Perturbation of the idiotypic network. I. Induction with multiple alloantigen stimulation

The effect of hyperimmunization on the immune network with allostimulated syngeneic lymphocytes responding to different haplotypes was analyzed. Ten different haplotypes were used to stimulate syngeneic donor mice. Control mice were multiply immunized with incomplete Freund's adjuvant alone or with syngeneic mixed lymphocyte culture-generated lymphocytes. BALB/c mice were immunized consecutively with alloreactive blasts or allogeneically stimulated spleen cells at 10-day intervals. After a rest period of 2 months, the ratio of T helpers to T suppressors was determined by immunofluorescent staining. The functional network was probed by immunizing the mice with phosphorylcholine (PC) coupled to hemocyanin. The sera were analyzed for anti-PC antibodies and TEPC15 (T15) idiotypic expression. The results demonstrated (i) a decrease in the level of anti-PC antibody titer and T15 idiotypic expression; (ii) a decrease in the number of T helper cells and an increase in the number of T suppressor cells; (iii) a loss of PC epitope specificity; (iv) an increase of IgM antibodies expressing T15 without anti-PC specificity; and (v) an elevated level of preimmune lymphocyte proliferation and Ig secretion. These results reveal a functional network linkage in the regulation of alloreactivity and antigen response and show how repeated exposure to alloantigens can induce a perturbation of the idiotypic network controlling the response of a non-alloantigen-related BALB/c strain dominant idiotype (T15).     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Regulation of T15 idiotype dominance. I. Mice expressing the xid immune defect provide normal help to T15+ B cell precursors

The immune response to phosphocholine (PC) in many strains of mice is dominated by the T15 idiotype family of anti-PC antibodies. By introducing the CBA/N X-linked immune defect (xid gene) into these mice, one profoundly alters their ability to make a T15-predominant, IgM anti-PC response. This loss of T15 dominance in mice expressing the xid gene is not due to the presence of suppressor T cells or the lack of T15 idiotype-specific helper cells in these mice. Thus, one can reconstitute a T15 idiotype-dominant response in immune defective mice with B cells from normal mice, and in adoptive transfer assays the primed T helper cells from immune-defective mice provide qualitatively the same help to normal B cells as the T helper cells from normal mice. T15 idiotype dominance appears to be controlled by the expression and activation of Lyb-5+ PC-specific B cells. Thus, the majority of T15+ B cell precursors are restricted to this B cell subset, whereas the Lyb-5- B cell subset contains predominantly T15-, anti-PC B cell precursors, which produce mainly IgG antibodies after activation by PC-containing antigens.     

Helper T lymphocytes from xid and normal mice support anti-phosphocholine antibody responses with equivalent T15, 511, and 603 idiotypic composition

We have examined the idiotypic composition of secondary adoptive transfer antibody responses to phosphocholine (PC) supported by KLH-primed helper T cells derived from normal mice or xid mice. CBA/N x BALB/c F1 male xid mice have diminished anti-PC responses and virtually undetectable levels of the T15 idiotype; xid mice do express the 511 and 603 idiotypes. Nonetheless, we find helper T cells derived from such mice are indistinguishable from T cells primed in a normal environment in their ability to cooperate with B cells producing anti-PC antibody bearing the T15, 511, or 603 idiotype markers. This result is in contrast to a previously published report from this laboratory. T cells from xid mice did support more IgG PFC than normal T cells, but serum IgG anti-PC antibody levels were similar in both groups. The IgM anti-PC response was predominantly of the T15 idiotype, whereas the 511 idiotype was associated with a minor fraction of IgG1 antibodies. The majority of the secondary IgG "anti-PC" antibody response bore none of the idiotypic markers associated with PC-binding myeloma or hybridoma antibodies, and was directed against phenyl-PC rather than PC. The phenomenon of T15 clonal dominance in the anti-PC response therefore is largely confined to the IgM response. We would conclude that the idiotype levels in the T cell priming environment do not influence the subsequent ability of such primed T cells to support anti-PC antibody responses.     

