Antibody response to phosphorylcholine in vitro. II. Analysis of T-dependent and T-independent responses.

Spleen cells from BALB/c mice primed with keyhole limpet hemocyanin (KLH), were stimulated with heat-killed vaccine of rough Pneumococcus pneumoniae R36A (Pn) and/or phosphorylcholine (PC)-coupled KLH to induce an anti-PC response in vitro. The response to PC-KLH was found to be T-dependent while it is T-independent to Pn. The antibodies induced with either antigen had similar avidity and expressed the TEPC 15 idiotype exclusively; thus T cell involvement in the response to PC-KLH failed to alter these parameters of the anti-PC response. At the precursor cell level, Pn induced small clones with an average size of 10 plaque-forming cells (PFC), whereas PC-KLH gave rise to larger clones of 40-50 PFC. This difference in the proliferative potential of PC precursor B cells hinted at the possibility that Pn and PC-KLH were stimulating different precursors.This was corroborated by the observation that a) when Pn and PC-KLH were added to the same cultures a synergistic effect was seen, i.e. the number of plaques was greater than the sum of the responses induced by each antigen, and b) in microcultures, under conditions limiting B cells only, Pn plus PC-KLH induced a higher fraction of responding wells than either antigen on its own. We postulate that Pn and PC-KLH stimulate subpopulations of PC precursor cells which are T-independent and T-dependent, respectively.     

Impaired antibody response against T-dependent antigens in rhino mice.

The antibody response in rhino mice, which carry a mutant gene hrrh, to thymus-dependent (TD) or thymus-independent (TI) antigens was compared with that of phenotypically normal littermates. The magnitude of antibody response to TD antigens in rhino mice decreased as they grew up, whereas the antibody response to TI antigens in rhino mice was indistinguishable from that of littermates. A transfer of thymus cells from littermates to rhino mice resulted in the partial restoration of the responsiveness to TD antigens. The experiments employing adoptive transfer of spleen cells from rhino mice to the irradiated normal mice suggested that the hyporesponsiveness of TD antigens of adult rhino mice was mainly due to the defect in the T helper cell activities rather than either the increase of the suppressor cells or defects in other cell types.     

Effects of a crystalline silica on antibody production to T-dependent and T-independent antigens in Balb/c mice

A crystalline silica (standard quartz DQ12 with particle size less than 5 micrometers) is able to stimulate in Balb/c mice the production of antibodies of various isotypes to the T-dependent antigen trinitrophenylated keyhole limpet hemocyanin. Under the same experimental conditions silica was not able to stimulate antibody response to the T-independent antigen trinitrophenylated Ficoll. These results support the possibility that adjuvanticity of silica is correlated to its effects on one or more of the various functions carried out by macrophages on immune responses.     

Effect of saikosaponin derivatives upon the immune response against T-dependent and T-independent antigens in mice

Effect of water soluble extracts and purified derivatives from the root of the plant Bupletum falcatum L. upon the immune response of BALB/c mice was investigated using heterologous erythrocytes and bacterial lipopolysaccharide. This Chinese Medicine is famous for its reputed anti-inflammatory, anti-tumor and other biological activities, and has been widely used alone or in a complexed form since ancient times in Oriental Countries. The effect on immune responsiveness of this drug was judged by the measurement of antibody secreting cells causing localized hemolysis in agar gel. The purified derivatives, Saikosaponin (SS) a and d, suppressed anti-SRBC plaque-forming cells (PFC), but the other derivatives, b1 and b2 and c, did not. Moreover, derivatives a and d also enhanced anti-LPS PFC. From experiments establishing minimum effective doses for anti-SRBC PFC in mice, one mg/kg was optimum for BALB/c mice. The effect of the drug on antibody formation against heterologous erythrocytes was nonspecific because treated mice had greatly depressed PFC responses against both sheep and rabbit erythrocytes. The phenotypic effect of this drug as judged by PFC against T-dependent or T-independent antigen was not identified. However, it was suggested that the purified derivatives SSa and d stimulated both T- and B-cells. The possible mechanism of effect against immunocompotent cells is discussed.     

