Idiotypic analysis of hybridoma antibodies to branched synthetic polymer (Tyr, Glu)-Ala-Lys: idiotypic relationship with antibodies to linear random polymer (Glu60, Ala30, Tyr10)

The expression of three anti-GAT idiotypes, CGAT, Gte, and GA-1, on 17 C57BL/10 and four C3H.SW hybridoma anti-(T,G)-A--L antibodies was analyzed. These hybridoma anti-(T,G)-A--L antibodies exhibited two patterns of fine antigen binding specificity. The majority of the hybridoma antibodies bound the (T,G)-A--L, GT, and GAT polymers but not the GA polymer, and were designated as GT-reactive hybridoma antibodies. A minor population of hybridoma anti(T,G)-A--L antibodies bound to (T,G)-A--L but not to GT, GAT, or GA, i.e., (T,G)-A--L-specific. A complete correlation between fine antigen binding pattern and the expression of CGAT idiotype was demonstrated. None of the 21 hybridoma anti-(T,G)-A--L antibodies expressed the GA-1 idiotype. All of the GT-reactive and none of the GT-nonreactive hybridoma anti-(%,G)-A--L antibodies expressed the CGAT idiotype. Furthermore, the Gte idiotype was found on the majority of CGAT+-bearing C57BL/10 hybridoma anti-(T,G)-A--L antibodies. These results indicate that C57BL/10 anti-(T,G)-A--L antibody repertoire can be grouped into a minimum of three families; i.e., CGAT+ Gte+, CGAT+ Gte-, and CGAT- Gte- families, with the CGAT+ Gte+ family as the major compartment. This is confirmed by the high percentage idiotype binding of serum anti-(T,G)-A--L antibodies with anti-CGAT idiotypic antisera. Finally, anti-idiotypic antisera made against CGAT+ hybridoma anti-GAT or anti-(T,G)-A--L antibodies crossreact extensively with other CGAT+ hybridoma anti-GAT and anti-(T,G)-A--L antibodies. However, additional experiments demonstrated that CGAT+ hybridoma anti-(T,G)-A--L antibodies also possess private idiotypes.     

Production of antisera specific for idiotype(s) of murine anti-(T,G)-A--L antibodies.

Antibodies to the synthetic polypeptide (T,G)-A--L were raised in C57BL/10 and C3H.SW mice. For each strain, the anti-(T,G)-A--L antibodies from 10 animals were pooled, affinity purified on a (T,G)-A--L-Sepharose column, and used to immunize Lewis rats. The resulting rat antisera were adsorbed with insolubilized normal mouse globulin in order to remove anti-isotypic and anti-allotypic antibodies. The residual antibodies specifically inhibited the binding of (T,G)-A--L by anti-(T,G)-A--L as measured by a radioimmunoassay. The specificity of this inhibition was demonstrated as follows: 1) failure of the anti-(T,G)-A--L anti-idiotype to inhibit the binding of nuclease to anti-nuclease antibody of the same allotype; 2) failure of Lewis anti-[B10 anti-(T,G)-A--L] to inhibit C3H.SW anti-(T,G)-A--L and vice versa; 3) ability to absorb anti-C3H.SW anti-idiotypic activity on insolubilized C3H.SW anti-(T,G)-A--L but not on B10 anti-(T,G)-A--L. The same or cross-reactive idiotype(s) was present in the majority of individuals of each of these strains.     