Mechanism of neonatal idiotype suppression. II. Alterations in the T cell compartment suppress the maturation of B cell precursors

The cellular mechanism in neonatally suppressed BALB/c mice, which maintains the chronic suppressed state of the TEPC-15 idiotype in the antibody response to phosphorylcholine (PC), was investigated. Cells taken from these suppressed mice cannot transfer suppression to adult BALB/c or affect the in vitro response to PC of adult BALB/c spleen cells. However, spleen cells or T cells from neonatally suppressed mice given to neonatal animals induce chronic suppression of the TEPC-15 idiotype in the anti-PC response. Co-transfer of T cells from neonatally suppressed cells with normal T cells prevented the induction of suppression in neonates. Transfer of T cells from normal or keyhole limpet hemocyanin-primed BALB/c increased the expression of TEPC-15 idiotype in chronically suppressed mice, whereas T cells from neonatally suppressed were ineffective. These findings show that T cells in neonatally suppressed mice can affect the development of immature but not mature cells. The restoration of TEPC-15 expression in neonatally suppressed animals by normal T cells and the failure to induce suppression in neonates by co-transfers of T cells from normal and chronically suppressed mice demonstrate the profound role of an altered T cell compartment in sustaining chronic idiotype suppression.     

Induced recovery of a suppressed idiotype by immunization with anti-idiotype

Neonatal Balb/c mice were suppressed forthe H8/T15 idiotype by injection of homologous anti-H8 (D. S. Strayer, D. A. Rowley, and H. Köhler, et al J. Immunol.114, 722, 1975. Six to eight weeks later groups of these suppressed mice were immunized up to three times with isologous anti-H8 raised in Balb/c. After a rest of 4 weeks each group was challenged with R36a vaccine and bleedings were obtained before and after this challenge. All sera were assayed for total anti-PC and H8 idiotype amounts by solid-phase radioimmunoassay. After two-preimmunizations with isologous anti-H8 no increase of the H8 levels was observed though these mice responded to R36a immunization with an increase of total anti-PC antibodies. After the third preimmunization, however, the H8 idiotype was increased in sera taken before and after challenge with R36a. These findings demonstrate that the state of neonatal idiotype suppression can be broken by immunization with complementary anti-idiotype.     

Perturbation of the autoimmune network. II. Immunization with isologous idiotype induces auto-anti-idiotypic antibodies and suppresses the autoantibody response elicited by antigen: a serologic and cellular analysis

We report on the humoral and cellular events following autologous immunization against an idiotype (Id62) borne on a murine monoclonal autoantibody to thyroglobulin, and their impact on the autoantibody response to thyroglobulin. BALB/c mice with a state of active auto-anti-idiotypic immunity and challenged with thyroglobulin in complete Freund's adjuvant 2 wk after the last immunization with idiotype were found to have a suppressed autoantibody response. This suppression could be adoptively transferred to syngeneic x-irradiated recipients by using whole spleen cells from idiotype-primed mice. Transfer of separate T and B lymphocyte populations proved instrumental in disecting humoral from cellular events and in establishing that whereas B cells were required for transferring an intact anti-idiotype antibody response, T cells from idiotype-primed mice were necessary to transfer suppression. These findings contribute to our understanding of the interrelationship between antigen, idiotype, and anti-idiotype in the immune response to self-antigens, and the role of certain idiotypes in regulating autoimmune responses.     

Mechanisms of idiotype suppression. V. Anti-idiotype antibody inhibits early B cell triggering induced by antigen

To investigate the relationship between antigen-mediated B cell commitment and induction of idiotype (id) suppression, anti-id antibody directed against the major id (TEPC-15 idiotype or T15id) of the anti-phosphorylcholine (PC) antibody was added at various time intervals to BALB/c spleen cell cultures stimulated with a T-independent PC antigen, R36a. The suppressive effect of anti-T15id antibody on the anti-PC response was rapidly decreased as addition of the antibody was delayed; when anti-T15id antibody was added 6 hr after the initiation of the cultures, only partial suppression was induced, whereas the addition of anti-id antibody after 24 hr did not result in significant suppression of the anti-PC response when compared with similar cultures treated with mock anti-id antibody. This acquisition of resistance to id suppression was completely inhibited by treatment with either sodium azide or colchicine, as well as at temperatures below 20 degrees C. The induction of resistance to id suppression during the preincubation period was dependent on the presence of an immunogenic level of specific antigen. This antigen-mediated B cell commitment did not appear to require macrophages because preincubation of macrophages with antigen did not affect the sensitivity of the B cells to anti-id antibody. These results support the possibility that anti-id antibody inhibits early B cell triggering, which involves an energy-dependent, epitope-mediated, lateral mobility of antigen receptors possibly followed by repolymerization of microtubules.     

