of DNA synthesis of spleen cells was determined by 3H-thymidine incorporation into spleen cells. The viability of spleen cells treated with ZrOCl2 exhibited similar changes at concentrations of 1-40μM and decreased at concentrations of 80-400μM, as compared with that of control cells.On the other hand, no effects of ZrO2 and ZrSiO4 on the viability of spleen cells were recognized at concentrations of 1-400μM. The degree of 3H-thymidine incorporation into spleen cells treated with ZrO2 or ZrSiO4 was not different from that of control cells.In the case of spleen cells treated with ZrOCl2, it was enhanced at concentrations of 1-20μM and was inhibited at concentrations of 40-400μM as compared with the control cells.The results of these studies in vitro suggest the following:1) The effect of Zr on the cytotoxicity of mouse spleen cells depends upon the solubility of Zr salts.2) ZrOCl2 was found to be weakly mitogenic for lymphocytes at a narrow concentration (1-20μM; max, about 10μM) range.

We have previously reported that mouse spleen and thymus cells in short term culture release an hitherto undescribed complement inhibitor that prevents the activation of C2. Here it is shown that the inhibitor is released by B lymphocytes and may be produced by lymphocytes. Release of the inhibitor takes place at 37 °C and in the presence of metabolic inhibitors, but not at 4 °C. Thus the release of the inhibitor could be related to the shedding of structures located on the lymphocyte membrane.

The biosynthesis of a 12000 dalton protein species released into the medium by mouse spleen was resistant to the inhibitory effect of hypertonic medium on protein syn­ thesis. This protein fraction comprised 10% or more of the total radioactive protein in the medium but could hardly be detected in the spleens. It was labeled very often to a higher extent in diseased animals. It is not related antigenically to either β-microglobin, mouse-interferon, or to mouse immuno-globulins. A preferential uptake into cells of certain organs, which, when themselves incubated in organ cultures did not synthesize and release the protein, was observed.

Normal or increased amounts of series III porphyrins with greater amounts of series I were observed on incubation of PBG in hemolysates of congenital erythropoietic porphyria vs. normal erythrocytes, human or bovine. Correlation with reticulocyte percentage was poor, in the aggregate a general trend toward increased values of both isomers I and III was noted with increasing reticulocytes. When the percent of type III was low the net amount was increased as compared with normal. Hemolysates of non-porphyric, reticulocyte-rich red cells (hemolytic or posthemorrhagic anemia) formed only minute amounts of type I porphyrin but at the same time no more, or even less type III than the porphyric hemolysates, although representing red cells of greater reticulocyte content. No evidence of deficient heme synthesis was observed in porphyric hemolysates incubated with [14 C] -porphobilinogen or 59Fe. Other studies of porphyric hemolysates incubated with and without added mouse spleen synthetase failed to reveal evidence of an absolute UPG-III cosynthetase (Co-S) deficiency. The large increases of type I porphyrin with normal or increased formation of type III, both in the disease and in the hemolysates, are believed due to a primary increase of ALA-S or UPG-S activity rather than a decrease of Co-S. Possible mutations which might be responsible for this increase are considered.

Mouse spleen lymphocytes to which uncleaved human C3 had been attached were interacted with two EBV− lines (RAMOS and BJAB) and six EBV+ sublines (A.W-RAMOS, 11.W.A.RAMOS, EHR.A.RAMOS, EHR.B.RAMOS; GC-BJAB, BJAB/HRIK), which were obtained by superinfection of the EBV− lines with EBV. Depending on the presence of C3, the EBV− sublines except for EHR-A.RAMOS formed rosettes with the mouse spleen lymphocytes. The interaction was inhibitable by DFP. The experiments are interpreted as indicating the presence of proteolytic, C3-cleaving activity on the surface of the EBV+ sublines. The expression of this activity on the EBV+ lines only, correlates it to the transformation of EBV− lines into EBV+ lines.

A comparative study of two BCG vaccines, using one vaccine as the reference preparation, has led to similar conclusions in laboratory tests and in controlled human vaccinations. The results of the multiplication test in the mouse spleen suggest that the detected difference is not merely due to the difference in the number of culturable particles between the vaccines. The importance of using the seed lot system is illustrated. As small differences in some laboratory tests may be indicative of important differences in human vaccinations, precise quantitative assays, using a reference vaccine, are needed.

