Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. III. Single histocompatibility antigens dominate the male antigen

Immunization of mice with multiple non-H-2 histocompatibility antigens results in the generation of cytolytic T lymphocytes that are specific for a limited number of immunodominant antigens. The experiments presented in this communication were designed to reveal immunodominance in pairwise combinations of autosomal and sex-linked non-H-2 histocompatibility (H) antigens. Priming and boosting responders with the male antigen, H-Y, paired with the H-4.2, H-7.1, or H-3.1 antigens, resulted in the generation of cytolytic T cells specific for the autosomal H antigens but not the H-Y antigen. Furthermore, co-immunization and boosting of C57BL/6 female responder spleen cells with BALB.B male cells resulted in the generation of cytolytic T cells specific for the BALB.B immunodominant antigens but not H-Y. No dominance was observed in H-4-plus H-7-incompatible combinations. Co-immunization of three different H-3 congenic strains with H-3.1 plus H-Y demonstrated that an efficient anti-H-3.1 T cell response is required for observing H-3.1 immunodominance over H-Y. Co-expression of H-3.1 and H-Y on the same priming and boosting cells was required for immunodominance. In fact, immunization with H-3.1 and H-Y presented on different cells resulted in normal generation of H-Y-specific cytolytic T cells, but no generation of H-3.1-specific cytolytic T cells resulted unless H-Y-specific cells were stimulated in the mixed lymphocyte cultures. These observations suggest that in vitro T cell responses to paired, non-H-2 H antigens may be independent, competitive, or synergistic, depending on the identity of the antigens and the priming and boosting conditions.     

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.     

Modulation of lymphoproliferation and oxidative burst by herpes-transformed tumors.

In this study, herpes simplex virus Type 2 (HSV-2)-transformed cells (H238) and conditioned medium (CM) from H238 cell cultures were studied with respect to their effects on lymphoproliferation and the chemiluminescent oxidative burst of phagocytic cells. The H238 cells expressed a nuclear antigen detectable by fluorescent antibody testing using pooled sera from tumor-bearing mice, but no HSV-1 or HSV-2 cell membrane antigens could be found using specific monoclonal antibodies. BALB/c mice subcutaneously injected with 1 X 10(6) H238 cells developed progressively growing fibrosarcomas and depressed T lymphocyte blastogenesis in response to phytohemagglutinin (PHA) by 6 weeks post-injection when compared to non-injected controls. In contrast, oxygen radical production was increased by nearly 28-fold in the tumor-bearing subjects at this time. Incubation of normal mouse spleen cells in 100 microliters to 500 microliters of CM/ml resulted in significant dose-dependent suppression of PHA-induced lymphoproliferation. This was seen when the total spleen cell population was used, as well as after removal of the adherent cells, thereby suggesting that the inhibitory effect was not due to activation of adherent suppressor cells by the CM. However, the oxidative burst of total and adherent spleen cells from normal mice was significantly enhanced by the presence of either the H238 cells or their CM. In contrast, oxygen radical production by J774A.1 cells (a BALB/c mouse macrophage cell line) was depressed by H238 cells. Our results show that H238 tumors can alter lymphocyte as well as phagocytic cell functions both in vivo and in vitro. These tumor-induced modulations may occur via secretion of soluble factors or direct cell-to-cell interactions and, thus, may influence the outcome of immunotherapy in the tumor-bearing host.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus]

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.     

Immunodominance in the T-Cell response to multiple non-H-2 histocompatibility antigens

Immunization of C57BL/6 mice with BALB.B spleen cells in vivo and subsequent boosting in mixed lymphocyte culture result in the generation of cytolytic T lymphocytes (CTLs) which are specific for a limited number of immunodominant antigens. Experiments are described which suggest the existence of a hierarchy of immunodominance in this donor: host combination. Two antigens, CTT-1.3 and CTT-2.3, are dominant in the C57BL/6 anti-BALB.B CTL response. The distribution of these antigens among CXB recombinant inbred (RI) strains suggests that they segregate as single gene traits. Elimination of the CTT-1.3 and CTT-2.3 antigens by complementation in the responder, or elimination from the priming and boosting stages by the selection.of CXB RI strain mice as responders or stimulators, reveals a second level of immunodominant antigens which include CTT-3.3 and CTT-4.3. CXB mice which express one of the CTT-1.3 or CTT-2.3 antigens will produce CTLs specific for the other antigen upon priming and boosting with BALB.B cells. Expression of both antigens in responders results in the generation of CTLs specific for the second level, dominant antigens. Immunodominance is not confined to the C57BL/6 anti-BALB.B system but can also be observed in the BALB.B anti-C57BL/6 and B10.D2 anti-DBA/2 systems. Finally, generation of CTLs following priming and boosting with dominant and dominated antigens presented on different cells confirmed that immunodominance can only be observed when the dominant and dominated antigens are presented on the same cells. These observations suggest that immunodominance is revealed at the level of antigen-presenting cells primarily involved in vivo priming.     