Clonal analysis of the primary and secondary B cell responses of neonatal, adult, and xid mice to (T,G)-A--L

The splenic focus assay was used to clone B cells from neonatal, adult and xid mice in order to examine their primary and secondary responses to (T,G)-A--L. Adult precursor cell frequencies to (T,G)-A--L were achieved late in neonatal ontogeny. Primary xid B cells responded to DNP-HY but not to (T,G)-A--L in the splenic focus assay. The frequency of secondary B cells from (T,G)-A--L-primed xid mice was less than or equal to 10% that of secondary B cells from wild-type (non-xid or X/Xxid heterozygous) mice. Although xid B cells were poorly responsive to (T,G)-A--L in the splenic focus assay, (T,G)-A--L-primed xid mice could provide help as recipients for stimulation of wild-type primary and secondary B cells. It seems likely that the B2 subset contributes most of the splenic focus response to (T,G)-A--L. The fine specificities of antibodies produced by neonatal, xid, and adult (wild-type) B cell clones were analyzed using analogues of (T,G)-A--L. A specificity shift was observed between the adult primary and secondary antibody responses to (T,G)-A--L. Less than 10% of adult primary clones produced antibodies cross-reactive on (Phe,G)-A--L (recognizing A--L determinants or Phe,Glu determinants), whereas more than 70% of primary clones produced Tyr,Glu side-chain specific antibodies cross-reactive on GT. The percentage of clones producing GT-binding antibodies diminished in the secondary response, while the percentage of clones producing antibodies cross-reacting on (Phe,G)-A--L increased. Neonatal clones also produced mostly GT-binding antibodies but gave a higher percentage of (Phe,G)-A--L-cross-reacting antibodies than adult primary clones. The specificities of secondary antibodies produced by xid and wild-type B cell clones were dissimilar. First, xid secondary clones were "primary-like" in that no anti-A--L antibodies were detected. Second, clones whose antibodies bound side-chain determinants but not GT were produced in higher frequency by xid than by wild-type secondary B cells. The differential responsiveness of B cell subsets to antigen and regulatory signals may influence memory B cell generation and the specificity of antibodies produced in the primary vs secondary response.     

Xid and normal mice express a light chain-associated cross-reactive idiotype in response to (T,G)-A--L and (T,G)-A--L-mBSA

Rabbit anti-idiotypic (Id) antibodies were prepared against purified ascites anti-(T,G)-A--L antibodies (TGB5) that had been absorbed to remove A--L-specific antibodies and were specific for (T,G)-side chain determinants. Purified rabbit anti-TGB5 Id antibodies detected an allotype-independent, light chain-associated cross-reactive Id expressed by the majority of individual mice immunized with (T,G)-A--L, (T,G)-A--L coupled to methylated bovine serum albumin (mBSA), or the linear terpolymer GAT. Primary and secondary monoclonal hybridoma protein (HP) antibodies from X/Xxid heterozygous (wild-type) mice immunized with (T,G)-A--L and/or (T,G)-A--L-mBSA were analyzed for isotypy and were grouped into eight antibody fine specificity sets defined by the patterns of direct binding to the antigens (T,G)-A--L, (Phe,G)-A--L, (T,G)-Pro--L, GT, and A--L. Analysis of these primary and secondary HP for TGB5 idiotypy showed a preferential expression of the TGB5 Id among GT+-binding HP (antibody fine specificity sets 1 through 3). All of the primary GT+-binding HP and the majority of secondary GT+-binding HP (sets 1 through 3) were TGB5 Id+. Most but not all of the TGB5 Id+ HP bound GAT. Of the side-chain-specific HP (sets 1 through 7), 78% of primary HP vs 49% of secondary HP bound GT. By these criteria, the primary HP response appears more restricted than the secondary HP response, consistent with the idea that Id diversification and antibody heterogeneity are regulated and selected events occurring during memory B cell generation. Although xid mice produce less antibody than wild-type mice to (T,G)-A--L, the TGB5 Id was produced early in the primary response by both xid and wild-type mice immunized with (T,G)-A--L or (T,G)-A--L-mBSA, and was maintained as a detectable Id in equivalent amounts in their secondary serum antibody responses. These results support the idea that distinct B cell subsets, including the xid B cell subset, share the same immunoglobulin gene repertoire.     