The inhibitory effect of various beta-lactams on antibody production in the spleen was evaluated in mice immunized with sheep red blood cells by Cunningham's method. With a few exceptions, cephem derivatives were more effective than the penicillins tested in inhibiting the production of antibodies in the spleen of mice. This tendency was especially marked with cefsulodin, cefmenoxime, cefotiam and ceftazidime. However, no effect was obtained with 6-aminopenicillanic acid or 7-aminocephalosporanic acid. Our findings suggest that the difference among various beta-lactams in inhibitory effect on antibody production resulted from the difference in activities of beta-lactams with different side chains at the 6- or 7-positions.

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.

Mouse spleen cells (0.5%) were bound specifically to DNP4-gelatin-coated dishes at 4 degrees C. The DNP-specific spleen cells can be recovered from the dishes by melting the gel at 37 degrees C. In contrast to spleen cells, thymocytes did not bind to DNP-gelatin layers. When dishes were pretreated with large numbers of thymocytes, the binding of splenocytes was reduced in a second adsorption step, depending on the number of thymocytes used for the pretreatment. The reduction of spleen cell binding by pretreatment of dishes with thymocytes could be inhibited by DNP18-rabbit-IgG, but not by ARS13-rabbit-IgG. These data show that, on the release of thymocytes during the incubation with DNP4-gelatin layers, thymocytes leave behind DNP-specific binding material which prevents a second adsorption of DNP-binding spleen cells.

IgG1 induction factor elevates the IgG1 response induced by lipopolysaccharide and suppresses the lipopolysaccharide-induced IgG3 and IgG2b responses in cultures of mouse spleen cells. We have developed new T cell lines secreting this factor by cloning mixed lymphocyte culture populations. Using supernatants of one of these T cell lines it was found that the assay is quantitative, reproducible and accurate, both when induction of IgG1 as well as reduction of IgG3 and IgG2b were measured. Using this analysis, different conditions to induce maximal production of the factor were tested. The cell line was thereafter used as fusion partner with a T cell lymphoma. The hybrids were selected in the presence of T cell growth factor and all of them secreted IgG1 induction factor.

The stimulatory effect of the combination of fetuin and 2-mercaptoethanol on the recovery of plaque-forming cells (PFC) in the absence of serum could be mimicked by the use of reduced alkylated fetuin. A correlation was found between the mitogenic and PFC-stimulating activities of reduced alkylated fetuin.

A relationship between delayed footpad reaction and antibody production was observed in hamsters immunized with erythrocytes of the mouse (MRC), sheep (SRC), or chicken (CRC). (i) In hamsters immunized with MRC in incomplete Freund's adjuvant (IFA), delayed reactions were positive in spite of high titers of IgM. Delayed reactions became negative with the appearance of IgG in hamsters pretreated with mouse spleen cells. (ii) In those immunized with SRC in IFA, positive delayed reactions were elicited only in the absence of IgG. Delayed reactions were converted from negative to positive by treatment with cyclophosphamide before elicitation in the presence of IgG. (iii) After immunization with SRC in complete Freund's adjuvant (CFA) or CRC in IFA or CFA, positive delayed reactions were elicited in the presence of IgG. There may exist an unstable form of delayed footpad reactions, which is regulated by antibody production, and a stable form, which is not regulated. Suppression in the former may be ascribed to some mechanism which is sensitive to cyclophosphamide and may be related to the production of IgG but not IgM.

The effects of erythropoietin and hypoxia on cyclic nucleotide concentrations in erythroid tissue were evaluated. A rabbit bone marrow culture system and a mouse spleen model provided evidence that erythropoietin and an hypoxic stimulus which increases erythropoietin production may enhance erythropoiesis by initiating reciprocal changes in erythroid cell cyclic nucleotide levels. Cyclic GMP appears to be the active signal in mediating the response to erythropoietin, whereas cyclic AMP may be a passive signal allowing full expression of the cyclic GMP response. Whether the type of response mediated by cyclic nucleotides is proliferative, differentiative or both is not clear, but our data and that of other investigators suggest that cyclic GMP mediates the proliferative actions of erythropoietin.