Differential L chain expression in the antibody responses to phosphorylcholine of adult bone marrow or peritoneum-derived B lymphocytes

Lethal irradiation of adult BALB/c mice followed by reconstitution with autologous bone marrow results in loss of T15 Id and IdX expression in the responses to phosphorylcholine (PC) and alpha(1-3)-dextran, respectively. T15 Id, but not IdX expression can be reconstituted with low numbers of syngeneic, T cell-depleted peritoneal resident cells. All three groups of mice produce comparable titers of specific anti-PC and anti-dextran antibodies. The inability of adult bone marrow-reconstituted BALB/c mice to produce T15 Id+ antibodies is not due to differential VH-gene expression in bone marrow or peritoneum-derived B cells. Thus, the levels of T15 VH in total serum Ig and in anti-PC antibodies are similar in all groups of mice. Furthermore, IEF patterns of T15 VH-associated L chains directly demonstrate differential Vk repertoire expression in bone marrow and peritoneum-derived B cells.     

Alteration of the B cell surface phenotype, immune response to phosphocholine and the B cell repertoire in M167 mu plus kappa transgenic mice

M167, mu plus kappa, transgenic mice have been analyzed for the expression of the transgene product as a cell surface, Ag-specific receptor and for their ability to respond to Ag. The vast majority of B cells in these H + L transgenics (97 to 99%) express large amounts of the transgene product on their surface and are capable of binding phosphocholine. A total of 4 to 30% of the B cells also express endogenous IgM and IgD H chain products. After immunization with phosphocholine (PC)-conjugated keyhole limpet hemocyanin, more than 1000 micrograms/ml of anti-PC antibody bearing the transgene IgMa allotype marker are produced. Surprisingly, significant amounts of anti-PC antibodies that express the endogenous, IgMb allotype, are also produced; however, these antibodies lack the T15-idiotype which dominates the anti-PC response in their nontransgenic littermate controls. The B cells producing these endogenous anti-PC antibodies also fail to switch to IgG anti-PC synthesis, whereas B cells producing anti-keyhole limpet hemocyanin antibodies readily undergo class switching. These last two observations may be due to the fact that the endogenous anti-PC antibody actually results from mixed mu a + mu b molecules in which the transgene encoded H and L chains are most likely responsible for the binding of PC. Thus, a switch of the endogenous isotype from mu b to IgG would result in a loss of specificity for PC in the IgG molecules produced using the endogenous VH-gene product(s), and mu a + gamma b hybrid molecules are not likely to be formed. This hypothesis is supported by the fact that the majority of (mu a + mu b) hybridomas have the mu b-allotype joined with a VH region other than the VH1 gene which is required for PC-binding and T15 idiotype expression.     

Regulation of T15 idiotype dominance. II. Genes unlinked to the Igh locus regulate T15 dominance of the secondary adoptive transfer response to phosphocholine

We have studied the idiotype and fine specificity of the secondary immune response to phosphocholine (PC) in C57BL (B10, B10.D2, and B.C8) and BALB (BALB/c, BAB-14, and C.B20) congenic strains of mice. In vivo IgM responses of mice from these two genetic backgrounds differed in their T15 idiotypic representation. BALB strains expressed the T15 idiotype on greater than 90% of their IgM, PC-specific plaque-forming cells (PFC), whereas C57BL strains expressed the T15 idiotype on approximately 50% of their IgM PFC. All strains examined expressed greater than 75% PC-inhibitable, VHPC idiotype-positive, IgM PFC. The IgG3 and IgA memory responses were similar to the IgM memory response; BALB strains produced a higher proportion of T15+ PFC than C57BL strains; however, the majority of IgG3 and IgA PFC in all strains were VHPC+, and PC-inhibitable. In contrast, the IgG1 memory response was not dominated by T15+, VHPC+, PC-inhibitable PFC in any of the strains tested. The IgG1 PFC required nitrophenylphosphocholine (NPPC) for efficient inhibition. The IgG2 memory response generally mimicked the IgG1 response with respect to idiotype and specificity. These data demonstrate that the representation of the T15 idiotype in the anti-PC immune response is determined by genes outside both the MHC and Igh genetic loci. Control of T15 expression in secondary IgM, IgG3, and IgA anti-PC responses was examined by using a cell-mixing protocol with primed T and B cells from BALB/c and B10.D2 mice. T15 representation in these responses was determined by the genotype of the B cell, not by the genotype of the helper T cell. Similarly, the B cell genotype was responsible for the idiotypic profile of a primary, in vitro, T-dependent, anti-PC response.     