Variable regions of antibodies to synthetic polypeptides. II. Analysis of variable region genes encoding antibodies specific for (T,G)-A--L

Monoclonal antibodies specific for the synthetic polypeptide antigen (T,G)-A--L have been produced in two strains of mice, C57BL/10 and C3H.SW. The genes encoding the variable (V) regions of these antibodies have been studied by using the DNA hybridization technique of Southern, as well as by gene cloning and sequencing. Hybridization of DNA from 14 different cell lines with a kappa-chain probe revealed that the different cell lines used one of two different gene rearrangements to encode the recombined V region gene. There was a perfect correlation between light chain rearrangement, idiotype expression, and fine specificity. Hybridization analyses of the heavy chain revealed a more complex pattern. Seven hybridomas had the rearranged heavy chain V region genes on a 4.4 kb EcoRI restriction fragment. Others were found on restriction fragments that differed in length by several hundred base pairs. The recombined heavy chain V region genes were cloned from three different hybridoma cell lines secreting anti-(T,G)-A--L antibodies, all of which express the same idiotype and fine specificity pattern. Restriction mapping and sequencing indicate that all three utilize the same V gene, identified as the 186-2 germline gene. However, different D and J genes are used to encode each of the antibodies. In contrast to the results seen in other antigen systems, heavy chain D and J genes do not have a major influence on idiotype expression and fine specificity of antibodies to the synthetic polypeptide (T,G)-A--L.     

Antigen-specific T-cell factor in cell cooperation and genetic control of the immune response.

Cell interactions between thymus-derived (T) and bone marrow-derived (B) lymphocytes in the antibody response appear to involve soluble T-cell mediators known as 'factors.' This paper describes the properties of a T-cell factor that has specificity for the inducing antigen, a synthetic polypeptide (T, G)-A--L, and is able to replace T cells in the thymus-dependent antibody response to (T, G)-A--L. Besides antigen specificity, the main features of the molecule are that it is nonimmunoglobulin; it has a molecular weight of about 50,000; and it is a product of the I-A subregion of the H-2 complex (the mouse major histocompatibility complex). These properties suggest that the factor is closely related to the T-cell receptor, which may, by inference, also be a product of the H-2 complex. The factor cooperates well with allogeneic B cells. It can also be absorbed by bone marrow cells and B cells. Studies on the genetic control of the immune response to (T, G)-A--L using the T-cell factor indicate that two immune response genes in the H-2 complex are involved in genetic control, one expressed in T cells and the other in B cells. This two gene hypothesis has been confirmed by showing that an F1 between two low responders to (T, G)-A--L can be a high responder.     

Genetic control of the immune response to (T,G)-A--L in C3H in equilibrium C57 tetraparental mice

When 15 C3H ? C57 tetraparental (allophenic) mice were analyzed for coat color, hemoglobin, and immunoglobulin allotype, all but two were shown to be chimeric. These 15 tetraparental mice were immunized with the synthetic polypeptide (T,G)-A--L, and the origin of the (T,G)-A--L-specific antibody produced was determined by using genetic markers (allotypes) on the immunoglobulin heavy chain constant region. Five tetraparental mice were high responders to (T,G)-A--L and had significant amounts of a (low responder) allotype antibody in their total serum. Three of these mice had significant amounts of anti-(T,G)-A--L antibody of the a (low responder) allotype. The antigen binding capacities of the a allotype fractions of these three were 4–5 times higher than the antigen binding capacities of immunized C3H (low responder) control mice. These results are compatible with the hypothesis that the inability of low-responder mice to produce significant amounts of anti-(T,G)-A--L antibody is a function of Ir-1A gene expression at the level of T cells.     

