Immunopotentiating effects of the adjuvants SGP and Quil A. I. Antibody responses to T-dependent and T-independent antigens

The present study was undertaken to compare the effects of two adjuvants, SGP (a starch-acrylamide polymer) and Quil A (purified saponin), with that of aluminum hydroxide (Al(OH)3) on murine primary antibody responses to T-independent (TI) and T-dependent (TD) antigens. All three adjuvants augmented the responses to the TD antigens, dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), and sheep erythrocytes (SRBC). SGP was the most potent adjuvant and increased the primary IgG response to DNP-KLH as much as 90-fold. Quil A and Al(OH)3 had comparable effects on the primary response to DNP-KLH, but Quil A was less effective than Al(OH)3 for augmenting the primary response to SRBC. Quil A and SGP both augmented the primary IgM and IgG responses to trinitrophenyl-lipopolysaccharide (TNP-LPS), TNP-Brucella (TI-1 antigens), and TNP-Ficoll (TI-2 antigens). Al(OH)3, like most commonly used adjuvants, had little or no effect on responses to TI antigens. The kinetics of the response to TNP-Ficoll was altered by SGP, since peak responses were maintained for at least 7 days, while the response to TNP-Ficoll alone peaked on Day 4 and had declined considerably by Day 7. Both SGP and Quil A could augment responses to both optimal and suboptimal doses of antigen. The adjuvant activity of SGP was diminished, but still effective, when smaller amounts of SGP were used with the immunizing antigen, and all three adjuvants were able to augment primary responses when given in separate injections from the antigen. These results demonstrate that SGP is a very effective adjuvant, and show that both Quil A and SGP have a unique ability to increase antibody responses to TI antigens, suggesting that their effects may be mediated at least partially through B cells.     

Effects of the adjuvants SGP and Quil A on the induction of experimental autoimmune thyroiditis in mice

In the present study, two adjuvants, SGP and Quil A, were assessed for their ability to induce experimental autoimmune thyroiditis (EAT) in mice. SGP (a synthetic copolymer of starch, acrylamide, and sodium acrylate) and Quil A (a plant saponin) were compared with lipopolysaccharide (LPS) and complete Freund's adjuvant (CFA) given together with mouse thyroglobulin (MTg) for their ability to induce EAT in CBA/J mice. Immunization with MTg and LPS, MTg and CFA, or MTg with SGP was effective in inducing anti-MTg antibodies and histologic EAT, while MTg with Quil A was ineffective in inducing either anti-MTg antibodies or EAT. MTg with LPS was able to prime mice for the development of an in vitro spleen cell proliferative response to MTg while MTg with SGP or with Quil A was unable to prime spleen cells to proliferate detectably in response to MTg. MTg with LPS given in vivo primes CBA/J spleen cells for further activation by in vitro culture with MTg to transfer EAT to naive CBA/J recipients. MTg with SGP was also effective in priming CBA/J spleen cells for in vitro activation and transfer of EAT while MTg with Quil A was ineffective. The effective adjuvant activity of SGP and its lack of toxicity relative to LPS should make it a useful agent for further studies in murine models of EAT.     

Identification of dendritic cell subsets responding to genital infection by Chlamydia muridarum

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum . Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naïve T cells in vivo . In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro . However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro . Our findings show that these two DC subsets likely have different functions in vivo . cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.     

Early programming of T cell populations responding to bacterial infection

The duration of infection and the quantity of Ag presented in vivo are commonly assumed to influence, if not determine, the magnitude of T cell responses. Although the cessation of in vivo T cell expansion coincides with bacterial clearance in mice infected with Listeria monocytogenes, closer analysis suggests that control of T cell expansion and contraction is more complex. In this report, we show that the magnitude and kinetics of Ag-specific T cell responses are determined during the first day of bacterial infection. Expansion of Ag-specific T lymphocyte populations and generation of T cell memory are independent of the duration and severity of in vivo bacterial infection. Our studies indicate that the Ag-specific T cell response to L. monocytogenes is programmed before the peak of the innate inflammatory response and in vivo bacterial replication.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. II. A correction

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of C4 by mouse peritoneal macrophages. II. Comparison of C4 synthesis by resident and elicited cell populations