Effect of colchicine derivatives on the antibody response inducedin vitro

Effect of 44 colchicine derivatives on the induction of antibody response in tissue cultures was tested. Lymphatic cells from the spleen of BALB/c mice were cultivated with antigen (sheep red blood cells) and the number of antibody forming cells was determined by the plaque technique. Most compounds with the immunoinhibitory effect are derived from the colchicine formula (I). The effect was increased by introducing ethyl, formyl or methylenedioxide groups. Colchinols exerted very good immunoinhibitory effect resulting by contraction of tropolone ring C into the aromatic one. A complete loss of the effectivity was detected in the case of glucoside of colchicine, colchiceine, isocolchicine, oxycolchicine, allocolchicine and in lumiderivatives of colchicine. No correlations between the immunoinhibitory effect, toxicity and stathmokinetic effect were detected: decrease of cell viability and arrest of mitoses were not observed in cultured lymphocytes within the range of the immunoinhibitory effect. The effect of colchicine derivatives was manifested as the inhibition of lymphocyte blastogenesis, which is probably the result of membrane transport blockade.     

Sex-related immunocompetence of BALB/c mice. I. Study of immunologic responsiveness of neonatal, weanling, and young adult mice.

The responses of lymphoid cells from the thymus, lymph nodes, and spleen of male and female BALB/c mice were evaluated to determine if sex-related variations in immune expression could be found. Immunologic assays used included blastogenic responses to mitogens, mixed lymphocyte responses, and direct and indirect measurement of plaque-forming cells against soluble and particulate antigens. The results indicated that responses of spleen cells from young adult female mice were higher than those of males in all comparative tests. Little or no differences between the sexes were observed in the mitogenesis of lymph nodes and thymuses. Newborn mice did not demonstrate the sex-associated immune differences. Among the weanling mice slight differences between male and female spleen cells responsiveness to mitogenic agents were observed.     

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).     

Enhancive and suppressive effects of cytomegalovirus on human lymphocyte responses in vitro.

Virus-specific lymphocyte proliferation in the presence of cytomegalovirus (CMV) without and with monocytes was studied in healthy persons. Three categories of lymphocyte response could be distinguished: seropositive low responders, naturally high responders, and lymphocyte populations responding well to CMV antigen in the presence of added CMV-incubated autologous monocytes. This latter category could be identified by preincubating autologous monocytes with CMV. CMV-seronegative persons were nonresponders. Early CMV antigens were produced in monocytes but not in lymphocytes by all CMV isolates. Infection of monocytes as detected by antibody to early viral protein did not appear to abort the antigen-presenting ability. The virus-specific responding lymphocytes were mainly of the T4+ phenotype. In contrast, addition of CMV to polyclonal mitogens significantly suppressed total lymphocyte DNA synthesis. CMV thus may have an enhanced virus-specific stimulatory effect on lymphocytes together with monocytes but a suppressive effect on the total lymphocyte population.     

Inhibition of the mixed lymphocyte culture response with anti-Lyb-4.1 serum 1,2,.

C57BL/Ks anti-L1210 serum, which recognized a non-H-2-linked B cell alloantigen, designated Lyb-4.1, specifically blocked the mixed lymphocyte culture (MLC) response to allogeneic cells that expressed the Lyb-4.1 determinant. Anti-Lyb-4,1 serum blocked the MLC response across H-2 and MLC disparities. To test that this effect was not the result of a toxic or nonspecific cell-coating action, the response of parental cells to F1 lymphocytes was studied in combinations in which only one parent expressed the recognized allele. MLC stimulation was blocked only when the responding parental cell recognized on the F1 cell H-2 or MLs disparities which were derived from the parent which possessed the Lyb-4.1 antigen. Several DBA/2 tumors were characterized by cytotoxic and quantitation absorption assays for the presence of the B cell antigen. The presence of the antigen correlated with the ability of a limited number of tumors to stimulate the MLC response of H-2d identical BALB/c lymphocytes. An increased representation of the B cell alloantigen was found on the transformed B lymphoblast cell line in comparison to splenic B lymphocytes.     