Cross-reactive idiotypic determinants on antibodies and antigen-specific helper T cell continuous lines

Anti-idiotypic antibodies produced in C57BL/6 mice (H-2b, Igh-1b) against (T,G)-A--L-specific antibodies of C3H.SW mice (H-2b, Igh-1j) were used to probe (T,G)-A--L-specific helper T cell lines and clones for the expression of idiotypic determinants on the cell surface of the monoclonal functional T cells. By using the fluorescence-activated cell sorter (FACS II), anti-idiotypic sera of individual mice that specifically bind C3H.SW anti-(T,G)-A--L antibodies were shown to stain significantly cells of the E-9M(+) continuous T helper line originated from C3H.SW (T,G)-A--L "educated" T cells. The same antisera did not react with a helper T cell line of C3H.SW origin specific to human gamma-globulin. They also did not stain a (T,G)-A--L-specific helper T cell line derived from CWB (H-2b, Igh-1b) mice, which differ from C3H.SW mice only in their heavy chain allotypes. Thus, the expression of the idiotypic determinants on the T cell lines appears to be antigen-specific and linked to the heavy chain allotypic marker as shown for the specific antibodies. Different clones derived from the E-9 M(+) line were tested their reactivity with the individual anti-idiotypic sera. All clones but one (1.11) were stained significantly. The clones were tested for their biologic activity and all of them except clone 1.11 were found to exert helper activity specific to (T,G)-A--L. Thus, individual anti-idiotypic sera against C3H.SW anti-(T,G)-A--L antibodies recognize cross-reactive idiotypic structures on the surface of antigen-specific monoclonal helper T cells.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Antigen-specific T cell factors: a fine analysis of specificity.

The specificity of T cell factors produced in presence of synthetic polypeptide antigens was studied. Factors prepared with either one of the three antigens: poly(Tyr,Glu)-poly(DLALa)--poly(Lys), (T,G)-A--L, poly(Phe,Glu)-poly(DLALa)--poly(Lys), (Phe,G)-A--L, and poly(His,Glu)-poly(DLALa)--poly(Lys), (H,G)-A--L, successfully cooperated with B cells for antibody production to the homologous as well as to the other two immunogens. Furthermore, the activity of a (T,G)-A--L-specific factor was removed after passage through immunoadsorbents built of Sepharose coupled to: (T,G)A--L, (Phe-G)-A--L and poly(Glu)-poly(DLAa)--poly(Lys), (G)-A--L, but not to poly (DLALa)--poly(LLys),A--L. No cross-reactivity was observed between (T,G)-A--L and poly(Tyr,Glu)-poly(Pro)--poly(Lys), (T,G)-Pro--L, at the level of T cell factors, as shown using the above approaches. These results lead to the conclusion that specificity of T cell factors, although not identical, is similar to that of antibodies.     

Regulation of helper cell activity by specifically adsorbable T lymphocytes.

Mice immunized with soluble proteins such as human serum albumin (HSA) or ovalbumin (OA) develop in their spleens antigen-specific T and B lymphocytes. These populations of lymphocytes can be separated from each other by different means; e.g. treatment with anti-theta-antiserum and complement removes selectively T lymphocytes, whereas passage through glass bead columns coated with mouse immunoglobulin (Ig): anti-Ig complexes creates a relatively pure population of T lymphocytes. During the course of such separation studies it was observed that the helper capacity of HSA (or OA) immune mouse spleen cells after Ig:anti-Ig column passage frequently was higher than expected from the enrichment in theta-positive cells. In addition, after adsorption onto antigen coated Bio-Gel beads this effect was even more pronounced, i.e., and increase in the relative helper capacity of about 3 or 4 times compared with an increase in the content of theta-positive cells from about 30% to 40 to 50% after adsorption. The present results will demonstrate that the increased helper capacity was a specific phenomenon which was regulated by theta-positive cells. The regulatory cells specifically adsorbed onto antigen-coated Bio-Gel beads have not been successfully eluted by EDTA or excess-free antigen so far, and they were still adsorbed after pre-incubation with anti-Ig antibodies under conditions where specific B lymphocyte adsorption was almost prevented.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. II. Requisite role of the monoclonal antibody-defined antigen systems in activation and proliferation of human and rat lymphocytes

