Anti-idiotypic Antisera Research Articles

Isotypic Discordance of Paraproteins and Lymphocyte Surface Immunoglobulins in Myeloma

B lymphocyte surface immunoglobulins (Smlg) were studied in 24 patients with multiple myeloma by means of anti-isotypic antisera, and their heavy and light chain isotypes were compared in each patient with those of the paraprotein. In 21 patients, lymphocyte Smlg consisted of only one light chain type, and in 16 of only 1 heavy chain type. However, the Smlg and paraprotein heavy and light chain types were identical in only 5 patients while in 6 they differed in heavy and light chain types, in 7 in light chain type, and in 4 in heavy chain type. In 2 patients with light chain myeloma, Smlg light chains were isotypically the same as the paraprotein. Isotypic discordance between paraprotein and Smlg may signify the proliferation of a second malignant clone with failure to differentiate into secreting plasma cells. Alternatively, it is conceivable that the lymphocyte Smlg could have the same idiotypic specificity as the paraprotein despite the isotypic differences, but this will require further studies using anti-idiotypic antisera.

Properties of a minor cross-reactive idiotype associated with anti-p-azophenylarsonate antibodies of A/J mice.

Experiments are described that further characterize an A/J anti-phenylarsonate (anti-Ar) antibody population, which is idiotypically cross-reactive within the strain but differs from the family of antibodies expressing the major cross-reactive idiotype (CRI). This population has been designated as expressing a "minor" cross-reactive idiotype. Chain recombination studies with hybridoma products (HP) revealed that 2 of 3 HP expressing minor idiotypic determinants contain L chains that are idiotypically closely related to L chains in anti-Ar antibodies bearing the major CRI. The results indicate the expression of the same or very similar L chains in antibodies that differ in idiotype. The minor idiotypic population in serum was investigated by separating antibodies with minor and major idiotypic determinants by use of an immunoadsorbent containing anti-idiotype directed to an HP expressing the major CRI. Studies with the nonadherent, minor idiotypic population showed that such molecules constitute about 8 to 10% of the anti-Ar antibodies in a pool of serum but that individual sera vary markedly in their content of anti-Ar antibodies bearing minor idiotypes. Preinoculation of mice with conventional anti-idiotypic antiserum before immunization with KLH-Ar was found to suppress the appearance of the minor idiotypes as well as the major CRI upon subsequent immunization. The suppression of the minor idiotypic population is presumably mediated by the small fraction of rabbit anti-id antibodies directed against such molecules.

Idiotypic analysis of anti-GAT antibodies. VIII. Comparison of interstrain and allotype-associated idiotypic specificities.

A guinea pig anti-idiotypic antiserum made against pooled specifically purified A/J anti-GAT antibodies was characterized. This antiserum contains anti-idiotypic antibodies specific to interspecies, interstrain, and allotype-linked idiotypic determinants. These idiotypic determinants are associated with the combining sites of idiotypic antibodies that are induced by GT-related but not GA-related antigenic moieties. Genetic and strain distribution studies indicated that the shared allotype-linked idiotypic determinants are controlled by Igh-1e- or Igh-1b-linked genes. The interrelationships of the allotype-linked and interstrain CGAT idiotypic specificities are described using monoclonal anti-GAT hybridoma antibodies. Four of 7 hybridoma anti-GAT antibodies of C57BL/6 origin expressed a major fraction of the idiotypic specificities of A/J anti-GAT antibodies. These 4 hybridoma antibodies also carried the common interstrain idiotype, termed CGAT, but not all CGAT-bearing anti-GAT hybridoma antibodies expressed the allotype-linked idiotypic specificities. The Ig-1b-positive, F17-167.1 hybridoma anti-GAT antibody was used as a ligand to selectively identify the major allotype-linked idiotypic specificities, which were designated Gte idiotype.

Mechanism of immune transfer by RNA extracts. Immune RNA induces the synthesis of idiotype-bearing antigen receptors in noncommitted cells.