Five elicited macrophage populations synthesized one-third to one-tenth as much hemolytically active C4 when compared with resident peritoneal macrophages. This decrease in functional C4 activity was not caused by inhibitors or protease activity in the elicited macrophage supernatants. Analysis of C4 antigen indicated a similar reduction in the elicited cells compared with the resident macrophages. A defect in precursor processing or secretion was deemed unlikely because the intracellular C4 precursor was appropriately reduced in the elicited cells. We postulate that mouse resident peritoneal macrophages are a pluripotent cell population with broad capabilities in regard to the initiation of the inflammatory response. In contrast, elicited or activated macrophages may be more specialized cells, with one manifestation of this being the "down" regulation of C4 synthesis.     

Functional activity in vivo of effector T cell populations. II. Anti-tumor activity exhibited by syngeneic anti-MoMULV-specific cytolytic T cell clones

The functional activity in vivo of murine tumor-specific cytolytic T lymphocyte populations and clones was studied. Tumor cell destruction induced after the i.v. injection of cytolytic effector cells was quantitated by monitoring the elimination of 131IUdR-labeled tumor cells in the peritoneal cavity by using whole-body counting techniques. Mixed leukocyte-tumor cell cultures were established by using spleen cells from C57BL/6 regressor mice that had rejected an intramuscular tumor induced by the injection of MSV-MoMuLV virus. This effector cell population was observed to eliminate syngeneic MoMuLV-induced tumor cells in a dose-dependent manner. Treatment of the effector cell population with monoclonal anti-Lyt-2 antibodies plus complement totally abrogated their ability to induce tumor cell destruction in the peritoneal cavity. MSV-MoMuLV-specific Lyt-2+ cytolytic T cell clones derived by micro-manipulation of T lymphocyte-tumor cell conjugates were also tested for functional activity in vivo. Several clones induced a rapid, specific elimination of 131I-labeled MBL-2 tumor cells from the peritoneal cavity after i.v. injection, whereas others were inactive. Both active and inactive clones were highly cytolytic and secreted MAF/IFN-gamma lymphokines. In contrast to previous results obtained in a tumor allograft model, the MSV-MoMuLV-specific cytolytic T cell clones that were active in vivo did not proliferate in vitro in response to stimulation with irradiated tumor cells plus filler spleen cells in the absence of an added source of interleukin 2.     

DNA synthesis in rabbit spleen cell populations stimulated by various doses of Concanavalin A: II. Equilibrium density sedimentation analysis

Autoradiographic studies have revealed that stimulation of rabbit spleen lymphocytes by various ConA doses involves two subpopulations responding successively when ConA concentration rises from 0.5 to 5 μg/ml. Equilibrium density sedimentation analysis of DNA replicated in the presence of BUdR is performed after stimulation by these different ConA concentrations. It is shown that at optimal doses to which each of these subpopulations responds, profiles of sedimentation correspond to a normal replication followed by cellular division and a second round of replication. After stimulation by minimal doses, sedimentation profiles indicate a discontinuous process of replication in which initiating events are numerous, but elongations partially blocked. This pattern suggests that the number of stimuli necessary to produce elongations is higher than that necessary to initiate replication. Comparison with effects produced by drugs like FUdR or HU on replication in continuous cultures suggests also that DNA synthesis in lymphocytes cultures could be regulated by a precise balance between stimulating and inhibiting factors. Finally equilibrium density analysis supports the autoradiographic finding that the duration of the S phase varies with the doses of ConA used. Different problems concerning replication in lymphocytes are discussed.     

Immunopotentiation of the humoral response by liposomes: effect of a T cell polyclonal activator

In this paper, we analyzed the influence of surface-linked Con A on the secondary response to liposome-associated antigen via either encapsulation or covalent linkage at the liposomal surface. The study was carried out on BALB/c mice using bovine serum albumin as antigen. The humoral response was evaluated by measurements of antibody-producing cells (total, IgM, and IgG) and serum antibody titers. The results indicate that Con A at submitogenic concentrations does not potentiate the overall effect of liposomes but drastically changes the isotype distribution pattern obtained in response to encapsulated antigen without however affecting that obtained in response to surface-linked antigen. In all situations where Con A and/or BSA was covalently linked, IgG and IgM isotypes were produced in equal quantity, while in response to encapsulated BSA, IgG was by far the dominant isotype produced as expected for a thymo-dependent antigen. These results suggest that the quality of an immune response and the mechanisms of activation may be profoundly influenced by the nature of antigen association with liposomes as well as by the presence at the liposomal surface of immunomodulators such as Con A.     

