Perturbation of the idiotypic network. II. Transfer of suppression to neonatal mice

The cellular mechanisms which are responsible for idiotype perturbation induced by repeated stimulation with either allogeneic mixed lymphocyte culture-generated syngeneic blasts or allogeneically stimulated syngeneic spleen cells were investigated as described in the preceding article. Using the splenic fragment culture system, the precursor frequencies of T and B cells for anti-phosphorylcholine (PC) antibodies and T15 idiotypic antibodies were determined in allogeneically challenged mice. Adoptive transfers of T cells to neonatal BALB/c mice induced suppression of the T15+ anti-PC responses. In addition, the effect of immunization with internal image-bearing anti-idiotopes on the level of anti-PC antibodies and T15 idiotype was determined. Results from this study demonstrated (i) a decrease in the precursor frequency in the PC-specific and idiotype-specific B cell repertoire; (ii) a decrease in the precursor frequency of T helper cells, which recognize idiotypes and anti-idiotypes; (iii) the possibility to transfer T15 suppression to neonatal mice; and (iv) the possibility to restore T15 dominance by anti-idiotypic antibody immunization. These data indicate that both the T and B compartments are involved in the maintenance of suppression induced by repeated exposure to alloantigen-sensitized syngeneic cells. Collectively these findings show that a nonspecific general suppression induced by allohyperimmunization can perturb the T15+ anti-PC response.     

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.     

IgA isotype-restricted idiotypes associated with T15 Id+ PC antibodies

Idiotypes are believed to be due to the structural conformation of the variable region of immunoglobulins (Ig). We have found an idiotype (C3-24) that requires both variable and constant regions of the heavy chain to be expressed. C3-24 Id is associated with both the T15 variable region from anti-phosphorylcholine (PC) antibodies and the constant region for the alpha-heavy chain. High titer anti-PC serum from a variety of inbred strains of different Ig haplotypes failed to express C3-24 Id. However, when IgA but not IgG or IgM fractions were isolated from a pool of anti-PC serum from BALB/c mice, more than 70% of the molecules expressed C3-24 Id. The high frequency of the expression of C3-24 Id in IgA anti-PC hybridoma proteins from mice of different Ig haplotypes and in the IgA fraction of normal anti-PC antibodies from BALB/c and presumably other strains of mice suggests that idiotypic determinants produced by the three-dimensional product of VH and CH regions may not be unusual.     

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.     

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.     

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.     

Detection of the dominant expression of the TEPC-15 idiotypic determinant(s) on phosphorylcholine-specific suppressor T lymphocytes in vitro

Phosphorylcholine-(PC) specific suppressor T lymphocytes, induced by immunization with PC-coupled syngeneic spleen cells and capable of suppressing antibody production in an in vitro system, were successfully obtained by removal of a PC-nonspecific, i.e., diazo-phenylstructure-directed, suppressor cell population using an immunoadsorbent column coupling an unrelated hapten with a diazo phenyl structure such as azobenzene arsonate (ABA). Column-purified PC-specific suppressor T cell activity was completely abrogated by treatment of the cells with anti-TEPC-15 (T-15) anti-idiotypic antibody and complement, or by the continuous presence of that antibody in the culture, whereas nonpurified suppressor cell activity was resistant to such treatment. Thus, the column-purified PC-specific suppressor T lymphocytes in BALB/c mice have a very homogeneous T-15 idiotypic determinant(s) on their functional receptors for antigen similar to those present on PC-specific antibody and/or B lymphocytes. Because of these results, we envision the growing importance of analysis of the fine specificity of the idiotype repertoire of T lymphocytes after purification of a hapten-specific population.     

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.     

Structural evidence for a polymorphic or allelic form of the heavy chain variable region.

The heavy (H) chains of anti-phosphocholine (PC) antibodies from C57BL/6J and CBA/J were sequenced through the N-terminal 36 residues and compared with previously published sequences of A/J anti-PC antibody and BALB/c PC-binding myeloma proteins T15, M603, and M511. Each of these antibody preparations contained molecules having light (L) chains and idiotypic determinants of T15, M511, and M603 indicating the presence of at least three different anti-PC antibodies in each pool. The structures of the C57BL/6J and CBA/J H chains each revealed a single sequence from positions 1 to 36 (which includes the first complementarity determining region (CDR), and they were identical. The first CDR was identical to that previously found for BALB/c and A/J indicating that this portion of these antibody molecules is highly conserved throughout inbred mice and is probably critical to PC-binding. A surprising finding was that both C57NL/6 and CBA sequence differed from the BALB/c and A/J sequences at two positions, residue 14 and 16. Since each of these strains differs at the allotype locus, the data indicates that the evolution of allotypy in mice occurred after variable region diversity for the particular genes.     

