Modulation of SLE induction in naive mice by specific T cells with suppressor activity to pathogenic anti-DNA idiotype.

T cells (CD8+) with specific suppressor activity against anti-dsDNA antibody (16/6 Id+) were generated in vitro. The cells were established from BALB/c-enriched T cells exposed in vitro to silica beads coated with the pathogenic anti-DNA idiotype, 16/6. The idiotype specificity of the suppressor cells was demonstrated by (a) specific induction of a decrease in proliferative response of T helper cell lines specific for the pathogenic idiotype (16/6 Id), when exposed to the idiotype, with no effect on T cell lines with other specificities, e.g., against human IgM or synthetic polypeptide. (b) Effectively suppressing in vitro antibody production of anti-16/6 antibody, employing 16/6-primed B cells and specific helper T cell line. The 16/6 Id-specific Ts cells were found to be MHC restricted. Weekly intravenous injections of 10(7) 16/6 Id-specific Ts cells given to BALB/c mice at different stages of experimental SLE disease prevented the clinical, serological, and pathological manifestations. This effect was characterized by decreased titers of autoantibodies (e.g., anti-DNA, anti-Sm antibodies) in the sera, by abolishment of the proteinuria, leukopenia, and the increased ESR, followed by decreased immunoglobulin deposition in the kidneys. Treating the mice with control IgM-specific T cells did not affect the above parameters. These studies demonstrate the ability to generate Ts cells specific for pathogenic idiotypes. The method might be employed therapeutically to modulate the course of autoimmune conditions.     

Systemic autoimmune disease caused by autoreactive B cells that receive chronic help from Ig V region-specific T cells

B cells present BCR V region-derived Id-peptides on their MHC class II molecules to Id-specific CD4+ T cells. Prolonged Id-driven T-B collaboration could cause autoimmune disease, but this possibility is difficult to test in normal individuals. We have investigated whether mice doubly transgenic for an Id+ Ig L chain and an Id-specific TCR develop autoimmune disease. Surprisingly, T cell tolerance was not complete in these mice because a low frequency of weakly Id-reactive CD4+ T cells accumulated with age. These escapee Id-specific T cells provided chronic help for Id+ B cells, resulting in a lethal systemic autoimmune disease including germinal center reactions, hypergammaglobulinemia, IgG autoantibodies, glomerulonephritis, arthritis, skin affection, and inflammatory bowel disease. Inflamed tissues contained foci of Id-driven T-B collaboration, with deposition of IgG and complement. The disease could be transferred with B and T cells. The results demonstrate a novel mechanism for development of autoimmune disease in which self-reactive Id+ B cells receive prolonged help from Id-specific T cells, thus bypassing the need for help from T cells recognizing conventional Ag.     

Expression and regulation of a recurrent anti-erythrocyte autoantibody idiotype in spleen cells from neonatal and adult BALB/c mice.

BALB/c spleen cells depleted of CD8+ T cells generate an autoantibody response to mouse RBC (MRBC) when cultured 5 days in the presence of syngeneic RBC. More than 80% of the cells secreting anti-MRBC antibody are blocked by an antiidiotypic mAb that recognizes the G8 Id. This G8 Id was originally identified in an autoimmune NZB mouse derived anti-MRBC mAb and later characterized as a dominant Id in NZB anti-MRBC autoantibodies. Furthermore, the CD8+ regulatory T cells that control this autoimmune response in BALB/c mice are specifically eliminated by cytotoxic treatment with the G8 mAb + C, suggesting that the regulatory cells recognize the G8 Id. Spleen cells from neonatal BALB/c mice, which lack those regulatory cells can generate an in vitro antibody response to MRBC without depletion of CD8+ cells. More than 80% of these AFC were also found to express the G8 Id. We propose that Id determinants on autoantibodies that are produced neonatally induce Id-specific regulatory cells that maintain peripheral tolerance to self-RBC throughout the life of normal animals.     

Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.     