Initiation of humoral immunity. II. The effects of T-independent and T-dependent antigens on the distribution of lymphocyte populations

The effect of injecting T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens on the redistribution of lymphocyte populations in immature male chickens was investigated. In the blood, percentages of total T-cells (CD3+), T-helper cells (CD4+), and T-cytotoxic/suppressor cells (CD8+) significantly decreased post-LPS injection (PLI) but not post-BSA injection (PBI), while percentages of monocytes/thrombocytes (K1+) significantly increased PLI. Interleukin-1 receptor expression on blood lymphocytes increased significantly PLI and PBI. In the spleen, the percentages of total T-cells (CD3+) increased significantly PLI and PBI, macrophage (K1+) percentages increased significantly PLI, while B-cell percentages decreased significantly PLI. These results indicate that following antigen injection, there is a redistribution of peripheral blood lymphocytes (specifically T-lymphocytes) to secondary lymphoid organs and the kinetics and magnitude of the changes can differ according to the type of antigen used.     

Age-dependent changes of isotype and idiotype expression in the antibody response to the phosphorylcholine hapten in BALB/c mice

The distribution of antibodies among different isotypes in an immune response to a given antigen reflects the immunoregulatory linkage of T and B cell compartments, the genetic background of an individual and the functional state of its immune system. In addition, idiotypes are markers for the clonal origin of antibodies and their genetic relationship. Therefore, we have analyzed the isotype patterns and development of the major idiotype (T15) in BALB/c mice of different ages, immunized with a T-cell-dependent phosphorylcholine conjugate. The response was dominated by mu, gamma 3 and gamma 1 isotypes. The proportion of these antibodies changed in the progress of immunization but not with age in the primary response. An age-dependent change of the isotype distribution was observed only in the memory response. The T15 idiotype was dominantly expressed in the primary response and decreased in the memory response by 80-95% independently of the age of the mice. The results demonstrate that 2 populations of anti-phosphorylcholine antibodies which both prefer particular isotypes are expressed according to the functional state of the T and B cell compartments with age.     

Antibody response elicited by T-dependent and T-independent antigens in gene targeted {kappa}-deficient mice

Animal models substantially contribute to the understandingof the pathogenesis of various human diseases, including thoseassociated with genetic defects. Our study investigated thecharacteristics of antibody responses elicited by T-dependentand T-independent antigens in mice rendered k-deficterrt bytargeted deletion of the J_kC_k gene segments. It is known thatin normal murine species the k repertoire dominates the antibodyrepertoire (k/ratio = 95:5). Our results indicate that the kgene deletion causes the alternative usage of 1 (93%) and 2(7%) light chains, confirming previous studies demonstratingthat in k-deficlent mice all B cells express IG receptors. Theanti-trinitrophenylbenzene (TUP) response in K–/–mice was compensated for by 1 and 2 bearing Igs. However, isoelectricfocusing analysis of anti-TNP antibodies showed a considerablymore restricted pattern of anti-TNP antibodies in K–/–as compared with antibodies in normal mice. No major differenceswere observed in the affinity for the hapten of or1 or 2 mAbsobtained from 129/Sv and K–/– mice. Furthermore,1 and 2 chains can reconstitute the expression of an Idiotype(460ld) borne on anti-TNP antibodies. The 460ld was detectedboth in polyclonal and monoclonal anti-TNP antibodies obtainedfrom K–/– mice. Our results clearly showed thatthe anti-TNP repertoire is compensated by the repertoire eventhough the latter is clonally restricted in K–/–mice.     

Cell preparation in immune response by immune ribonucleic acid. II. Independence of T lymphocytes in immune response against T-independent antigens.

The immune response of congenitally athymic (nude) mice induced by immune ribonucleic acid (iRNA) to lipopolysaccharides of Escherichia coli 0-55 (LPS) was studied. The thymus-independent nature of the immune response of mice to LPS was confirmed and nude mice responded to LPS in a manner similar to normal mice. An iRNA preparation extracted from the spleen of nude mice immunized with LPS could induce the proliferation of rosette-forming cells (RFC) in nude mice. iRNA preparations were insensitive to treatment with deoxyribonuclease and pronase, but were inactivated by ribonuclease treatment. The active fraction of the iRNA preparation had sedimentation values in a sucrose density gradient between 7 and 16 S and comprised only a small fraction of the total RNA present in the spleen cells, thereby indicating that the active moiety was one or more species of RNA. The anamnestic response was induced by treatment with iRNA made from the spleen of nude mice immunized with LPS. An increase in the number of rosette-forming cells (RFC), plaque forming cells (PFC) and formation of humoral antibody to LPS was seen after injection with a small amount of LPS 4 weeks after iRNA treatment.     