Immune RNA (I-RNA) was extracted form lymphoid organs of BALB/c mice immunized with AKR lymphoid cells. Incubation of normal BALB/c spleen cells with this I-RNA (but not with normal RNA) resulted in leukocyte migration inhibition reactions (LMIR) against AKR extracts but not against purified protein derivative or BALB/c sarcoma extracts. This transfer was abolished by pretreating I-RNA with RNAse but not with pronase. The active fraction of I-RNA was retained by and could be eluted from Poly-U Sepharose columns. Normal cells pretreated with I-RNA also reacted in the presence of an anti-idiotypic anti-serum of anti-(BALB/c anti-AKR) specificity. Pretreatment of cells with anti-idiotypic serum plus complement did not inhibit the subsequent transfer of LMIR with I-RNA. Idiotypic receptors were expressed on I-RNA treated cells less than one hour after I-RNA treatment. Using an I-RNA of double specificity, the results suggested that I-RNA entered into and acted on the cells through a nonspecific mechanism. Finally, I-RNA could induce BALB/c anti-AKR idiotypic markers in C57Bl/6 cells, genetically committed for different idiotypes, while RNA extracted from C57Bl/6 immune cells could not induce in BALB/c cells their own genetically acquired idiotypes. This series of data would prove that I-RNA acting as a mRNA is able to induce in normal noncommitted cells the de novo synthesis of antigen receptors similar or identical to those present in the surface of in vivo immunized lymphoid cells of the same strain.

The role of immunoglobulin receptors and T cell mediators in B lymphocyte activation. I. B cell activation by anti-immunoglobulin and anti-idiotype reagents

The possibility that the failure of anti-mouse immunoglobulin (Ig) antibody to induce antibody synthesis by B cells might be due to reversible receptor blockade was investigated. Murine spleen cells were cultured for 3 days in the presence of minute quantities of intact of (Fab') fragments of rabbit anti-mouse Ig antibody. Thereafter, the cells were washed and either trypsin treated or not before reculturing for 18 hr. Only cells that had been trypsinized after culturing with either intact or fragments of anti-Ig gave a vigorous polyclonal antibody response. This response was extremely T dependent, since T cells or culture supernatants from Con A-activated T cells were required for the B cell response. Moreover, anti-delta was much more effective than anti-mu in inducing antibody synthesis. Finally, the use of three different anti-idiotypic antisera rather than anti-Ig reagents selectively activated the specific idiotype in each instance. The findings demonstrate that anti-Ig reagents can potentiate the response of B cells to signals delivered by T cells.

The role of immunoglobulin receptors and T cell mediators in B lymphocyte activation. I. B cell activation by anti-immunoglobulin and anti-idiotype reagents.

Abstract The possibility that the failure of anti-mouse immunoglobulin (Ig) antibody to induce antibody synthesis by B cells might be due to reversible receptor blockade was investigated. Murine spleen cells were cultured for 3 days in the presence of minute quantities of intact of (Fab') fragments of rabbit anti-mouse Ig antibody. Thereafter, the cells were washed and either trypsin treated or not before reculturing for 18 hr. Only cells that had been trypsinized after culturing with either intact or fragments of anti-Ig gave a vigorous polyclonal antibody response. This response was extremely T dependent, since T cells or culture supernatants from Con A-activated T cells were required for the B cell response. Moreover, anti-delta was much more effective than anti-mu in inducing antibody synthesis. Finally, the use of three different anti-idiotypic antisera rather than anti-Ig reagents selectively activated the specific idiotype in each instance. The findings demonstrate that anti-Ig reagents can potentiate the response of B cells to signals delivered by T cells.

Idiotypic analysis of anti-GL phi antibodies. I. Identification and strain distribution of GL-1 idiotypes.

We utilized both the inhibition of antigen binding and direct idiotype binding methods to identify a new set of common idiotype determinants on anti-GL antibodies of various mouse strains. Three anti-idiotypic antisera, each prepared against individually purified B10.WB anti-GL phi antibodies, were able to detect antibody-combining site-associated common idiotypic determinants, designated GL-1 idiotype(s), in antisera with GLT-binding activity obtained from all mouse strains except strains bearing Igh-1e allotype. Anti-GL phi antisera obtained from rabbits, guinea pigs, and rats did not express detectable levels of GL-1 idiotypes. Nonresponder mice to GL phi, upon immunization with GL-F gamma G or GL phi-F gamma G produced anti-GL antibodies expressing GL-1 idiotypes. Although the magnitude of the immune response to various GL-containing polymers is controlled by distinct Ir genes, the common GL-related antigenic determinants on these polymers are able to induce anti-GL antibodies with GL-1 idiotypic specificities.