Vaccination with membrane-associated idiotype provides greater and more prolonged protection of animals from tumor challenge than the soluble form of idiotype

The purpose of this work was to compare the efficacy of immunizing mice with a soluble vs a cell-associated form of a tumor Ag. A murine B cell tumor (2C3), which displays an Id on its cell surface, grows progressively and gives rise to Id-negative tumor variants in nonimmunized animals. We previously reported that the tumor variants arise as a consequence of Id-specific T cell suppression of the Id+ tumor. The Id-specific effector T cells are CD4+, CD8-. Based upon these findings vaccination protocols have been designed and tested to determine whether the expansion of tumor-specific effector T cells would eliminate the Id+ tumors and prevent the subsequent generation of Id- tumor variants in vivo. MHC-restricted T cells typically recognize soluble Ag subsequent to modification by an APC, and APC may ultimately express a processed form of the Ag that is different from that expressed on the surface of the tumor cells. Based upon this assumption, the efficacy of immunizing mice with cell-associated 2C3 Id was compared to immunization with a soluble form of the same Id. Mice were immunized with either irradiated 2C3 cells or syngeneic spleen cells to which 2C3 protein was covalently linked. These immunization protocols provided a complete and lasting protection against a tumor challenge of up to 1 x 10(6) tumor cells. In contrast, most mice hyperimmunized with a soluble form of the Id did not survive this level of tumor challenge in spite of the production of significant levels of anti-Id antibodies. Mice immunized with the soluble form of the Id, which did survive, produced slowly progressing tumors expressing a 1000-fold less of the marker Id. These results illustrate the importance of understanding and properly exploiting a host's natural response to a tumor-specific Ag when designing effective immunization protocols for cancer therapy.     

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)     

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.     

Evidence for idiotypic- and antiidiotypic B-B cellular interaction with the use of cloned antiidiotypic B cell line

Immunization of BALB/c mice with MOPC104E myeloma protein induces antiidiotypic B lymphocytes that have Id-specific enhancing activity on antibody production. The B-B cell interaction was restricted to both Igh and class II MHC. However, anti-Thy-1 and C-treated splenic B cells were maintained for more than 1 y in a mixture of Con A-stimulated splenocyte culture supernatant and synthetic medium. In applying the long term culture method, we have established a cloned B cell line named B19-1d, B19-1d cells are specific to MOPC104E or J558 cross-reactive Id and they express surface mu, lambda but no Ly-1. B19-1d do not spontaneously secrete Ig but produce them upon stimulation with bacterial LPS. The effect of B19-1d cell line on idiotypic antibody production was tested. Addition of only 10 to 100 B19-1d cells into dextran-immune B cell culture greatly enhanced the Id+ antidextran antibody responses. On the contrary, the antidextran antibody production was suppressed by the higher doses of B19-1d cells. The effective cooperation between dextran-immune B cells and B19-1d cloned B cells was restricted to class II MHC. The role of idiotypic- and antiidiotypic B-B cell interaction in immune regulation and repertoire generation was suggested.     

The role of T cells in IgG production; thymus-dependent antigens induce B cell memory in the absence of T cells.

B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.     

Polyclonal activation of the murine immune system by an antibody to IgD. X. Evidence that the precursors of IgG1-secreting cells are newly generated membrane IgD+B cells rather than the B cells that are initially activated by anti-IgD antibody

