scholarly journals Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS.

1975 ◽  
Vol 142 (6) ◽  
pp. 1488-1508 ◽  
Author(s):  
B J Skidmore ◽  
D C Morrison ◽  
J M Chiller ◽  
W O Weigle
Keyword(s):  
B Cells ◽  
Mouse Strain ◽  
Structural Integrity ◽  
Mitogenic Activity ◽  
Bacterial Lipopolysaccharide ◽  
Mouse Strains ◽  
Striking Difference ◽  
Spleen Cells ◽  
Destructive Effect

The C3H/HeJ mouse strain, previously shown to be a nonresponder to bacterial lipopolysaccharide (LPS)-induced mitogenesis in vitro, was demonstrated by the present studies to be competent to respond mitogenically to LPS, but only to LPS preparations obtained by selected extraction methods. These preparations appear to be confined to LPS isolated by mild extraction techniques, such as TCA or butanol. In contrast, those obtained by techniques utilizing phenol were only weakly stimulatory or completely nonstimulatory for spleen cells from the C3H/HeJ. All LPS preparations tested, on the other hand, were highly stimulatory for cells from another mouse strain, namely the C3H/St. The critical importance of the method of extraction of LPS on its mitogenic activity for C3H/HeJ cells was stressed by experiments in which LPS was prepared from Escherichia coli K235 using either of two procedures. In these experiments, phenol-extracted LPS, although mitogenic in the C3H/St, was completely nonstimulatory in the C3H/HeJ; whereas, butanol-extracted LPS was highly stimulatory in both strains of mice. This striking difference was attributed to a destructive effect of phenol on LPS, as demonstrated by the fact that treatment of butanol LPS with phenol resulted in a total loss of its mitogenic activity in the C3H/HeJ, but in only a partial loss in the C3H/St. In general, the mitogenic response observed with selected LPS preparations in the C3H/HeJ was quantitatively lower and more transient than that seen with the C3H/St, although qualitatively these responses appeared to be similar. This was evidenced by the observation that in both mouse strains LPS was a specific mitogen for B cells, a property which was also attributed in both strains to the same distinct structural region of the LPS molecule, that is lipid A. A preparation of LPS that failed to stimulate B cells from the C3H/HeJ nonetheless had the capacity to block activation of these B cells by a stimulatory preparation of LPS. These results strongly suggest that mitogenic stimulation of B cells by LPS is a function of the structural integrity of both the LPS molecule and putative B-cell receptors for LPS.

scholarly journals INDUCTION OF A HEMOLYSIN RESPONSE IN VITRO

1971 ◽  
Vol 133 (6) ◽  
pp. 1325-1333 ◽  
Author(s):  
Klaus-Ulrich Hartmann
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Close Contact ◽  
Precursor Cells ◽  
Spleen Cells ◽  
High Concentrations ◽  
Thymus Cells ◽  
Bone Marrow Chimeras

Spleen cells of bone marrow chimeras (B cells) and of irradiated mice injected with thymus cells and heterologous erythrocytes (educated T cells) were mixed and cultured together (17). The number of PFC developing in these cultures was dependent both on the concentration of the B cells and of the educated T cells. In excess of T cells the number of developing PFC is linearly dependent on the number of B cells. At high concentrations of T cells more PFC developed; the increase in the number of PFC was greatest between the 3rd and 4th day of culture. Increased numbers of educated T cells also assisted the development of PFC directed against the erythrocytes. It is concluded that the T cells not only play a role during the triggering of the precursor cells but also during the time of proliferation of the B cells; close contact between B and T cells seems to be needed to allow the positive activity of the T cells.

scholarly journals IMMUNOLOGIC REACTIONS TO HAPTENS ON AUTOLOGOUS CARRIERS

1973 ◽  
Vol 137 (2) ◽  
pp. 411-423 ◽  
Author(s):  
John W. Moorhead ◽  
Curla S. Walters ◽  
Henry N. Claman
Keyword(s):  
T Cells ◽  
B Cells ◽  
Aminocaproic Acid ◽  
Single Amino Acid ◽  
Secondary Response ◽  
Spleen Cells ◽  
Activated T Cells ◽  
Thymus Cells