The exis tence of a soluble fac tor s ec r e t ed by lymphoid cel ls a f te r t r e a t m e n t with ant i lymphocyt ic globulin, and with the abil i ty to inhibit the development of exogenous colonies in the spleen of lethal ly i r rad ia ted mice a f te r t ransplanta t ion of syngeneic bone m a r r o w t r ea ted with this fac tor (called s t e m cell inhibition fac tor SCIF) in vi t ro , into them was d i scovered p rev ious ly [2, 3]. It has been shown that SCIF does not affect the d i rec t ion of s t e m cell differentiat ion, for the re la t ive numbers of e ry thro id , myeloid, and megakaryocy t i c foci r ema ined within no rma l l imi t s despi te a genera l reduct ion in the number of colonies [1].

The in vitro anti-trinitrophenyl (TNP) response induced by TNP-lipopolysaccharide (TNP-LPs) in mouse spleen cells was eliminated by passage through Sephadex G10 columns. Conditioned medium obtained from peritoneal macrophages restored the response, indicating the supportive role of such cells in the response. Conversely, polyclonal B-cell activation mediated by LPS was not affected by Sephadex G10 treatment, as judged by incorporation of 3H-thymidine, generation of specific anti-TNP plaque-forming cells, and induction of polyclonal IgM-secreting cells. The failure of the LPS moiety of TNP-LPS to induce B-cell activation without macrophage help suggests a restriction in the number of available anti-LPS receptor molecules when the Ig anti-TNP receptor interacts with the haptenic (TNP) groups. This restriction can be due to the attachment of the TNP molecules to residues located in the vicinity of the lipid A mitogenic region of the carrier.

Injection of 1×108 C57BL/6 mouse spleen cells into CBA mice 1 day before treatment with cyclophosphamide (CP) was shown to promote survival of 2×107 allogeneic or semiallogeneic cells injected later (3–6 h after CP). The criterion of survival was the ability of the donor cells to produce antibodies against sheep's red blood cells in recipients tolerant to this antigen. Injection of 1×108 allogeneic cells 2 days before CP treatment had no protective effect. After intravenous immunization with allogeneic cells, killer cells began to appear in the recipient's spleen as early as on the 2nd day, and their number reached a maximum on the 5th day. The results suggest that CP eliminates the recipient's lymphocytes responding to transplantation antigens, but the killer cells already formed are resistant to the action of CP.

A short term incubation of the mixture of established human T-lymphoid cells (MOLT) and sheep red blood cells (SRBC) resulted in the release of factors which nonspecifically suppressed the response of mouse spleen cells against heterologous erythrocytes in vitro. Neither human B-cell line (RPMI 1788), nor the supernate of MOLT cell suspension in the absence of SRBC had such suppressive effects. The supernate of the mixture of MOLT cells with chicken red blood cells (CRBC) did not suppress either anti-CRBC or anti-SRBC responses of mouse spleen cells. Since CRBC did not form rosettes with MOLT cells, it is suspected that the origin of the production of these factors might be MOLT cells forming SRBC rosettes. Some of these factors are dialysable.

Exogenously added N6, 02'-dibutyryladenosine-3'-5'-cyclic phsophate (DBcAMP) exerted a biphasic effect on the in vitro primary response of mouse spleen cells to sheep erythrocytes (SRBC). High concentrations of DBcAMP were markedly suppressive, whereas lower concentrations brought about significant increases in the peak numbers of antibody-forming cells. The kinetics of the response were not altered by DBcAMP. A direct effect of DBcAMP on bone-marrow derived lymphocytes was suggested because this nucleotide increased the in vitro response of "nude" mouse spleen cells to SRBC. The magnitude of the response, however, was considerably less than that mounted by spleen cells of normal mice. These results show that DBcAMP can have either enhancing or suppressive effects on antibody formation and these effects are dependent upon nucleotide concentration, antigen-dosage and time of assay. The effects of this cyclic nucleotide differed in two major respects from those of the synthetic polyribonucleotide adjuvant, polyadenylic acid-polyuridylic acid (poly A:U) complexes. Thus poly (A:U) decreased the time of peak response in vitro but did not increase the magnitude of the response.