Clonal regulation in the response to phosphorylcholine. II. Heterogeneity among T15 idiotype-positive antibodies in inbred and wild mice

Inbred stains of mice routinely respond to phosphorylcholine (PC)-containing antigens, S. pneumoniae and PC-keyhole limpet hemocyanin, with the production of three major families of antibodies. Two of these groups, the M 511 and M 603 idiotype (Id) families, are clearly heterogeneous, whereas the third, T15, normally exhibits much less heterogeneity and appears to be monoclonal in many strains. We report here on the occurrence of additional T15 Id⁺ antibodies which can be detected readily by isoelectric focusing. These antibodies have been observed in 9 of 12 strains examined at an average frequency of 10%. They occur in both the IgG₁ and IgG₃ isotypes. Collectively, the antibodies comprise a heterogeneous group although cofocusing antibodies were observed in individuals of a strain or between strains. Two H-2-congenic strains, BALB.B and BALB.G, regularly displayed multiple cofocusing T15 Id⁺ antibodies in each individual. Idiotypic analysis of these antibodies in all strains showed that each contained H, L and H-L-specific variable region determinants common to T15. A surprising finding was the consistent expression of a heterogeneous T15 Id response in two wild Mus musculus lines. Most of these antibodies cofocused with T15 Id⁺ antibodies in BALB.G or C3H as well as contained strain-specific idiotypic determinants found among inbred mice. These data demonstrate that BALB/c and other inbred strains possess the genetic potential to generate a family of closely related T15 Id⁺ antibodies. The results further suggest that the restricted response usually seen among inbred strains is not normal and exists because of a highly selective regulatory system.     

Comparison of the immune response potential of newborn mice to T-dependent and T-independent synthetic polypeptides.

Newborn mice do not, in general, produce antibodies during the 1st week of life; this inability to respond immunologically has been attributed to lack of functional macrophages and T cells. To determine whether B cells of newborn mice are functionally mature and therefore capable of producing antibodies to thymus (T) independent antigens, the response of 1-9-day-old C3H/HeJ mice injected with a thymus-independent polypeptide, poly(DTyr,DGlu)-polyDPro- -polyDLys was compared to that of their littermates injected with a thymus-dependent immunogen, poly(LTyr,LGlu)-polyLPro- -polyLLys. No antibodies were detected in 1- or 2-day-old mice immunized with the T-dependent antigen, as revealed by haemagglutination and haemolytic plaque-forming cell assays, performed 6 days after injection of the antigen. Injection of 3-day-old animals with the thymus-dependent immunogen resulted in significant immune responses which increased with age. In contrast, 1- and 2-day-old mice responded to the T-independent immunogen with high antibody levels, however, in 3-day-old injected mice, the levels were lower. When 3-day-old nude mice were injected with this antigen, no decrease in the immune response was observed. Thus, newborn mice respond immunologically to a thymus-independent antigen injected at the first 2 days after birth and the antibody levels decrease with maturation of the thymus.     

Deficiency of antibody responses to T-independent antigens in gerbils---Meriones unguiculatus

Meriones unguiculatus commonly known as gerbils are widely used as animal models for a variety of parasitic infections such as Brugia malayi, Entamoeba histolytica, Giardia duodenalis, Toxoplasma gondi, Helicobacter pylori, Strongyloides stercoralis and Echinococcus multilocularis. Groups of BALB/c mice, gerbils and XID mice were studied for antibody responses to T-independent antigens. Gerbils were found to be significantly deficient in eliciting antibodies to both dextran and phosphorylcholine (PC) in comparison to BALB/c mice. The antibody response of gerbils to T-independent antigens was found to be similar to the response observed in Bruton's tyrosine kinase (Btk) deficient XID mice, which are known to be poor responders to T-independent antigens. Similar to XID mice, normal gerbil sera were found to be deficient in naturally occurring antibodies to single stranded DNA (SS-DNA), lipopolysaccharide (LPS) and phospholipids. This raises the possibility of a deficiency of CD5+ B-lymphocytes (also known as B-1 cells) in gerbils, since deficiency of this sub-population of B-lymphocytes has been attributed to the absence of such naturally occurring antibodies in XID mice. These results indicate the need to study immunogenicity of parasite T-independent antigens and their relationship to protective immunity in parasitic infections in gerbils.     

Seminal plasma inhibits lymphocyte response to T-dependent and -independent antigens in vitro.