A murine monoclonal antibody (MoAb) B3 to rat cells and MoAb HBJ127 and HBJ98 to human cells were found previously to recognize the homologous antigen systems (gp130 in the rat and gp125 in the human) which are predominantly distributed on the cell surface of proliferating cells of the respective species, and the expression of the antigen systems in lymphocytes were indicated previously to correlate closely with the activation and proliferation of the lymphocytes. In this respect, the in vitro effects of these MoAb on the nucleic acid synthesis, cell cycles, or proliferation of stimulated rat and human lymphocytes were examined by use of T cell-enriched and B cell-enriched cell populations. The addition of B3 MoAb to cultures diminished Con A-induced or allogeneic mixed lymphocyte culture-induced rat T cell proliferation and lipopolysaccharide-induced rat B cell proliferation, whereas B31 MoAb, which is unreactive with the gp130 antigen, did not inhibit these lymphocyte responses. Similarly, both HBJ127 and HBJ98 MoAb could inhibit the human lymphocyte proliferation in vitro, although HBJ127 MoAb showed about eight times greater inhibitory activity than did HBJ98 MoAb; HBJ127 MoAb almost completely inhibited the DNA synthesis of the Con A-stimulated lymphocytes at concentrations higher than 13 micrograms/ml. The flow cytometric analysis of the cellular nucleic acid contents with acridine orange-stained cells showed that when B3 MoAb and Con A were simultaneously added to unstimulated rat T cells, progression of the cell cycle was blocked at the G0 to G1 transition. In this culture condition, the appearance of the B3-defined antigen was arrested in a moderate level, as determined with fluorescein-stained cells. On the addition of B3 MoAb to the culture of the T cells after 24-hr Con A stimulation, the MoAb also strongly inhibited the cellular DNA synthesis, but it did not arrest the cell cycle at a certain phase and did not modulate the corresponding antigen. These data suggest that the B3 MoAb-defined antigen on the rat lymphocytes and the HBJ127/HBJ98 MoAb-defined antigen on the human lymphocytes may play some requisite roles not only in lymphocyte activation but also in the subsequent progression through the cell cycle to proliferate.     

Cell-mediated cytotoxicity against HLA-D-Region products expressed in monocytes and B lymphocytes

The cytotoxic effector cells that recognize HLA-D-region determinants and their precursors were characterized using monoclonal antibodies against human T lymphocytes and T-cell subsets. These studies were performed using MLC combinations giving rise to cytotoxic cells specific for both class I (HLA-A, B, C) and class 11 (HLA-D-region) antigens, and then tested against target cells displaying relevant antigens of only one class. Both class I and class II specific CTL (cytotoxic T lymphocytes) were inhibited by treatment with the OKT3 monoclonal antibody and complement, indicating that the effector cells were T lymphocytes. A major portion.of class II specific CTL, and their precursors, were inhibited by OKT4 and complement, while class I specific CTL from the same cultures were not. The T4+T8 — cell subset has previously been associated with helper or inducer functions, but not with cytotoxicity. The present findings indicate that class I and class 11 specific CTL, and their precursors, are different on the basis of the class of target antigen recognized and on the basis of surface phenotype detected by monoclonal antibodies.     

Idiotype-specific T lymphocytes. I. Regulation of antibody production by idiotype-specific H-2-restricted T lymphocytes