At nontoxic concentrations (1–10 μg/ml) rhodamine B (N-[9-(2-carboxyphenyl)-6-(diethylamino)-3 H-xanthen-3-ylidene]-N-ethylethanaminium chloride; D & C Red No. 19; CAS no. 81-88-9), a low-molecular-weight dye, suppressed the primary in vitro plaque-forming-cell response of BDF1 mouse spleen cells both to the thymus-dependent antigen, sheep red blood cells and to the thymus-independent antigen, E. coli 0127:B8 lipopolysaccharide. Suppression was effective when the D & C Red No. 19 was added at any time up to 48 hr of culture. Rhodamine B at concentrations from 1 to 10 μg/ml significantly suppressed mitogen-induced proliferation of both B- and T-lymphocytes in vitro. BDF1 mice exposed to 50 and 100 ppm dye in their drinking water for 14 days prior to splenectomy gave a depressed in vitro plaque-forming-cell response to sheep red blood cells. In contrast, a significant enhancement of antibody response resulted when mice were exposed to the dye and sheep-red-blood-cell antigen was administered in vivo. Mitogen-induced B- and T-lymphocyte proliferation was also enhanced in mice exposed to the dye for 14 days.

The localization of erythropoietin-responsive cells (ERCs) in the mouse spleen was investigated in vitro. A thin fibrous spleen ‘capsule’ obtained by squashing the organ against a steel mesh was cultured with erythropoietin. The heme synthesis rate in the capsule was compared with that in the spleen fragments from the inner or the outer part of the organ. A far greater heme synthesis was found in the ‘capsule’ than in the other parts of the spleen. On the contrary, the heme synthesis rate in the spleen from anemic mice was higher in the outer part than in the capsule. It is suggested that a larger number of ERCs are located in the ‘capsular’ fragments than in the other portions of the organ. The ERCs may have affinity for the fibrous capsular structure when they are in the undifferentiated stage. The capsular tissue may provide a favorable microenvironment for the differentiation of the ERC.

Natural killing (spontaneous cell-mediated cytotoxicity) by either human blood cells or mouse spleen cells is reversibly inhibited by nordihydroquaiaretic acid or BW755C, agents that antagonize the lipoxygenation of arachidonic acid. Cultured mouse natural killer cells are similarly inhibited. These studies suggest that natural killing may be dependent on the lipoxygenation of arachidonic acid.

The binding of [ 3 H]dexamethasone and the in vitro effects of steroid on [ 3 H]uridine incorporation were determined in cell subpopulations from murine spleen separated by nylon filtration or bovine serum albumin gradient centrifugation. The results showed that the number of glucocorticoid receptors was lower in the T-cell-enriched fractions compared with the fractions depleted in T cells. This difference was not associated with a change in in vitro steroid effects.

Lipopolysaccharide (LPS)-induced plaque-forming cells secreting IgM (IgM-PFC) and antibodies against bromelain-treated mouse erythrocytes (anti-BrMRBC PFC) on Days 1 and 2 of cultures were quantitatively estimated in spleen cells from mice of various ages. The concentrations of the four groups of PFC changed independently with age. The LPS dose dependency of the PFC response was markedly different between PFC on Days 1 and 2, but not different between anti-BrMRBC PFC and IgM-PFC or between 2- and 10-week-old mice. In a second experiment, spleen cells from 2- and 10-week-old mice were separated into subpopulations with or without Fc receptors, C3 receptors, or Ia antigens, and the LPS-induced PFC responses were quantitatively assessed in each subpopulation. Both the receptor-bearing and -lacking populations included LPS-reactive B cells, and the percentages of the LPS-reactive B cells recovered in the receptorbearing population increased with age. However, the percentages of anti-BrMRBC PFC recovered in each receptor-bearing or -lacking population were different from those of IgM-PFC. In Iat populations, the percentages of IgM-PFC on Day 2 were obviously higher than those on Day 1, and both of the percentages increased with age. These results suggest that the four groups of LPS-reactive B cells can be discriminated from each other by their LPS dose dependency and their cell surface markers, and that they develop differently during ontogeny.