Both thymus-derived (T) and bone marrow-derived (B) lymphocytes participate in the response to a hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), coupled to a nonimmunogenic isologous carrier, mouse gamma globulin (MGG). Spleen cells from mice immunized with NIP-MGG show increased DNA synthesis in vitro when cultured with NIP-MGG. The participation of and requirement for T cells in the response was demonstrated by treating the spleen cells with anti-θ serum. This treatment resulted in a 77% inhibition of the antigen response. Furthermore, adoptively transferred normal thymus cells could be specifically "activated" by NIP-MGG in vivo and they responded secondarily to the antigen in vitro. The active participation of B cells in the secondary response was demonstrated by passing the immune spleen cells through a column coated with polyvalent anti-MGG serum. Column filtration reduced the number of NIP-specific plaque-forming cells and NIP-specific rosette-forming cells (both functions of B cells) and produced a 47% inhibition of the NIP-MGG response. The ability of the cells to respond to phytohemagglutinin (PHA) was not affected by column filtration showing that T cells were not being selectively removed. The participation of B cells in the in vitro NIP-MGG response was also shown by treatment of the spleen cells with antiserum specific for MGG and MGG determinants. B cells were removed by treatment with anti-IgM or polyvalent anti-MGG serum plus complement, resulting in a respective 46 and 49% inhibition of the response to NIP-MGG. (Treatment with anti-IgM serum had no effect on T cells.) The contribution of the hapten NIP to stimulation of T cells was investigated using NIP-MGG-activated thymus cells. These activated T cells responded in vitro very well to the NIP-MGG complex but not to the MGG carrier alone demonstrating the requirement of the hapten for T cell stimulation. The response was also partially inhibited (41%) by incubating the activated cells with NIP coupled to a single amino acid (epsilon-aminocaproic acid) before addition of NIP-MGG. These results demonstrated that T cells recognize the hapten NIP when it is coupled to the isologous carrier MGG.

scholarly journals Lack of mature B cells in nude mice with X-linked immune deficiency.

1982 ◽  
Vol 155 (3) ◽  
pp. 903-913 ◽  
Author(s):  
H H Wortis ◽  
L Burkly ◽  
D Hughes ◽  
S Roschelle ◽  
G Waneck
Keyword(s):  
Immune Deficiency ◽  
B Cells ◽  
B Cell ◽  
Nude Mice ◽  
Normal Serum ◽  
The Other ◽  
Spleen Cells ◽  
Virtual Absence ◽  
Immunoglobulin Levels

Mice were bred that simultaneously expressed the mutations nude and x-linked immune deficiency (xid). These doubly deficient animals had less than 10% of normal serum immunoglobulin levels. Their spleen cells did not respond to thymus-independent antigens in vitro nor did they respond to lipopolysaccharide. There was a virtual absence of cells with surface mu, kappa, or lambda 1, as detected by fluorescence. Sections of lymphoid organs revealed an absence of primary B cell follicles. Taken together, these results indicate a lack of mature B cells in nude xid mice. The possibility is considered that mature B cells belong to two subpopulations representing two lineages, one controlled by alleles at the xid locus and the other by alleles at the nude locus.

scholarly journals MATURATION OF THE HUMORAL IMMUNE RESPONSE IN MICE

1974 ◽  
Vol 139 (2) ◽  
pp. 249-263 ◽  
Author(s):  
Patricia G. Spear ◽  
Gerald M. Edelman
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Function ◽  
Spleen Cells ◽  
Con A ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Humoral Antibodies

In spite of the prenatal appearance of immunoglobulin-bearing lymphocytes and θ-positive lymphocytes in the spleens of Swiss-L mice, these mice are not able to produce detectable levels of humoral antibodies in response to antigen until after 1 wk of age. Adult levels of response are not achieved until 4–8 wk of age. In the presence of bacterial lipopolysaccharides, which can substitute for or enhance T-cell function, the B cells from young Swiss-L mice were found to be indistinguishable in function from adult B cells, both with respect to the numbers of plaque-forming cells (PFC) produced in vitro in response to antigen and with respect to the kinetics of PFC induction. The spleen cells from young Swiss-L mice are significantly less sensitive than adult spleen cells, however, to stimulation by the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA). Very few Con A-responsive cells could be detected at birth but the numbers increased sharply with age until 3 wk after birth. On the other hand, PHA-responsive cells could not be detected in the spleen until about 3 wk of age. The latter cells were found to respond also to Con A, but at a lower dose (1 µg/ml) than that required for the bulk of the Con A-responsive cells (3 µg/ml). The cells that respond both to PHA and to Con A appear in the spleen at about the time that Swiss-L mice acquire the ability to produce humoral antibodies, and these cells can be depleted from the spleen by the in vivo administration of antithymocyte serum. The development of humoral immune responses in these mice therefore appears to be correlated with the appearance of recirculating T lymphocytes that are responsive both to PHA and to Con A.

scholarly journals In vitro tolerance induction of neonatal murine B cells.