Cytotoxic T lymphocytes (CTL) specific for MOPC-104E myeloma cells of BALB/c origin could be induced in BALB/c, (BALB/c X BALB.B)F1, and (BALB/c X BALB.K)F1 mice. (BALB/c X BALB.B)F1 CTL activity specific for MOPC-104E was effectively inhibited by anti-H-2d but not by anti-H-2b alloantiserum. However, the activity was hardly blocked by specific anti-idiotypic antibodies to MOPC-104E. For further analysis of the recognition of idiotype on target cells by CTL, the effect of those lymphocytes on anti-dextran B1355S antibody-producing B lymphocytes, which have a cross-reactive idiotype to MOPC-104E, was investigated. Lymphocytes from the CTL population did inhibit antibody production by dextran-immune spleen cells, but those from the CTL population specific for irrelevant myeloma cells (MOPC-167) did not. The (BALB/c X BALB.K)F1 CTL population suppressed the antibody production of BALB/c but not of BALB.K. This indicates that F1 cells can preferentially see H-2 antigens of immunizing myeloma cells on target B lymphocytes. The inhibition of antibody production was antigen specific and was only restricted to the PFC that were inhibitable by anti-idiotypic antibodies. The surface phenotypes of the cells that inhibited the antibody production were Thy-1+, Lyt-1-, Lyt-2+, and I-J-. These results strongly suggest that CTL specific for MOPC-104E recognize self H-2 antigens simultaneously with idiotypic determinants on B lymphocytes. Possible immunoregulatory roles of idiotype-specific CTL on antibody production systems are also suggested.     

Specificity of H-2-linked Ir gene control in mice: demonstration of T helper cells recognizing branched synthetic polypeptides in low responder mice

This report provides evidence for the presence of T helper cells capable of recognizing the polypeptide antigens T6-A--L and (H,G)-A--L in low responder mice of H-2k and H-2b haplotypes, respectively. Mice were primed in vivo with the T6-A--L-avidin-(H,G)-A--L complex or, in the case of T6-A--L in H-2k mice, with the cross-reactive and permissive antigen T6-S--L. T helper cells cooperating with DNP-primed B cells could be rechallenged in vitro with the DNP-conjugates of T6--A--L or (H,G)-A--L, although the cells were of low responder type with respect to these antigens. This implies that T cell-macrophage interaction required for restimulation is apparently not defective in these low responders. The implications of these results for the concept of Ir gene control are discussed.     

Fine specificity of antibodies to the synthetic polypeptide poly(L-tyrosine, L-glutamic acid)-poly(DL-alanine)--poly(L-lysine) and its ordered analogs as followed by solid-phase radioimmunoassay

The fine specificity of antibodies against (T,G)-A--L and its ordered analogs (T-T-G-G)-A--L and (T-G-T-G)-A--L was studied. Fifty percent of the antibodies against (T,G)-A--L are directed toward the T-T-G-G determinants and 19% against T-G-T-G-like determinants. The rest of the antibody response to (T,G)-A--L is directed against determinants which exist in (T,G)-A--L but are not cross-reactive with either T-T-G-G- or T-G-T-G-like determinants. Although (T-T-G-G)-A--L and (T-G-T-G)-A--L differ only in the sequence of tyrosine and glutamic acid in their side chains, no crossreactivity was observed between antibodies toward the two ordered polypeptide antigens.     

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

The studies described in this paper were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes.     

Genetic control of immune responsiveness to poly (LPro)--poly (LLys)-derived polypeptides by histocompatibility-linked immune response genes in the rat.

In rats responsiveness to branched synthetic polypeptides carrying a Pro--L backbone, such as (T,G)-Pro--L or (Phe,G)-Pro-L and to Pro--L itself is controlled by Ir genes which are linked to the major histocompatibility genes. The level of antibody production to these polypeptides does not fall into strict high or low responder categories but covers the range in between. (T,G)-pro--L and Pro--L elicit a very similar response pattern which, however, differs from that obtained with (Phe,G)-Pro--L. Anti-(T,G),Pro--L antibodies do not cross-react with (T,G)-A--L, but do so extensively with Pro--L. Anti-(Phe,G)-Pro--L antibodies show cross-reactivity to (Phe,G)-A--L only when the antibody-producing strain is a high responder to (Phe,G)-A--L. These results when considered in view of data obtained in mice on genetic control of the immune response to (T,G)-Pro--L suggest that at least two unlinked Ir genes are involved in controlling anti-Pro--L responsiveness.     