The effect of seminal plasma, epididymal spermatozoa, or whole semen on antibody producing cells was examined in vitro after (i) direct culture with spleen or B cells, and (ii) cocultivation of B cells with T cells previously incubated with seminal plasma. Seminal plasma, and not epididymal spermatozoa, have an inhibitory effect on the direct hemolytic plague forming cell response. This was mediated by a direct inhibitory effect on the B cell and not through the generation of suppressor T cells as demonstrated by responses to T-independent and -dependent antigens. Thus, the mode of suppressive action of seminal plasma in vitro is probably different from that previously reported in vivo.     

B-cell subsets responsive to fluorescein-conjugated antigens. III. Differential effect of E. Coli lipopolysaccharide on T-dependent and T-independent responses in vivo.

The effect of E. coli lipopolysaccharide (LPS) on the induction of both hapten-specific immunity and tolerance was studied in an in vivo system utilizing putative T-cell dependent (TD) or T-cell independent (TI) challenge antigens. The administration of LPS 1 day prior to challenge preempted the response of C3D2 mice to a TD antigen (FL-KLH) but had little effect on the response to FL-Ficoll, a TI antigen. LPS did not affect the responsiveness of C3H/HeJ mice, an LPS-unresponsive strain, to either antigen. The reduction of the response to a putative T-dependent antigen by LPS pre-treatment was only temporary since mice challenged 7 days after LPS responded normally in vivo. We also confirmed that LPS administered shortly after a tolerogen prevented FL-specific IgG tolerence induction and produced B-cell priming to a subsequent T-dependent antigenic challenge. LPS, however, did not significantly interfere with tolerance induction in terms of the IgM responce to either challenge antigen. These results suggest that LPS acts either directly or indirectly on a subpopulation of B cells responsive to a TD antigen. Our data further reflect the heterogeneity of B-cell subpopulations responsive to various polyclonal activators.     

Mechanism of lipopolysaccharide-induced immunosuppression: immunological activity of B cell subsets responding to T-dependent or T-independent antigens in lipopolysaccharide-preinjected mice.

Spleen cells from mice preinjected with high doses of bacterial lipopolysaccharide did not generate anti-trinitrophenyl (TNP) plaque-forming cells in vitro to the T-dependent antigen, TNP-sheep erythrocytes, but did generate fully plaque-forming cells to the T-independent antigens, TNP-Ficoll and TNP-Brucella abortus. The immunological activity of B cells from such lipopolysaccharide-preinjected mice was analyzed in the present study. T cell-depleted spleen cells from mice injected with 30 micrograms of lipopolysaccharide 3 days previously did not respond to combined stimulation with TNP-sheep erythrocytes and concanavalin A-induced T cell-replacing factor and had no suppressive activity on normal T cell-depleted spleen cells. Splenic B cells, which were separated from T cells and macrophages from mice injected with 30 micrograms of lipopolysaccharide 3 days previously, responded only partially (about 25% of the control response) to combined stimulation with TNP-sheep erythrocytes and concanavalin A-induced cell-replacing factor in the presence of normal macrophages, but responded fully to TNP-B. abortus, regardless of the presence of normal macrophages. These results indicate that B cells responding to the T-dependent antigens are rendered unresponsive to antigenic stimulation in mice preinjected with lipopolysaccharide, whereas B cells responding to the T-independent antigens are kept intact.     

The immune response in NZB mice of different ages to thymus-dependent and thymus-independent phosphorylcholine antigens.

This study was designed to examine aberrations of immune responses in autoimmune NZB strain mice during ageing, at the level of individual B cell clones. The response to phosphorylcholine (PC) was chosen because murine responses to PC are restricted to a few B cell clones and can be characterized with idiotypic markers. Responses to both thymus-dependent (TD) and thymus-independently (TI) PC-containing antigens were measured in mice ranging from 1 to 62 weeks of age. We found that: (1) TD responses to phosphorylcholine keyhole limpet haemocyanin (PC-KLH) decreased markedly (about 17-fold) between 28 and 62 weeks of age. TI responses to the R36a strain pneumococcus decreased only slightly during the same period. (2) The PFC responses to both antigens became markedly prolonged in mice older than 24 weeks. (3) The NZB response to either antigen is essentially monoclonal, as measured by inhibition of PFC with specific anti-idiotype serum and PC hapten. No age-related alteration in avidity or idiotype expression was observed. Our results demonstrate that no aberrant PC-reactive B cell clones appear in old NZB, and lend support to the notion that the abnormalities observed are due to defective regulatory mechanisms.     