1976 ◽  
Vol 143 (6) ◽  
pp. 1327-1340 ◽  
Author(s):  
E S Metcalf ◽  
N R Klinman
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Specific Interaction ◽  
Tolerance Induction ◽  
Antigen Receptors ◽  
Spleen Cells ◽  
Protein Conjugates ◽  
Immature B Cells

The susceptibility of neonatal and adult B lymphocytes to tolerance induction was analyzed by a modification of the in vitro splenic focus technique. This technique permits stimulation of individual hapten-specific clonal precursor cells from both neonatal and adult donors. Neonatal or adult BALB/c spleen cells were adoptively transferred into irradiated, syngeneic, adult recipients which had been carrier-primed to hemocyanin (Hy), thus maximizing stimulation to the hapten 2,4-dinitrophenyl coupled by Hy (DNP-Hy). Cultures were initially treated with DNP on several heterologous (non-Hy) carriers and subsequently stimulated with DNP-Hy. Whereas the responsiveness of adult B cells was not diminished by pretreatment with any DNP conjugate, the majority of the neonatal B-cell response was abolished by in vitro culture with all of the DNP-protein conjugates. During the 1st wk of life, the ability to tolerize neonatal splenic B cells progressively decreased. Thus, tolerance in this system is: (a) restricted to B cells early in development; (b) established by both tolerogens and immunogens; (c) achieved at low (10(-9) M determinant) antigen concentrations; and (d) highly specific, discriminating between DNP- and TNP-specific B cells. We conclude that: (a) B lymphocytes, during their development, mature through a stage in which they are extremely susceptible to tolerogenesis; (b) the specific interaction of B-cell antigen receptors with multivalent antigens, while irrelevant to mature B cells, is tolerogenic to neonatal (immature) B cells unless antigen is concomitantly recognized by primed T cells; and (c) differences in the susceptibility of immature and mature B lymphocytes to tolerance induction suggest intrinsic differences between neonatal and adult B cells and may provide a physiologically relevant model for the study of tolerance to self-antigens.

scholarly journals The "Ly-1 B" cell subpopulation in normal immunodefective, and autoimmune mice.

1983 ◽  
Vol 157 (1) ◽  
pp. 202-218 ◽  
Author(s):  
K Hayakawa ◽  
R R Hardy ◽  
D R Parks ◽  
L A Herzenberg
Keyword(s):  
B Cells ◽  
Normal Mouse ◽  
Cell Subpopulation ◽  
Mouse Strains ◽  
Cell Surface Molecules ◽  
Spleen Cells ◽  
Culture Studies ◽  
Surface Molecules ◽  
Old Mice ◽  
Small Subpopulation

A small subpopulation of normal murine splenic B cells carrying all of the classic B cells markers (IgM, IgD, Ia, and ThB) also carries Ly-1, one of the major T cell surface molecules. This "Ly-1 B" subpopulation (identified and characterized by multiparameter FACS analyses) consists of relatively large, high IgM/low-IgD/low-Ly-1 lymphocytes that represent approximately 2% of the spleen cells in normal animals and, generally, 5-10% of spleen cells in NZB mice. Ly-1 B are clearly detectable in all normal mouse strains tested as well as NZB, CBA/N, other X-id mice and nude (nu/nu) mice. They are found primarily in the spleen; are either absent or very poorly represented in lymph node, bone marrow, and thymus; appear early during ontogeny, and comprise about a third of the small number of lymphocytes present in 5-d-old mice. NZB and (NZB x NZW)F1 mice have more Ly-1 B than all other strains and, furthermore, have a unique Ly-1 B population that secretes IgM when cultured under usual conditions in the absence of added antigen. The IgM secretion by these Ly-1 B cells accounts for the previously reported "spontaneous" IgM secretion by NZB spleen cells in culture. Studies with FACS-sorted cells show that the presence of Ly-1 on these IgM-secreting cells distinguishes them from the (Ly-1 negative) IgM-secreting "direct" plaque-forming cells generated in NZB mice after stimulation with sheep erythrocytes.

scholarly journals Isotypic and allotypic specificity of mouse rheumatoid factors.