"Nonspecific" immunoenhancing T cells in tumor-bearing mice include anti-idiotypic subsets

Splenocytes from DBA/2 mice inoculated 3 wk earlier with syngeneic P815 mastocytoma tumor cells produce increased numbers of antibody plaque-forming cells (PFC) when stimulated with either sheep red blood cells (SRBC) or phosphorylcholine (PC) on Streptococcus pneumoniae R36a in vitro. The nature of this nonspecific hyperreactivity was investigated in mixed cultures of purified splenic T and B cells. The addition of T cells from P815 tumor-bearing mice (TP815) into the cultures of normal B cells produced a significant enhancement of the PFC response to both SRBC and PC, when compared with the effect of normal T cells added to control cultures. The idiotypic profile of the enhanced anti-PC response was studied by a PFC-inhibition assay with monoclonal antibodies against two distinct idiotopic determinants (Id) of the T15 family. Normal B cells produced greater than 90% of T15 Id-positive (Id+) PFC. Addition of normal T cells diminished the proportion of T15 Id+ PFC to approximately 60%, whereas the rest of PFC were Id-. Addition of the immunoenhancing TP815 cells into the normal B cells cultures elevated the number of both T15 Id+ and Id- PFC responses, proportionally. However, when TP815 cells were first incubated on T15 protein-coated dishes and the non-adherent fraction was added to B cell cultures, the anti-PC PFC response remained enhanced but consisted of predominently T15 Id- PFC. These observations suggest that the early stage of P815 tumor growth activates various populations of specific helper/amplifier T cells including subsets with anti-idiotypic activity and that the generalized increase of antibody response to various antigens in tumor-bearing mice may be regarded as a polyclonal activation of specific T cells.     

A new surface antigen (PC.2) expressed exclusively on plasma cells

Somatic cell hybridization of NS.1 nonsecretor myeloma cells with spleen cells of (DBA/2 X C57BL/6)F1 mice immunized against the myeloma MOPC 70A of BALB/c mice led to the establishment of five hybridoma clones which continuously secrete anti-MOPC 70A cytotoxic antibodies. The respective antigen detected by each of the five monoclonal antibodies is expressed both on plasmacytomas and on antibody-secreting cells as the only normal cell type. The tissue distribution of this new antigen is different from that reported for the alloantigen PC.1, and we have therefore designated it as PC.2. On the basis of immune elimination of direct and indirect plaque-forming cells, all mouse strains tested express PC.2 determinants, identifying PC.2 essentially as an autoantigen. Conventional anti-PC.1 alloantiserum contains antibodies to the PC.2 determinant, and these antibodies are distinguishable from the anti-PC.1 antibodies proper by the fact that only the latter are absorbed by liver cells. Monoclonal anti-PC.2 antibodies are not directed against MuLV-(murine leukemia virus)--associated antigens as over 20 ecotropic, several MCF (mink colony forming recombinant, and xenotropic viruses failed to react in immunofluorescence assays.     

Idiotype shifts caused by neonatal tolerance to phosphorylcholine

The injection of as little as 0.5 microgram phosphorylcholine-(PC) conjugated mouse immunoglobulin into BALB/c neonates within 48 hr of birth results in complete unresponsiveness to PC for 3 to 4 wk. Thereafter, anti-PC responses can be detected in tolerized animals, but these responses differ significantly from those of normal BALB/c mice. First, the magnitude of responsiveness does not approach normal levels even 9 mo after birth. Second, although the initial responses as tolerance is broken can be T15+, idiotypic dominance is not established; instead, a heterogeneous T15- population eventually emerges, which includes clones with higher and with lower avidity than T15. Unirradiated unresponsive mice will help transplanted normal B cells to produce T15+ responses to thymus-dependent PC antigens. The responses of animals recovered from tolerance are stable upon adoptive transfer. We have, moreover, found no evidence of either loss of idiotype-specific T cell help or generation of suppression. Therefore, neonatal exposure to PC tolerogen can effect profound, permanent changes in the antigen-specific B cell compartment independent of any influence on conventional T cell regulatory mechanisms.     

A new surface antigen (PC.2) expressed exclusively on plasma cells

Somatic cell hybridization of NS.1 nonsecretor myeloma cells with spleen cells of (DBA/2 × C57BL/6)F₁ mice immunized against the myeloma MOPC 70A of BALB/c mice led to the establishment of five hybridoma clones which continuously secrete anti-MOPC 70A cytotoxic antibodies. The respective antigen detected by each of the five monoclonal antibodies is expressed both on plasmacytomas and on antibody-secreting cells as the only normal cell type. The tissue distribution of this new antigen is different from that reported for the alloantigen PC.1, and we have therefore designated it as PC.2. On the basis of immune elimination of direct and indirect plaque-forming cells, all mouse strains tested express PC.2 determinants, identifying PC.2 essentially as an autoantigen. Conventional anti-PC.1 alloantiserum contains antibodies to the PC.2 determinant, and these antibodies are distinguishable from the anti-PC.1 antibodies proper by the fact that only the latter are absorbed by liver cells. Monoclonal anti-PC.2 antibodies are not directed against MuLV-(murine leukemia virus) —associated antigens as over 20 ecotropic, several MCF (mink colony forming) recombinant, and xenotropic viruses failed to react in immunofluorescence assays.Abbreviations used in this paper PFC plaque-forming cell(s) - PC plasma cell - SRBC sheep red blood cell - B6 C57BL/6 - MuLV murine leukemia virus     

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.     