Injection of BALB/c mice with an affinity-purified goat antibody to mouse IgD (GaM delta) stimulates T cell-independent B cell activation as well as later T cell activation. Activated T cells then induce polyclonal differentiation of B cells into IgG1-secreting cells, which results in an approximately 100-fold increase in serum IgG1 level. It is not known whether the same B cells that are initially activated by GaM delta are the progenitors of the IgG1-secreting cells. To investigate this issue a system was developed in which CB20 mice, which are congenic to BALB/c mice but express Ig of the beta allotype rather than the BALB/c alpha allotype, were injected with GaM delta and simultaneously or subsequently also received BALB/c B cells. The IgG1 response generated by the donor BALB/c B cells was quantitated by an assay specific for IgG1 of the alpha allotype. Our experiments with this system indicate that: 1) BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta generate a much larger IgG1 response than do BALB/c B cells transferred simultaneously with GaM delta antibody; 2) B cells that express membrane IgD generate the great majority of this response; 3) differences in the magnitudes of the responses of BALB/c B cells transferred at different times after CB20 mice were injected with GaM delta antibody cannot be explained by differences in homing of the donor B cells to the host spleen or by short survival of donor BALB/c B cells after their transfer; and 4) the response made by donor BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta is proportionate to donor cell representation in the host spleen 1 day after their transfer, whereas the response made by donor cells transferred simultaneously with GaM delta is disproportionately small. These observations suggest that most of the IgG1 antibody made by GaM delta-injected mice is generated by newly produced, mIgD+ B cells that appear approximately 2 days after GaM delta injection, rather than by those B cells that are present in the spleen at the time of GaM delta injection, and support the view that signals that induce B cell secretion of Ig require an interaction with at least partially activated Th cells.     

A comparison of the IgG isotype expression in the so-called thymus-independent immune responses to alpha(1----3) and alpha(1----6) dextran

This paper presents data on the IgG antibody response against two "thymus-independent" dextran (Dex) antigens from Leuconostoc mesenteroides, alpha(1----3) Dex B 1355S and alpha(1----6) Dex B 512F in BALB/c and C57BL/6 mice, which are considered to be responders or low responders to the respective antigen. The data point toward three common rules governing the two anti-Dex responses despite immunogenetic and antigenic disparities: (1) age dependency of the IgG isotype regulation of the response; (2) down-regulation of IgG isotype expression by T cells; and (3) individually determined preposition for IgG isotype formation in a given animal.     

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.     

Analyses of Idiotypes on Anti-α-1, 6-Dextran Antibodies in Mice

When mice of strains C57BL/6, C3H/He, and BALB/c were immunized with native dextran B512, only a small amount of IgM antibody was produced, but a substantial amount of anti-dextran antibody of IgG class was produced after immunization with a conjugate of dextran T10 and keyhole limpet hemocyanin regardless of the mouse strain used. Isoelectric focusing (IEF) spectra revealed limited heterogeneity of anti-dextran antibody of IgG class with strict consistency in all individual sera from C57BL/6 mice, even after secondary immunization, whereas antibodies from C3H/He and BALB/c mice showed more heterogeneous IEF spectra with some individual variations. Rabbit anti-idiotypic (Id) antibodies were raised by immunization with a subfraction of anti-dextran antibody of IgG class from C57BL/6 mice, which showed major bands focused at around pH 7.7 upon IEF. It was found by using the anti-Id antibodies that virtually all anti-dextran antibody molecules of both IgG and IgM classes from C57BL/6 mice possessed common Id determinants which can be classified into two specificities, one specific for antibody from C57BL/6 mice and the other cross-reactive with antibodies from BALB/c and C3H/He mice. About 80% of the antibody molecules from BALB/c and less than 20% of those from C3H/He mice were positive for the interstrain cross-reactive Id. Both Id determinants seemed to be closely related to the antigen binding sites, or at least to reside in the vicinity of the antigen binding sites of anti-dextran antibody.     

T cell-induced Ig allotypic suppression in mice. II. Both CD4+ CD8- and CD4- CD8+ T cell subsets from sensitized Igha mice are required to induce suppression of Igh-1b allotype expression