1983 ◽  
Vol 157 (3) ◽  
pp. 1006-1019 ◽  
Author(s):  
J L Van Snick ◽  
V Stassin ◽  
B de Lestré
Keyword(s):  
Normal Mouse ◽  
Mouse Strains ◽  
Igg Subclass ◽  
Rheumatoid Factors ◽  
Spleen Cells ◽  
Polyclonal Activation ◽  
Old Mice ◽  
Reactive Antibody

The specificity of polyclonal mouse rheumatoid factors (RF) was analyzed by competition experiments with heat-aggregated mouse IgG subclasses. The RF spontaneously produced by three normal mouse strains (129/Sv, CBA/Ht, and C57Bl/6) and by two strains with autoimmune diseases (MRL/l and NZB) were found to consist of distinct non-cross-reactive antibody subpopulations each specific for one IgG subclass. The sera of the normal strains contained IgG1- and IgG2a-specific RF. The autoimmune strains produced an additional variety of RF that was specific for The autoimmune strains produced an additional variety of RF that was specific for IgG2b. Also, the RF secreted by spleen cells of various normal strains after in vitro polyclonal activation with lipopolysaccharide could be resolved into distinct subpopulations specific for IgG1 or IgG2a. These results were confirmed by the analysis of monoclonal RF derived from BALB/c, C57Bl/6, CBA/Ht, and 129/Sv mice: of 73 hybridomas with RF activity, 71 displayed a strict subclass specificity. The subclass predominantly recognized depended on the origin of the spleen cells used to generate the hybridomas. After polyclonal activation in vitro, a broad spectrum of different specificities was obtained with 16 RF specific for IgG1, 13 for IgG2a, and 4 for IgG2b. In contrast, 27 of 28 monoclonal RF derived from 129/Sv and BALB/c mice without prior polyclonal activation were specific for IgG2a, and of these 75% were allotype specific since they failed to react with IgG2a of the b allotype. These results demonstrate the importance of subclass specificity in the production of RF in vivo. With the exception of the IgG2b-specific clones, all these monoclonal RF reacted preferentially with heat-aggregated or antigen-bound IgG. Among the hybridomas generated by the fusion of in vitro polyclonally activated spleen cells of 4-wk-old mice, the frequency of clones with RF activity was at least 40 times higher than that of clones specific for mouse IgM, human IgG, ovalbumin, and hen lysozyme.

scholarly journals INDUCTION OF IMMUNITY AND TOLERANCE IN VITRO BY HAPTEN PROTEIN CONJUGATES

1972 ◽  
Vol 135 (4) ◽  
pp. 735-753 ◽  
Author(s):  
Marc Feldmann
Keyword(s):  
B Cells ◽  
Inhibitory Effect ◽  
Spleen Cells ◽  
Protein Conjugates ◽  
Normal Spleen ◽  
High Concentrations ◽  
Induced Tolerance ◽  
Thymus Cells ◽  
Relationship Of

Of many dinitrophenylated (DNP) protein conjugates tested, only DNP conjugated to polymerized flagellin (DNP-POL) (or the structurally related bacterial flagella) elicited a primary anti-DNP response in vitro. Other DNP proteins, such as DNP-monomeric flagellin (DNP-MON), were capable of inducing secondary responses in vitro. The capacity of DNP-POL to immunize spleen cell suspensions devoid of thymus-derived cells was the reason for the greater immunogenicity of DNP-POL, since even large numbers of flagellin-reactive activated thymus cells did not increase the anti-DNP response of normal spleen cells immunized with DNP-POL, whereas the thymus-dependent response to DNP-MON was markedly increased. The capacity of various batches of DNP-POL to immunize normal spleen cells in vitro varied markedly, depending on the number of DNP groups conjugated. DNP-POL with few DNP groups conjugated was immunogenic, but even at very high concentrations did not induce tolerance. In contrast, highly conjugated DNP-POL did not immunize, but readily induced tolerance. DNP-POL with intermediate degrees of conjugation were, like unconjugated polymeric flagellin, capable of inducing both immunity and tolerance. Since DNP-POL immunizes bone marrow-derived lymphocytes (B cells) directly the reduced response was not due to a masking of carrier determinants, necessary for cell collaboration. By using mixed DNP-5-(dimethylamino)-1-naphthalyl (dansyl)-POL conjugates, it was found that the inhibitory effect of a high degree of hapten conjugated was hapten specific. Depolymerization of DNP-POL to DNP-MON, which does not induce primary anti-DNP responses, was excluded by centrifugation analysis and electron microscopy. The relationship of the degree of hapten conjugation on DNP-POL to the capacity to induce tolerance and immunity in B cells has clarified the mechanism of immunological triggering of these cells. A model of the mechanism of "signal" discrimination between immunity and tolerance in B cells, based on these findings, is proposed.