Structural studies on induced antibodies with defined idiotypic specificities. IX. Framework differences in the heavy- and light-chain-variable regions of monoclonal anti-p-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype.

Amino terminal amino acid sequence analyses have been performed on the heavy and light chains of induced monoclonal antibodies with specificity for the hapten p-azophenylarsonate. Four of the eight antibodies react with conventional antisera to the previously described A/J anti-arsonate cross-reactive idiotype (CRI). Of the 16 chains analyzed, all but one contain sequence differences in their first framework segment (residues 1-30) that distinguish them from the heavy- and light-chain sequences found in anti-arsonate antibodies isolated from A/J serum or ascites fluid. The presence of such framework differences appears to be independent of whether or not the hybridoma antibodies bear the CRI. In spite of the framework substitutions, all four of the CRI-positive hybridoma antibodies have variable (V)-region frameworks that are very similar to each other and to the CRI-positive molecules found in A/J serum. Two of the four CRI-negative molecules are also structurally similar to the serum antibodies. Two others, however, are strikingly different from any serum anti-arsonate antibody thus far described and appear to reflect a completely separate repertoire of anti-arsonate antibodies in the A/J MOUSE. In addition, serological analyses with an anti-idiotypic antiserum generated against a CRI-positive hybridoma product suggest that each monoclonal antibody may possess individual antigenic specificities different from the determinant(s) detected with the conventional rabbit anti-CRI. The consistent appearance of framework substitutions in what has been thought to be a homogeneous antibody population has important implications for our understanding of the generation of antibody diversity and for the precise chemical definition of an idiotype.

Isolation and properties of antigenic-specific receptors from rabbit and pig lymphoid cells

In order to study the character and properties of antigen-specific receptors on lymphocyte surfaces, an immunoadsorption method based on the principle described by Kiefer (1973, 1975) has been developed. This method permits isolation of receptors from rabbit and pig spleen and lymph node cells in quantities sufficient for basic analyses. Isolated receptors represent a mixture of two groups of antigen binding molecules. Both groups react with corresponding anti-idiotypic antisera but only one of them reacts with antisera to serum immunoglobulins. These two fractions can be separated using an immunoadsorbent with bound antibodies to serum immunoglobulins. In the fraction bearing immunoglobulin determinants, the presence of IgM and IgG but not IgA has been detected. The IgG receptors and IgG antibodies in the serum of the same animal had different isoelectrofocusing spectra. The origin of the other antigen-binding fraction remains to be established. The results obtained with antisera to the ‘constant part’ of this molecule confirmed that it does not carry conventional immunoglobulin determinants.

Comparison of antigen-specific I-region-associated cell interaction factors.

Two basic types of factors reacting with anti-I region (anti-Ia) antisera are compared, those derived from macrophage-like antigen presenting cells and others derived from T-lymphocytes, of either the suppressor or helper type. Despite the common property of reacting with anti-Ia antisera, the two sets of factors differ by many criteria. Macrophages, upon culture with antigen, release complexes of Ia antigen and a fragment of the original immunogen. This material is only produced by responder macrophages and thus appears to be a soluble Ir gene product. The genetic restriction of the T-macrophage interaction was investigated in chimeras, and it was found that the host environment as well as the donor genotype was of importance in determining restrictions, which were thus not really directed to "self." There was no evidence for intrinsic T-cell Ir genes, as nonresponder stem cells developed into responder T-cells in a (responder X nonresponder) F1 environment. However, these cells only responded in the presence of responder macrophages. Specific T-cell factors are different in nature. These all react with anti-Ia antisera, but the nature or function of the T-cell Ia is unknown. The basic structure involves a VARIAble region" responsible for antigen binding which, as it reacts with anti-idiotype antisera and anti-variable region framework antisera is an immunoglobulin variable region. There is also a "constant region," defined by its biological properties as well as by specific rabbit antisera. This two-region nature of specific factors is reminiscent of immunoglobulin structure and it is a reasonable hypothesis that the constant region is linked to the Ig cluster of genes.