We established the phenotype of T splenocytes (Ts) from Igha/a BALB/c mice sensitized against B splenocytes from the Ighb/b CB20 congenic mice that induce Igh-1b (IgG2a of the Ighb haplotype) suppression. This was achieved by studying the action of anti-T cell subset mAb on the capacity of Ts to induce this chronic allotypic suppression in Igha/b (BALB/c x CB20)F1 mice. The Ts were treated with cytotoxic anti-mouse CD4 or anti-mouse CD8 rat mAb in vitro before their injection into the Igha/b newborns or in vivo after their injection into the Igha/b newborns. Exposure to either anti-CD8 or anti-CD4 mAb in vitro or in vivo leads to loss of the capacity of Ts to induce Igh-1b allotypic suppression. Mixing CD4+-cell-depleted Ts and CD8+-cell-depleted Ts preparations restored the capacity of the cells to induce Igh-1b suppression. Thus, both CD4+ CD8- Ts and CD4- CD8+ Ts are required for the induction of this allotypic suppression. Bone marrow cells and B splenocytes from Igh-1b-suppressed adult Igha/b mice were shown to be able to durably express Igh-1b when transferred into irradiated Igha/a BALB/c hosts whereas whole spleen cells from such donors failed to do it. Abrogation of Igh-1b suppression by in vivo anti-CD8 mAb treatment was achieved in adult Igha/b heterozygotes but with a lower efficiency than in adult Ighb/b homozygotes, all being chronically Igh-1b suppressed. The CD4- CD8+ cell population essential for maintaining this suppression is resistant to in vivo 600 rad irradiation and seems to be slightly inhibited by in vivo administration of free Igh-1b.     

Interaction of idiotype-specific T suppressor factor with the hapten-specific third-order T suppressor subset results in antigen-nonspecific suppression

The interaction between the third-order T suppressor (Ts3) cell and the idiotype (Id)-specific second-order Ts factor (TsF2) was studied in the phenyltrimethylamino (TMA) hapten system. The experimental system which we used allowed the independent analysis of induction and activation requirements of Ts3. The procedure consisted of inducing the Ts3 in vivo and activating the enriched T-cell populations containing Ts3 in vitro with TsF2. The suppressive potential was then tested in mice previously primed for delayed-type hypersensitivity responses which were also treated with cyclophosphamide to deplete Ts3 and other drug-sensitive Ts cell types. Using this experimental system, it was found that the Id-specific TsF2 was required for the in vitro activation of Ts3. Furthermore, the TsF2 activated only the homologous and not heterologous antigen-primed Ts3-containing T cells and moreover, the target of TsF2 was found to be the Ts cells bearing hapten-specific receptors. Once the TMA hapten-specific Ts3 was activated with TsF2, the ensuing suppression was antigen nonspecific. The data demonstrate that the Ts3 represents a final effector Ts cell type in the TMA system.     

T cell regulation of immunoglobulin class expression in the B cell response to TNP-Ficoll: characterization of the T cell responsible for preferential enhancement of the IgG2a response

Syngeneic T cells injected into athymic nu/nu mice cause a preferential enhancement in the amount of IgG2a anti-TNP Ab produced by these mice to TNP-Ficoll. This enhancement appears to be caused by T cell effects on the IgG switching pathway. Through the use of F1----parent chimeras, the helper T cells were shown to affect TNP-Ficoll-responsive B cells in an H-2-unrestricted manner. The ability of T cells to mediate this IgG2a enhancement did not appear to be unique to any particular murine genetic background, because it was observed with T cells and nu/nu mice of C57BL/10, BALB/c, CBA/Ca, and B10.D2 strains. Priming of T cell donors with Ficoll or TNP-Ficoll did not increase the ability of splenic T cells, on a per cell basis, to enhance the IgG2a Ab response to TNP-Ficoll. The T cell population responsible for modulating the isotypic response was found to be sensitive to C-mediated cytotoxicity with both anti-Lyt-2 and anti-Lyt-1 hybridoma Ab. Although T cells from both the thymus and the spleen expressed enhancing activity, splenic T cells were more effective, on a per cell basis, than were thymocytes. The observations suggest that T cells that appear to enhance the switch to IgG2a in TNP-Ficoll-responsive B cells are not effectively primed by the antigen and interact with TNP-Ficoll-activated B cells through an H-2-unrestricted mechanism.     

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.     