scholarly journals SELECTIVE ROLES OF THYMUS-DERIVED LYMPHOCYTES IN THE ANTIBODY RESPONSE

1974 ◽  
Vol 140 (1) ◽  
pp. 239-252 ◽  
Author(s):  
Tomio Tada ◽  
Toshitada Takemori
Keyword(s):  
T Cells ◽  
B Cells ◽  
Antibody Response ◽  
Suppressive Effect ◽  
Antibody Responses ◽  
Cell Transfer ◽  
Spleen Cells ◽  
Igg Antibody ◽  
Igm Antibody

Passively transferred thymocytes and spleen cells from donors primed with keyhole limpet hemocyanin (KLH) exerted differential suppressive effect on IgM and IgG antibody responses of syngeneic recipients immunized with DNP-KLH depending primarily on the time when KLH-primed cells were transferred. This was demonstrated by the decrease in the numbers of DNP-specific direct and indirect PFC in the spleen of the recipients given KLH-primed cells at different times during primary and secondary immunization. Whereas the cell transfer simultaneously with or 2 days after the primary immunization produced only slight suppression of the peak IgM antibody response, it caused profound suppression of late IgM and IgG antibody responses. By contrast, the cell transfer 3 days after the immunization produced immediate suppression of the ongoing IgM antibody response resulting in its earlier termination, while being unable to prevent the induction of IgG antibody response. KLH-primed cells could moderately suppress the secondary anti-DNP antibody response, in which IgG antibody response was found to be slightly more sensitive than IgM antibody response to the suppressive influence of KLH-primed cells. The suppressive effect of the KLH-primed spleen cells was completely eliminated by the in vitro treatment of the cells with anti-θ and C before cell transfer, indicating that cells responsible for the suppression are, in fact, T cells. The suppression of DNP-specific antibody response by KLH-primed T cells was achieved only if the recipients were immunized with DNP-KLH but not with DNP-heterologous carrier, suggesting that direct interaction between T and B cells is necessary for the suppression of the antibody response. It is concluded that susceptibility of B cells to the specific suppressive influence of T cells is inherently different depending on the differentiation stage of B cells and on the immunoglobulin class they are destined to produce.

scholarly journals Regulation of T-cell-mediated lympholysis by the murine major histocompatibility complex. I. Preferential in vitro responses to trinitrophenyl-modified self K- and D-coded gene products in parental and F1 hybrid mouse strains.

1979 ◽  
Vol 149 (6) ◽  
pp. 1379-1392 ◽  
Author(s):  
R B Levy ◽  
G M Shearer
Keyword(s):  
T Cell ◽  
T Cell Response ◽  
Mouse Strains ◽  
Response Patterns ◽  
Spleen Cells ◽  
F1 Hybrid ◽  
Ctl Response ◽  
Histocompatibility Complex ◽  
D Region

Spleen cells from H-2b,k,d C57Bl/10 congenic mice were sensitized in vitro to trinitrobenzenesulfonate (TNBS)-modified autologous spleen cells. Cold target competition studies at the lytic phase demonstrated three distinct patterns of cytotoxic responsiveness: (a) H-2b spleen cells generated approximately equivalent CTL responses against Kb and Db modified self products, (b) H-2d spleen cells generated preferential responses against Dd modified self products, and (c) H-2k spleen cells generated cytotoxic responses which could only be detected against Kk self products in association with TNP. F1 spleen cells were sensitized against autologous TNBS-treated cells. The results showed that, although H-2b parental cells generated approximately equivalent Kb-TNP- and Db-TNP-specific CTL, the presence of the H-2b haplotype did not result in the generation of (a) Dk-TNP CTL response by (H-2b x H-2k) spleen cells, nor (b) a Db CTL response by (H-2b x H-2a) F1 spleen cells. Additionally, (H-2d x H-2k) F1 cells failed to generate detectable Dd-TNP-specific CTL, although H-2d parental cells generated D-regional-specific CTL. The findings demonstrated that these F1 response patterns paralleled those of the H-2k and H-2a parents, i.e. weak or no D-region TNP-specific CTL were induced. Because (H-2d x H-2a) F1 responders stimulated with H-2d TNBS-treated cells did generate good Dd TNP responses, the results illustrated that the presence of responder genes was not sufficient to result in a D-region TNP CML. It is suggested that the absence of Kk alleles on the stimulating population is necessary for the generation of D-region TNP CTL in these F1's. Mechanisms which could account for these response patterns in parental F1 mice are discussed including immunodominance, suppression, T-cell response , and Ir-gene defects.

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