Initiation of humoral immunity. I. The role of cytokines and hormones in the initiation of humoral immunity using T-independent and T-dependent antigens

The early events during the initiation of immune responses following the injection of T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens were studied in immature male chickens. Specifically, the role of cytokines and hormones in the initiation of humoral immunity against these antigens was investigated. Both interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) increased significantly post-LPS but not post-BSA injection. While interleukin-2 (IL-2) significantly decreased post-LPS injection, IL-2 significantly increased post-BSA injection. Furthermore, corticosterone levels significantly increased and tri-iodothyronine (T(3)) levels significantly decreased post-LPS but not post-BSA injection. In this study, the results indicate that although LPS and BSA can induce a humoral antibody response in chickens, they activate different cytokines and neuroendocrine network systems.     

CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens

CD59a is the primary regulator of membrane attack complex in mice. Recently, we have shown that CD59a-deficient (Cd59a-/-) mice exhibit enhanced CD4+ T cell responses. Here, we explored the effects of CD59a on B cell function and antibody production. Contrary to our expectations, Cd59a-/- mice showed a decreased humoral immune response to a T cell dependent antigen, sheep red blood cells. We found that the decreased humoral immune response was associated with a reduction in plasma cell number in vivo and reduced ability to respond to stimuli during in vitro culture experiments. Using MLR studies in which purified wild type or Cd59a-/- CD4+ T cells were mixed with purified B cells from each source, we found that the reduced B cell activation was largely due to the absence of CD59a on CD4+ T cells. Furthermore, a CD59a fusion protein bound specifically to mouse B cells, and enhanced B cell proliferation in a MLR, demonstrating that B cells express an as yet unidentified ligand for CD59a that aids in B cell activation.     

Evidence for the presence of idiotype-bearing regulatory T cells in which idiotype expression does not show linkage to either IgH alleles or the MHC.

An anti-idiotypic antiserum was raised in rabbits to a monoclonal antibody with specificity for one of the two antigenic determinants on the ferredoxin (Fd) molecule. The monoclonal antibody (Fd-B2) was derived from fusion of spleen cells from Fd-immune B10.BR (H-2k, Ighb). Examination of an extensive number of samples of Fd-immune serum from B10.BR and other mouse strains established that the Fd-B2 idiotype is essentially never present in such sera in detectable concentrations (greater than 30 ng/ml). Administration of the anti-idiotypic antibody (anti-Fd-B2) i.v. to B10.BR mice, or treatment of B10.BR T cell-enriched populations with anti-Fd-B2 + C prior to adoptive transfer to irradiated B10.BR recipients followed by challenge with Fd resulted in a significant increase in the production of anti-Fd antibodies. This effect was specific and was not reflected by a change in expression of the Fd-B2 idiotype in the antibody produced. Similarly, injection of 10 micrograms of Fd-B2 into B10.BR mice resulted in an enhanced anti-Fd response. When similar experiments were carried out using B10.D2 mice (H-2d, Ighb), which are genetic non-responders to Fd, it was observed that treatment which anti-Fd-B2 followed by challenge with Fd resulted in production in treated animals of significant levels of antibody to Fd. Again, the antisera thus produced did not contain detectable levels of the Fd-B2 idiotype. Further experiments using high responder (H-2k) mice with Igh allotypes differing from the B10 strains (C57/BR, Igha, and RF/J, Ighc), showed that treatment of these animals with anti-Fd-B2 also resulted in a highly significant enhancement of the anti-Fd response. These data imply that the anti-idiotypic antiserum (anti-Fd-2B) is exerting its influence by acting on an id + population of T cells and that the expression of this id is not dependent on genetic linkage to either the H-2 or the Igh loci.     

Immune response to phosphorylcholine. I. Characterization of the epitope-specific antibody

The antibodies to phosphorylcholine induced in BALB/c mice were isolated and studied with a variety of biochemical and immunochemical methods. Analysis of the idiotype and the hapten-binding specificities showed no differences to the phosphorylcholine-binding myeloma protein TEPC 15, indicating a high degree of homogeneity. However, disc electrophoresis, isoelectric focusing and amino acid composition data indicated large differences in the structure of anti-phosphorylcholine antibody and TEPC 15. By these criteria the anti-phosphorylcholine anti-bodies from BALB/c mice are of oligoclonal origin.