Representation of heavy but not light chain Ig idiotypes on T cell receptors for alloantigens.

We have analyzed idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants by the use of different anti-idiotypic antibodies. Such antisera were produced in (Lewis X DA) F1 rats against Lewis anti-DA alloantibodies (= B cell product) and Lewis T lymphocyte receptors with the same specificity. We found that B lymphocytes bear unique idiotypic determinants which are not present on the corresponding T lymphocytes. T cell unique (not shared by B lymphocytes) idiotypes were so far not detected. T cells idiotypic determinants which are present on heavy but not light chains of the corresponding alloantibodies.     

Thymus-independent induction and antigen-dependent recovery of idiotype-specific suppression

BALB/c nude (nu/nu) mice and euthymic (nu/+) littermates were treated as neonates with anti-T15 antibody and challenged at various ages with either a thymus-independent, PC-Brucella abortus (PC-BA), or thymus-dependent, PC-keyhole limpet hemocyanin (PC-KLH), form of phosphorylcholine (PC). Nu/nu mice challenged with PC-KLH received KLH-primed splenic T cells prior to immunization. Neither neonatally anti-idiotype-treated nu/+ nor nu/nu mice responded with the production of T15-positive anti-PC antibodies after challenge with either form of PC antigen. It is concluded that neither induction nor maintenance of a state of T15-specific suppression requires thymus-matured T cells. Recovery of anti-PC responsiveness in suppressed nu/+ or nu/nu mice was similar and was found to be related to the form of antigen used to elicit the response. Immunization with PC-KLH revealed a long-lasting unresponsiveness (up to 16 weeks). In contrast, immunization with PC-BA elicited a full anti-PC response as early as at 6.5 weeks of age.     

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.     

Idiotypic-anti-idiotypic B cell interactions generated against a protective antigen of a morbillivirus in mice.

The idiotypic network theory (N. K. Jerne, Ann. Immunol. 125, 373-389, 1974) predicts that any antibody that can be made by an individual would have its preexisting specific complementary B cells in its germline repertoire. We transplanted syngeneic BALB/c mice with live hybridoma cells and demonstrated the simultaneous presence of interacting idiotypic and anti-idiotypic B cells in an individual animal by immuno-cytoadherence assays. Furthermore, we demonstrate that interacting B cells displaying idiotypic and anti-idiotypic antibodies are subjected to lysis by complement. It is therefore tempting to speculate that this complement-sensitive interaction between idiotypic and complementary anti-idiotypic B cells in vivo may provide a mechanism for the regulation of B cell populations.     

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.     

Immunologic memory to phosphocholine. IV. Hybridomas representative of Group I (T15-like) and Group II (non-T15-like) antibodies utilize distinct VH genes

The anti-phosphocholine (PC) memory response elicited in BALB/c mice by phosphocholine-keyhole limpet hemocyanin (PC-KLH) contains two groups of antibodies distinguished by their fine specificity for PC and p-nitrophenylphosphocholine (NPPC). Group I antibodies are inhibited by both PC and NPPC, while Group II antibodies are inhibited appreciably only by NPPC; only Group I antibodies are dominated by the T15 idiotype. Anti-PC hybridomas representative of the memory response to PC-KLH were produced to examine the variable region genes expressed by memory B cells. Two IgM hybridomas were of the Group I type, because they were inhibited by both PC and NPPC and they bound to the pneumococcus R36A. However, only one of these antibodies (PCM-2) expressed a T15 idiotope, while the other (PCM-1) did not express any of three T15 idiotopes. Despite its negative T15 idiotype profile, N-terminal amino acid sequencing of PCM-1 purified heavy chain and Southern blots of the hybridoma DNA indicated that it utilizes the T15 VH and JH1 genes. Three hybridomas, IgG1, IgM, and IgE, typical of Group II antibodies, were examined; these were negative for three T15 idiotopes and displayed measurable avidity only for NPPC in a PC-protein binding inhibition assay. These three hybridoma antibodies, like serum Group II IgG1, did not measurably bind to the bacterium R36A. The heavy chain amino termini of all three of these antibodies were inaccessible for Edman degradation. Southern blots of DNA from the IgG1 hybridoma revealed the T15 VH gene to be in the germ line configuration only and unassociated with any JH segment, indicating that this Group II antibody utilizes a VH gene different from the T15 family. These results signify that, whereas some diversity of the (anti-PC) memory response may be generated by somatic diversification of variable regions important in the primary response, a significant contribution to the overall heterogeneity of memory antibodies originates in the expression of additional variable region genes.