Idiotypic Analysis of Anti-GAT Antibodies

Abstract Previous studies demonstrated that anti-GAT antibodies of all inbred strains of mice share a common idiotype, termed CGAT idiotype. The CGAT idiotype is present on a major fraction of anti-GAT antibodies and is induced by the “GT”-related antigenic determinants on GAT molecules. The present paper describes another common idiotype of anti-GAT antibodies, termed GA-1 idiotype, the identification of which is based on a heterologous idiotypic interaction between guinea pig anti-idiotypic antiserum made against pooled specifically purified D1.LP anti-GAT antibodies and a monoclonal hybridoma anti-GAT antibody from the F9-102.2 cell line. The homologous anti-F9-102.2 idiotypic antiserum detected private idiotypic specificities. GA-1 and CGAT are distinct idiotypes induced upon GAT immunization. Unlike CGAT idiotype, GA-1 idiotype is expressed on a minor fraction of the anti-GAT antibodies and “GA”-related antigenic determinants are responsible for the inducation of GA-1 idiotype. Thus, anti-GA, anti-GAT and anti-GLA40 antisera from 21 different inbred strains of mice expressed GA-1 idiotype. However, both the CE/J anti-GAT and anti-GA antisera lack detectable levels of GA-1 idiotype.

Genetic control of the immune response to the terpolymer l-glutamic acid 60- l-alanine 30- l-tyrosine 10 (GAT)—IV : Heterogeneity of idiotype GAT-715

A major idiotype GAT-715 was defined in mice by a rabbit anti-idiotypic antiserum raised against BALB/c anti-poly (Glu 60-A!a 30-Tyr 10) (GAT) antibodies. Corresponding idiotypic specificities were found to be present on anti-GAT antibodies from responder and non responder mice regardless of their allotypic markers. Some of the idiotypic specificities that we have termed highly conserved idiotypic specificities are expressed by rat and guinea pig after immunization with GAT. In this paper we report experiments that show that idiotype GAT-715 can be further analysed and subdivided into public and private idiotypic specificities. All mice tested express the public specificities that represent 80% of idiotype GAT-715. Some strains also express private specificities; no genetic linkage is observed between the inheritance of heavy chain allotypic markers and private specificities. In addition, public and highly conserved specificities have been compared. A structural model for GAT-715 idiotype is discussed.

Immunoregulatory effects of a covalent antigen-antibody complex.

It is becoming increasingly clear that any specific immune response must be subject to a control mechanism of the same order of specificity. The basis of such specificity in control almost certainly lies in the antibodies themselves, whether free or cell bound. The response to a given antigen might then be regulated by antibodies specific either for the antigen (‘Ab 1’) or for the variable regions (idiotypes) of Ab 1. Whereas the latter can interact directly with the binding sites of cellular receptors for antigen, the former can only do so if the antigen is also present to act as a bridge. Furthermore, because an anti-idiotype antiserum is usually prepared against a monoclonal antibody, it will only be able to influence directly those clones of cells which carry the chosen idiotype, and thus may leave unaffected other clones which are specific for the same antigen but which carry different idiotypes. An immune complex, on the other hand, should influence all clones sensitive to the antigen in question. Experimentally, the injection of antigen–antibody complexes can either increase1,2 or decrease3,4 the subsequent response to antigen. These effects often require the presence of an intact Fc region4 and may differ according to the class (or isotype) of the antibody5. The injection of anti-idiotype antisera can have striking effects on the expression of particular idiotypes, but usually has little effect on the overall response. Again, these effects require the participation of the Fc region of the anti-idiotype antibody6, and can vary according to its isotype7,8. To study the regulatory effects of antibody more closely, we have attempted to prepare immune complexes which could influence the overall response, and yet have effects as powerful as those obtained with anti-idiotype antibodies. The immune complexes obtained were of the defined constitution Ag2Ab, in which stability was ensured by linking antigen to antibody through a covalent bond. We report here that, depending on the conditions used, these complexes induced either priming or tolerance of the response of mice towards subsequent injections of antigen alone. We consider that such complexes could be useful not only as tool for studying immunoregulation, but also as a practical means of influencing immune responses.

Hapten-specific T-cell responses to 4-hydroxy-3-nitrophenyl acetyl. II. Demonstration of idiotypic determinants on suppressor T cells.