Spleens of mice treated with 9-β-D-[2,8-3H]arabinofuranosyladenine (araA), 0.5–30.0 mg/kg showed rapid dose-dependent accumulation of 3H, which peaked at 2.5 mg/kg. Lymphocytes from spleens showed linear uptake when incubated with 0.68, 1.36, and 5.03 μM araA over 120 s. With 1.0 mM araA, uptake was reduced in rate but was not saturated. Tritiated metabolites identified from these lymphocytes were araA which predominated after brief incubations, its deaminated form (araH), and some phosphorylated product in small amount. Inhibition of deaminase increased intracellular araA. Although potent inhibitors of nucleoside transport inhibited araA uptake marginally, adenosine competed with araA for about 50% of the uptake capacity. The data suggest that the uptake of araA by mouse lymphocytes, which is not simple diffusion, occurs by a mechanism distinct from typical nucleoside transport.

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).

Human peripheral lymphocytes and mouse spleen cells were frozen under controlled rates in the presence of polyethylene glycols (PEG) of high polymerization grade, stored in liquid nitrogen and tested, following thawing and mitogen-stimulation, for their capacity to incorporate 3H-thymidine and to form colonies in soft agar. We found that human lymphocytes, frozen with 10% PEG 20,000 and PHA-stimulated afterwards, showed an 8 fold higher incorporation rate versus 7.5% dimethylsulfoxide, whilst colony growth was unaffected. The potentiating effect by PEG was also seen with mouse spleen cells. PHA-prestimulated human lymphocytes also revealed an increased incorporation rate. Due to their lower toxicity the PEG offer, as cryoprotecting agents, a useful alternative to dimethylsulfoxide.

Interferon production in cocultures between lymphoma cells and lymphocytes was previously found to be caused by mycoplasma contamination of the tumor cells. Here we have shown that M. arginini caused interferon production in spleen cells of C57BL/6 mice. The producer cell in spleen cell cultures was insensitive to treatment with anti-theta serum plus complement and it was present in spleen cell cultures of homozygous nude mice. Macrophage depletion did not adversely affect interferon production whereas interferon production was abolished in cultures of cells obtained after passage through nylon wool columns. Collectively, these data suggest, but cannot definitively prove that interferon is produced by B cells. By the use of specific antisera it was found that by all probability the interferon produced represented interferon alpha.

Both cortical and medullary thymocytes could give rise to both allospecific and hapten-specific cytotoxic T lymphocytes (CTLs) if the cultures contained the T-helper-cell-replacing hormone, interleukin 2 (IL-2). The H-2 restriction specificity of these CTLs was tested by using three different specificity determinants that are known to be influenced by the H-2 genotype of the thymus (rather than by the H-2 of the precursor cell itself): the cross-killing of trinitrophenyl (TNP) coupled allogeneic targets in anti-TNP self responses; the dominance of H-2k-restricted CTLs over H-2d-restricted CTLs in the response of (H-2k X H-2d)F1 mice to TNP-self; and the capacity of alloreactive CTLs to cross-reactively lyse TNP-coupled syngeneic cells. The experiments show that, with regard to all of these specificity determinants, CTLs derived from these intrathymic populations are similar to CTLs derived from the adult mouse spleen. These data suggest that the H-2 restriction specificity is dictated before or at the same time that the cell acquires the capacity to respond to allogenic or haptenated syngeneic cells.

12-O-Tetradecanoyl-phorbol-13-acetate, a comitogen for lymphocytes, suppresses the concanavalin A-induced accumulation of 32 P-phosphatidylinositol, in mouse spleen lymphocytes incubated with 32 P-orthophosphate. The comitogenic tumor promoter does not affect the rate of de , novo synthesis of phosphatidylinositol, as measured by [ 3 H]-glycerol incorporation. Mitogen stimulates the incorporation of [ 32 P]-phosphate into phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, but the tumor promoter suppresses only the increased labelling of the phosphatidylinositol, while it enhances phosphorylation of the polyphosphoinositides.