A study of autologous anti-idiotypic antibody-forming cells in mice of different ages and genetic backgrounds.

Antibody response to phosphorylcholine, an immunodominant epitope of Streptococcus pneumoniae R36a (Pn), is characterized by a public idiotype, T15, that is expressed on a large proportion of antibody molecules produced by all mouse inbred strains. The ability of the immune system to produce an autologous antibody to T15 upon immunization with Pn vaccine was investigated using a modified ELISA plaque assay for detection of single antibody-forming cells (AFC). The limit of ELISA assay for detection of specific anti-T15 AFC is approximately 300 cells/spleen. However, our studies failed to detect any autologous anti-T15 AFC in the course of the primary antibody response to Pn vaccine in young/adult (2-4 months) BALB/c and C57BL/6 mice. Aged mice (20-22 months) also failed to develop any specific auto-anti-T15 AFC upon the primary Pn immunization, despite the fact that the anti-Pn response in these animals changes both quantitatively and qualitatively. In order to generate specific anti-T15 AFC, BALB/c mice had to be immunized repeatedly with Pn vaccine (four weekly injections) or immunized directly with T15 protein in CFA. Different results were obtained with D1.LP mice that are low responders to Pn and express lower levels of T15 Id as compared to BALB/c. Young D1.LP mice produced high numbers of auto-anti-T15 AFC of both IgM and IgG isotypes following a single immunization with Pn vaccine. The kinetics of auto-anti-T15 response in D1.LP mice was similar to that of the antigen-specific response. These results demonstrate that the ability of the immune network to produce autologous antibody to a shared Id depends on the genetic makeup of the host, and that this response may be regulated by the level of Id expression.     

Regulation of idiotope expression. III. H-2 influences the magnitude and the idiotypy of a T-independent antibody response in mice of certain genetic backgrounds

Antibody response to the phosphocholine (PC) epitope on Streptococcus pneumoniae R36a (Pn), a T-independent Ag type 2, was studied in H-2 congenic mouse strains. The PC-specific antibody plaque-forming cells (PFC) were enumerated in the spleen at various intervals after the primary Pn injection, and the proportion of PFC that produced antibody expressing the AB1-2 idiotope (Id) was determined by using the corresponding monoclonal anti-Id. AB1-2 is a cross-reactive Id, detectable on germline-encoded PC antibody of the T15 family, and on most, but not all, somatic variants of that antibody. The specific PFC responses in BALB/c (H-2d) and BALB.B (H-2b) strains were of comparable magnitude and most, if not all, PFC were ABl-1 Id-positive (AB1-2+). This was not the case in the responses of the B10D2 (H-2d) vs C57BL/10 (H-2b) strains and the D1.C (H-2d) vs D1.LP (H-2b) strains (on DBA/1 background). In each of these pairs, the H-2d mice were high responders, and the response was dominated by AB1-2 Id (greater than or equal to 80% AB1-2+ PFC at the peak, on day 5). The H-2b mice were low responders, and only a minor proportion of PFC (less than or equal to 30%) were AB1-2+; an increase of AB1-2+ was seen later in the response (d.10). The results of PFC assays were confirmed by measuring the PC-binding antibody and AB1-2 Id in the sera of D1.C and D1.LP mice immunized repeatedly with Pn. Moreover, D1.LP mice that had very low levels of AB1-2 Id had higher serum levels of antibody expressing two other T15 Id, B36-82, and B24-44. The B36-82 and B24-44 Id have been previously found on somatic variants of PC antibody expressed independently of the Ab1-2 Id. The concentrations of these two Id in D1.LP mice after repeated immunization approached those in D1.C. These results indicate that 1) the H-2 allelism may have a significant effect on TID antibody response in mice of a certain genetic background, but not in the BALB/c; and 2) the idiotypic repertoire of the response may be influenced by H-2 at the level of clonal variants of PC-reactive cells.     

Soluble OX40L favors tumor rejection in CT26 colon carcinoma model

OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice.We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.