The ability of NP-coupled syngeneic spleen cells to induce antigen-specific T-suppressor cells capable of binding to NP-BSA-coated Petri dishes and mediating transfer of specific suppressive activity to NP was demonstrated. Furthermore, in strains of mice bearing the Ig-1b allotype, including SJL, and in (non-Ig-1b x Ig-1b)F1 hybrids, the NP-specific suppressor cells also interferes with expression of immunity after priming with NIP-BGG. Anti-NPb anti-idiotype antiserum plus complement treatment effectively abrogated the ability to transfer suppression. Formal genetic mapping of the fine specificity of cross-reactivity with Ig-1 allotypic congenic mice implies that expression of this trait is linked to the Ig-1b heavy chain linkage group. The sensitivity of NP-suppressor cells of appropriate strains to anti-idiotype treatment was also consistent with the formal mapping data. These experiments suggest that there are shared V-region structures on antibody and T cells that are crucial in the suppression pathway for the same antigen.

Alloantigen-specific idiotype-bearing receptors on mouse T lymphocytes. I. Specificity characterization and genetic association with the heavy-chain IgG allotype.

The present study describes the qualitative reactions of a xenogeneic anti-idiotype (Id) antiserum produced in a mouse-gamma-globulin-tolerant rabbit (5,936) against B6 anti-CBA IgG antibodies. The results showed that such an anti-Id antiserum reacts specifically against anti-H-2k antibodies and against H-2k alloantigen-activated T cells from the following pairs of congenic mice: B10 (H-2b) and B10.D2 (H-2d); and A.BY (H-2b) and A.SW (H-2s), but not against C3H.SW (H-2b) and C3H.OH (H-2o); and BALB/b (H-2b) and BALB/c (H-2d). CB 20 (BALB/c mice with the Ig-1b allotype) anti-CBA T blasts also express idiotypic determinants that react with rabbit 5,936 antiserum. Thus, positive reactions are obtained between rabbit 5,936 anti-Id antiserum and anti-H-2k IgG preparations and T blasts from mice carrying the Ig-1b or Ig-1e allotype, but not from mice carrying the Ig-1a allotype. These reactions are qualitatively independent of the H-2 genotype of the Id-producing mice. Such a finding strongly suggests that the Id-bearing receptor molecules on mouse T cells are coded for by genes that are associated with the Ig heavy-chain-linkage group and not to the mouse histocompatibility complex. Furthermore, the anti-Id antibodies studied react preferentially against anti-H-2k antibodies or T cells with specificity toward the IAk-region-associated serological specificities. Thus, genes associated with the Ig heavy-chain-linkage group seem to be structural genes for at least T-cell receptors with specificity for IA-region-coded membrane antigens.

Hybridoma antibody immunoassays for the detection of parasitic infection: development of a model system using a larval cestode infection in mice.

A prototype immunodiagnostic assay has been developed using chronic infection with the larval cestode, Mesocestoides corti, as a model system in mice. The assay is highly sensitive, it appears to be absolutely specific for M. corti infection, and is based on the inhibition of binding (by sera from infected mice) of a radiolabelled anti-M. corti hybridoma antibody to a crude M. corti antigen extract. The hybridoma antibody binds to living M. corti larvae and is an IgG1 protein. In large scale experiments no false positives were detected and the only M. corti-infected mice not detected by the assay were hypothymic nude (nu/nu) mice. Only limited success has been achieved in attempts to convert the assay to one not requiring parasite antigen and based on the inhibition of binding of radiolabelled anti-parasite hybridoma antibody and a large pool of anti-idiotype antiserum. Monoclonal antibodies derived from anti-parasite antibody-secreting hybridoma cell lines will be of particular use in the development of new, highly specific, immunodiagnostic reagents for the detection of parasite infection, exposure and disease.

Mechanisms of regulation of cell-mediated immunity. IV. Azobenzenearsonate-specific suppressor factor(s) bear cross-reactive idiotypic determinants the expression of which is linked to the heavy-chain allotype linkage group of genes.