Lymphoid cell subpopulations. II. Characterization of cell populations responsible for syngery in the mixed lymphocyte interaction.

Mixtures of isogeneic lymph node cells (LNC) and thymocytes (TC) exhibit far greater responsiveness in the murine MLI, as measured by proliferation and development of cytotoxic effector cells, than either cell type cultured alone. Pretreatment of either lnc or TC with mitomycin-C or ultraviolet irradiation completely abolished their synergistic interaction. Administration of cortisone acetate to cell donors 20 hr before sacrifice reduced the capacity of LNC and enhanced the capacity of TC to synergize. The LNC and TC populations participating in synergy, were found to be thymus dependent. LNC were shown to be responsible for the bulk of proliferative and effector activity observed in synergizing cultures, whereas TC appeared to amplify the activation of LNC. These findings provide the basis for a three cell model of MLI responsiveness.     

Identification of various testicular cell populations in pubertal and adult cockerels

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.     

Distinctions between rabbit lymph node cell populations responding to primary and secondary injections of keyhole limpet hemocyanin

Rabbits were injected once or twice into the hind foot pads with alum-precipitated keyhole limpet hemocyanin. At the height of the primary or secondary responses individual rabbits were sacrificed for the preparation of lymph node cell suspensions from the regional lymph nodes. These cells were employed for the in vitro study of antibody synthesis by the incorporation of ¹⁴C-leucine and the secretion of antibody by the time of appearance of radioactive antibody in the medium. The primary response cells rapidly synthesized and secreted IgM and IgG antibodies. The secondary response cells rapidly synthesized IgM and IgG antibodies, secreted IgG antibody promptly, but secreted the IgM antibody with a lag of six to ten hours. Microsomal fractions could not be prepared from the primary response cells, but were readily produced from the cells of the secondary response. The primary response cells contained mainly free ribosomes, those of the secondary response predominantly membrane-bound ribosomes. It was postulated that IgM antibody was not secreted until it was glyco-sylated in the Golgi apparatus and that the lag in secretion entailed the time for this rate-limiting step to occur.     

Growth regulation of the interstitial cell population in hydra. II. A new mechanism for the homeostatic recovery of reduced interstitial cell populations

The interstitial cells of hydra comprise a stem cell population, producing at least two classes of terminally differentiated cell types, nerve cells and nematocytes. Exposure to hydroxyurea (HU) results in selective depletion of interstitial cells from the tissue. The surviving cells subsequently recovered to normal levels, and the mechanisms involved in this repopulation were examined. Hydra were treated for varying times with HU such that interstitial cell numbers were reduced to 7 or 35% of normal. Subsequent growth of the epithelial and interstitial cell populations in these animals was monitored. The results indicate that the growth rates of these two cell types were only slightly different from untreated controls during the 4 weeks after HU exposure, implying that repopulation should not have occurred. However, recovery of the interstitial cell population was observed. Further analysis revealed that the interstitial cells in HU animals, unlike normal hydra, were not uniformly distributed in the body column, and were especially reduced in the budding region. In normal animals a constant fraction of the interstitial and epithelial cells are lost into buds. However, as a consequence of this nonuniform distribution a smaller fraction of the interstitial cells are displaced into HU buds, thereby retaining a higher proportion in the adult tissue. Calculations indicate that this mechanism of increased retention is of sufficient magnitude to account for 40-60% of the observed recovery after HU treatment.     

Fractionation of Saccharomyces cerevisiae cell populations by centrifugal elutriation.

An exponential population of Saccharomyces cerevisiae cells was fractionated by centrifugal elutriation, using water as the elutriating liquid. Evidence that the population had been fractionated according to age in the cell cycle was obtained by examining the fractions for their size distribution, their microscopic appearance after Giemsa staining, and their ability to initiate synchronous growth.