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.     

Stimulation of lymphoid cells from normal and immune mice by syngeneic BALB/c plasma cell tumors.

Lymphoid cells from normal and immunized BALB/c mice could be stimulated in vitro by syngeneic PCT contrasted with an absence of response to a number of other tumors. Maximal responses of normal cells to PCT were found to occur 5 days after the initiation of the cultures at an optimal responding:stimulation cell ratio of 1:2. MLTI activity of normal cells could not be blocked or enhanced by PCT myeloma protein products indicating that MLTI reactivity was directed against non-idiotypec cell surface determinants. Lymphoid cells from immunized mice demonstrated increased MLTI responses to cells of the immunizing tumor but not to other PCT, indicating that the post-immunization MLTI responses were primarily to individual rather than shared tumor cell surface antigens. Activity of both normal and immunized spleen cells was found to involve thymus-derived lymphocytes. The persistence of residual MLTI activity after treatment with anti-theta serum and complement, however, implicated participation of non-theta antigen-bearing cells in MLTI reactivity. From these data, we conclude that lymphoid cells from un-immunized mice are capable of T cell-dependent reactivity to syngeneic PCT-associated antigens and that elevations in these reactivities after immunization may reflect specific cellular immune responses.     

Specific neonatal induction of functional tolerance to allogeneic Mls determinants occurs intrathymically and the tolerant state is Mls haplotype-specific

The studies described show that functional Mls specific tolerance, which we previously reported in peripheral spleen cells of mice injected within 24 h of birth with Mls incompatible spleen cells, is observable in the thymus on day 6. At this time a significant positive response is not detectable in spleen cells of normal mice. In the limiting dilution assay, we are able to detect a more profound depletion than others have found with anti-TCR antibodies. The tolerance in the thymus is not due to active suppression or simple dilution of responders by nonresponsive cells of the neonatal inoculum. By tolerizing BALB/c (Mls(b,c] mice with spleen cells from Mls(a) congenic mice, we show that Mls(a) incompatibility alone is sufficient for tolerance induction. Data from these experiments also show that the T cells seen responding at high frequency to stimulators from mice expressing Mls(a) determinants, as well as many other non-H-2 encoded incompatibilities, are indeed responding to Mls(a) determinants. In addition, experiments involving neonatal injection of Mls(b) mice with Mls(a) and Mls(c) spleen cells show no cross-reactivity of tolerance between Mls(a) and Mls(c) haplotypes. Our findings also show coexpression of determinants common to both Mls(a) and Mls(c) haplotypes by the Mls(d) haplotype. In all, the described experiments elucidate a pattern of Mls determinant specific hyporesponsiveness, in mice neonatally injected with appropriate allogeneic spleen cells, which bears all the hallmarks of functional, alloantigen specific, clonal deletion type tolerance.     

The influence of glutamine, its decomposition products, and glutaminase on the transformation of human and mouse lymphocytes.

The extent of blast transformation for human and BALB/c mouse lymphocytes has been examined over a wide range of glutamine concentrations with several agents which initiate blastogenesis. Maximum [3H] thymidine incorporation was seen at 0.5 mM glutamine for lymphoid tissues stimulated in the following manner: human and BALB/c splenic and peripheral blood lymphocytes with phytohemagglutinin, BALB/c splenic lymphocytes with lipopolysaccharide, and BALB/c vs C3H/HeJ two-way mixed lymphocyte cultures. The inhibition of blastogenesis exerted by glutamine concentrations greater than 0.5 mM could not be reversed by washing and reculturing the cells at 0.5 mM glutamine. To elucidate the reason for inhibition by higher glutamine concentrations, the products of spontaneous glutamine decomposition, L-2-pyrrolidone-5-carboxylic acid and ammonia were tested for their in vitro influence on BALB/c splenocyte blastogenesis. Pyrrolidone-carboxylic acid, in concentrations up to 5 mM, was without effect. In contrast, ammonia concentrations exceeding 1 mM became increasingly more inhibitory. The genesis of inhibitory levels of ammonia in culture medium was confirmed and has been considered as primarily responsible for inhibiton by high glutamine. Addition of Escherichia coli glutaminase (pH optimum 4.9) to cultures of BALB/c splenocytes or human peripheral blood lymphocytes had no effect on either the extent of blastogenesis of these tissues or the glutamine levels in their culture medium.     

Endogenous MMTV proviruses induce susceptibility to both viral and bacterial pathogens

Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.     

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.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.