T-cell derived suppressor factor(s) (SF) specific for azobenzenearsonate (ABA) were prepared by the mechanical disruption of suppressor cells. Such suppressor factors were adsorbed to and recovered from immunoadsorbents prepared from the F(ab')2 fragments of rabbit immunoglobulin directed against the cross-reactive idiotype of A/J anti-ABA antibodies. These ABA-suppressor factors were not retained on Sepharose 4B immunoadsorbent columns which had been coupled with F(ab')2 fragments or normal rabbit immunoglobulins prepared from prebleeds of rabbits used to make anti-idiotypic antiserum. The specificity of the F(ab')2 rabbit anti-idiotypic serum was established by direct idiotypic-binding assays and by affinity purification over an immunoadsorbent consisting of CRI+ anti-ABA immunoglobulin from A/J mice. ABA-suppressor factors were shown to be specifically absorbed and eluted from F(ab')2 anti-idiotypic columns. Futhermore, the eluted suppressor factor can be specifically reabsorbed and recovered from a second anti-idiotypic immunoadsorbent. The concordance between antigen-binding specificity and the presence of idiotypic determinants was demonstrated by adsorbing ABA SF to antigen columns and then fractionating the ABA-specific factor on anti-idiotypic immunoadsorbents. ABA-suppressor factors were shown to be specifically retained on immunoadsorbents directed against major histocompatibility complex (MHC) determinants. Factor eluted from anti-MHC columns could then be specifically adsorbed to anti-idiotypic immunoadsorbents. This suggests that the same molecular complex that is recognized by the H-2 alloantiserum is specifically adsorbed to an anti-idiotypic immunoadsorbent. Genetic analysis of the expression of CRI+ suppressor factor was performed using the C.AL-20 mouse strain which has the AL/N allotype and produces CRI+ anti-ABA immunoglobulins. The implication of these findings to the nature of T-cell-derived regulatory molecules is discussed.

Demonstration of rheumatoid factor idiotypic antigens on peripheral blood B and T lymphocytes from patients with rheumatoid arthritis.

Antisera were raised against three polyclonal IgM rheumatoid factors (RF). After adequate absorptions, the antisera were rendered idiotype-specific, as assayed by haemagglutination technique. By using the anti-idiotype antisera in indirect immunofluorescence on peripheral blood lymphocytes from the patients used as donors for the immunizing RF, it was demonstrated that 3-14% of the lymphocytes were stained, and thus had membrane-bound structures with idiotypic antigens similar to those of the circulating IgM RF of the same patients. While most of these idiotype-positive lymphocyte were B lymphocytes, it was demonstrated in one patient that about 7% of the T lymphocytes also had the same idiotypic antigens.

Shared idiotypic determinants on antibodies and T-cell-derived suppressor factor specific for the random terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10.

T-cell derived suppressor factors (TsF) specific for the random copolymers L-glutamic acid60-L-alanine30-Ltyrosine10 and L-glutamic acid60-L-alanine40, referred to as GAT and GA, respectively, were prepared and partially purified on the approprate antigen immunoadsorbents. GAT-TsF obtained from nonresponder DBA/1 (H-2q) and SJL (H-2s) mice were passed over immunoadsorbents prepared from normal guinea pig serum (NGPS) or guinea pig anti-idiotype antiserum (anti-CGAT) specific for a common cross-reactive idiotype found on most anti-GAT antibodies in all mouse strains tested. Both the directly suppressive activity of the GAT-TsF and the ability of GAT-TsF to induce new suppressor T cells (Ts2) in vitro were adsorbed to and fully recoverable from the guinea pig anti-CGAT-Sepharose immunoadsorbent, while the TsF passed through the control NGPS-Sepharose without appreciable binding. The SJL GAT-TsF specifically eluted from anti-CGAT-immunoadsrobents was shown to still posses I-J determinants. These data provide evidence suggesting a sharing of V region structures between B-cell antibody and T-cell suppressor factor specific for an antigen (GAT) under Ir gene control, in agreement with earlier studies on T and B-cell alloreceptors, T-cell helper factors, and T and B-cell receptors for conventional antigens.

Phosphorylcholine-specific helper T cells in mice with an X-linked defect of antibody production to the same hapten.

F1 male mice with the CBA/N X-linked defect that are unable to produce plaque-forming cell responses to phosphorylcholine (PC) provide normal PC-specific helper T-cell activity when compared to F1 female littermates. Inhibition of helper activity with anti-idiotypic antiserum indicates that PC-specific T cells from both NBF1 female and male mice possess predominantly BALB/c myeloma protein HOPC-8 idiotypic determinants. Therefore, the CBA/N defect cannot be explained as a deletion of genes coding for V-region anti-PC specificities. The demonstration of helper activity in NBF1 male mice, which occurs in the absence of anti-PC antibody synthesis, also demonstrates the endogenous origin of the T-cell receptor.