T lymphocyte-enriched murine peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation with anti-Ia antisera.

The recent development of a reliable murine T lymphocyte proliferation assay has facilitated the study of T lymphocyte function in vitro. In this paper, the effect of anti-histocompatibility antisera on the proliferative response was investigated. The continuous presence of anti-Ia antisera in the cultures was found to inhibit the responses to the antigens poly (Glu58 Lys38 Tyr4) [GLT], poly (Tyr, Glu) ploy D,L Ala-poly Lys [(T,G)-A--L], poly (Phe, Glu)-poly D,L Ala-poly Lys [(phi, G)-A--L], lactate dehydrogenase H4, staphylococcal nuclease, and the IgA myeloma protein, TEPC 15. The T lymphocyte proliferative responses to all of these antigens have previously been shown to be under the genetic control of major histocompatibility-linked immune response genes. The anti-Ia antisera were also capable of inhibiting proliferative responses to antigens such as PPD, to which all strains respond. In contrast, antisera directed solely against H-2K or H-2D antigens did not give significant inhibition. Anti-Ia antisera capable of reacting with antigens coded for by genetically defined subregions of the I locus were capable of completely inhibiting the proliferative response. In the two cases studied, GLT and (T,G)-A--L, an Ir gene controlling the T lymphocyte proliferative response to the antigen had been previously mapped to the same subregion as that which coded for the Ia antigens recognized by the blocking antisera. Finally, in F1 hybrids between responder and nonresponder strains, the anti-Ia antisera showed haplotype-specific inhibition. That is, anti-Ia antisera directed against the responder haplotype could completely block the antigen response controlled by Ir genes of that haplotype; anti-Ia antisera directed against Ia antigens of the nonresponder haplotype gave only partial or no inhibition. Since this selective inhibition was reciprocal depending on which antigen was used, it suggested that the mechanism of anti-Ia antisera inhibition was not cell killing or a nonspecific turning off of the cell but rather a blockade of antigen stimulation at the cell surface. Furthermore, the selective inhibition demonstrates a phenotypic linkage between Ir gene products and Ia antigens at the cell surface. These results, coupled with the known genetic linkage of Ir genes and the genes coding for Ia antigens, suggest that Ia antigens are determinants on Ir gene products.     

Antigen presentation by hapten-specific B lymphocytes. V. Requirements for activation of antigen-presenting B cells

Splenic B cells specific for the haptens, 2,4,6-trinitrophenyl (TNP) or fluorescein isothiocyanate (FITC) were cultured with a range of concentrations of unmodified or TNP- or FITC-conjugated conalbumin and the conalbumin + I-Ak-specific, interleukin (IL) 1-dependent helper T cell clone, D10 . G4, in the presence and absence of IL-1. Lymphokine secretion, T cell proliferation, and antibody secretion by B cells all exhibited identical antigen dose responses. Thus, hapten-binding B cells presented low concentrations of haptenated conalbumin for activation of both the T and the antigen-presenting B cells. Whereas proliferation of D10 . G4 required the addition of IL-1, both lymphokine production and stimulation of B cells to antibody secretion occurred without exogenous IL-1. These results demonstrate that when B lymphocytes function as presenting cells for antigens that bind to their immunoglobulin receptors, activation of the responding T cells and the B cells themselves occur at similar concentrations of antigen. Moreover, for functional T-B interactions, antigen-presenting B and responding T lymphocytes constitute a complete system that requires no other accessory stimuli, whereas clonal expansion of T cells is dependent on accessory factors such as IL-1. Finally, since D10 . G4 secretes IL-4 but neither IL-2 nor interferon-gamma, our results demonstrate that differentiation of B cells as a consequence of direct ("cognate") interactions with helper T cells as well as of bystander B cells can occur in the absence of IL-1, IL-2, and interferon-gamma.