Coexistence of antigen-specific and idiotype-specific suppressor T cells in mice injected neonatally with a mixture of antigen and anti-idiotype antibody

Specific tolerance to phosphorylcholine (PC) can be induced in BALB/c mice by neonatal injection with either pneumococcal C-polysaccharide (PnC) containing PC or anti-TEPC-15 idiotype (T15id) antibody which recognizes the predominant idiotype of anti-PC antibody of BALB/c mice. Suppressor T cells (Ts) induced after treatment with anti-T15id antibody react with the T15id and PnC-induced Ts cells appear to recognize PC. A brief incubation of anti-id-induced, T15id-specific Ts with PnC-induced, PC-reactive Ts resulted in complete cancellation of their suppressor functions. However, both types of Ts were present in mice neonatally injected with mixtures of PnC and anti-T15id antibody. Neutralization experiments using either PnC-induced or anti-id-induced suppressor T cells strongly suggest that only one of the Ts cell types is functionally dominant in those mice: most frequently, T15id-specific Ts cells. The suppressor function of the other population is detectable only when the predominant Ts cell population is removed by anti-id or monoclonal IgM anti-PC (SP45) plus complement. However, both suppressor activities are completely eliminated when one of the Ts populations is removed by adherence to either antigen or T15id. These results suggest that mice neonatally injected with a mixture of antigen and anti-id antibody possess both types of suppressor T cells, yet only one type is functionally dominant.     

Immunologic memory to phosphocholine. VII. Lack of T15 V1 gene utilization in Xid anti-PC hybridomas

CBA/N mice carrying the Xid defect fail to make antibodies expressing the T15 idiotype in response to immunization with PC-KLH. Antibodies predominating in the Xid response have binding properties characteristic of group II antibodies that emerge in the memory response in BALB/c; the prototype group II antibody utilizes a VH gene product distinct from the V1 gene product expressed by T15 idiotype-positive antibodies. To examine VH gene usage in the anti-PC response of Xid B cells, hybridomas were produced from Xid mice immune to PC-KLH. Four hybridomas possessing properties typical of the predominant group II antibody response in Xid mice and two representing minor components of the response were studied. Analysis of DNA by Southern blot hybridization revealed that none of the hybridomas utilized the T15 V1 gene segment, nor did they share use of a common VDJ gene product. These results indicate that Xid group II antibodies either make use of different VH gene segments or use the same VH in combination with various D and JH segments.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Predominance of the prototypic T15 anti-phosphorylcholine junctional sequence in neonatal pre-B cells.

The primary antibody response to phosphorylcholine (PC) is dominated by T15 antibodies. There are three families of anti-PC antibodies which can be made in mice: T15, 511, and 603. All use the same H chain V, D, and J segments, but each anti-PC family has a different L chain, as well as a family-specific Vh-D junctional sequence. Here we test the hypothesis that T15 antibodies are dominant because the prototypic T15 V-D junction is generated in pre-B cells more often than the alternative non-T15 V-D junctional sequences. Rearranged IgH genes from DNA derived from fetal or newborn liver pre-B cells and from adult bone marrow pre-B cells of BALB/c mice were amplified by polymerase chain reaction, cloned, and sequenced. DNA from adult splenic B cells was also amplified, for comparison. All V1-DFL16.1 and DFL16.1-Jh1 junctional sequences were analyzed. Fifty-three percent (9/17) of all neonatal pre-B cell V-D junctions with V1 and DFL16.1 had the prototypic T15 junctional sequence, which has no N regions. In contrast, no prototypic T15 V-D junctions were observed in adult pre-B cells, and each junctional sequence was unique. Adult splenic B cells contained an intermediate number of T15-type V-D junctional sequences (7/21). The prototypic D-J junctional sequence used in many anti-PC antibodies was also observed in a high percentage of sequences. The high frequency of T15 junctions in the neonatal pre-B cells can be explained by two observations: 1) N regions are absent in neonatal but not adult junctions and 2) in the absence of N regions, joining of V, D, and J segments may be targeted to short regions of sequence homology near the ends of the genes. This mechanism would preferentially give rise to the T15 V-D and D-J junctions. Preservation of the T15 V-D junction in adult splenic B cells is most likely due to antigenic stimulation of long lived precursors, because a high frequency of T15-type D-J junctions are coexpressed with T15 V-D junctions in splenic sequences. These results predict that T15 anti-PC precursors would be made at a very high frequency in the neonate, and at a much lower frequency in the adult. This may explain why the neonatal period is critical in establishing T15 dominance.     

Maturation of B cell subpopulations responding to phosphorylcholine

In this study, the order of appearance of B cell precursors responding to different phosphorylcholine (PC) antigens was investigated. Cells from neonatal BALB/c mice were transferred to male (CBA/N X BALB/c)F1 immunodeficient mice, and splenic fragment cultures were set up at different times after transfer. Because Xid F1 mice are unable to mount a primary anti-PC response, the time to prepare fragment cultures after cell transfer could be prolonged. This created an expanded maturation effect in which small differences in the response pattern of different precursor subsets are amplified. Splenic fragment cultures containing neonatal precursors were immunized with PG-TGG-HY (TD antigen), PnC (TI-2 antigen), or PC-TGG-LPS (TI-1 antigen), or with combinations of these antigens. The in vitro responses to these antigens were found to be additive, indicating the existence of different subpopulations. The detected precursors were also analyzed with respect to the T15 idiotype, which is the normally dominant idiotype of the response against PC in BALB/c mice. The analysis demonstrated a distinctly different maturation sequence of the three B cell subsets in which the order of appearance is: TI-1, TD, and TI-2 responding precursors. The T15 idiotype is expressed dominantly in all stages except the early stage of the TI-1 precursors.     

Immune response to levan. II. T independence of suppression of cross-reactive idiotypes by anti-idiotype antibodies.

Pretreatment of BALB/c mice with antisera to a cross-reactive idiotype (E109IdX) expressed on many anti-bacterial levan (BL) and anti-inulin (Inu) antibodies leads to a prolonged suppression in production of IdX-bearing molecules in response to BL immunization. There is a comparable suppression in numbers of plaque-forming cells secreting IdX-bearing anti-BL and anti-Inu molecules. Furthermore, spleen cells from anti-E109IdX pretreated mice are unable to transfer to irradiated recipients the ability to produce IdX-bearing anti-BL and anti-Inu antibodies. These results indicate that the suppressive effect is at the precursor level and not simply a clearance of antibodies bearing the IdX. Suppression of IdX production can be achieved by pretreating nu/nu BALB/c mice with anti-E109IdX antibodies. Furthermore, spleen cells from pretreated mice do not inhibit the capacity of spleen cells from normal mice transferred to irradiated recipients to produce E109IdX in response to BL. This indicates that the suppression of IdX production in the anti-BL system is T independent and probably represents direct inhibition of precursors by anti-IdX.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Delayed-type hypersensitivity to hen egg-white lysozyme. I. The surface phenotypes of suppressor T cells induced by intravenous injection of lysozyme-modified spleen cells, and their molecular interactions

Suppressor T cells (Ts) induced by lysozyme-modified syngeneic lymphocytes were characterized. Hen egg-white lysozyme (HEL)-specific delayed-type hypersensitivity (DTH) was suppressed when HEL-induced Ts were transferred into naive mice. These HEL-induced Ts had surface markers of both Thy-1 antigen, and I-J gene products. The suppression of HEL-specific DTH was greatly increased, when these Ts had been enriched with HEL-coated petri dishes. Isolated anti-HEL antibodies from B10.BR or A/Sn mice were inoculated into rabbits to induce anti-cross-reactive idiotype (CRI) antibodies. The rabbit antisera were extensively absorbed with normal B10.BR or A/Sn immunoglobulins (Igs) and MOPC 104E ascites Igs to render them idiotype (Id) specific. Using these anti-CRI antibodies, we observed that these Ts possessed Id receptors on their cell surface. Results of both fluorescence techniques and cytotoxicity tests revealed that about 10% of the enriched T cells containing these Ts were Id positive. Moreover, these enriched T cells were substantially killed by anti-I-J antiserum plus complement. However, this killing was completely blocked by HEL antigen. These results suggest that both Id receptors and I-J gene products might be forming the same molecular complexes or might coexist in the vicinity of the molecule.     

Resistance to Streptococcus pneumoniae is induced by a phosphocholine-protein conjugate

Previous studies demonstrated that naturally occurring antibodies to the pneumococcal cell wall hapten phosphocholine (PC) are important for the survival of mice against infection with Streptococcus pneumoniae, and that passively administered hybridoma antibody to PC results in added resistance. To determine if a PC-protein conjugate could elicit protective levels of anti-PC antibody, mice were immunized with PC-keyhole limpet hemocyanin (KLH) and tested for their ability to resist challenge with virulent S. pneumoniae. PC-KLH-immunized mice were observed to be resistant to 10- to 1000-fold more organisms than unimmunized control animals. The levels of protection were comparable to those induced with capsular polysaccharide antigens, but had the advantage of not being type-specific; immunization with PC-KLH protected mice against both type 1 and type 3 organisms. The induced immunity appeared to be antibody-mediated; it could be passively transferred with immune serum, and absorption of the immune serum with PC-Sepharose removed its protective capacity. Anti-PC antibodies in the serum of immunized mice were primarily IgM and IgG3 and possessed predominantly the T15 idiotype. Antibodies with these particular isotypes and this idiotype also arise after immunization with heat-killed rough pneumococci and recently were shown to be important in the resistance of mice to pneumococcal infection.