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Naturally occurring CD4(+)25(high)Foxp3(+) T regulatory (T-reg) cells are critical for maintaining tolerance to self and non-self Ags. The Foxp3 master-regulatory gene and CD28 costimulation are both required for thymic development and suppressogenic function of CD4(+)25(high)Foxp3(+) T-regs. Herein, we show that the sole CD28 stimulation of T-reg thymic precursors augments Foxp3 expression through the increase in Foxp3 mRNA life span by a mechanism involving p56(lck) and its binding motif on CD28 cytosolic tail, as well as the lipid rafts. We found that 1) the glycosphingolipids and cholesterol components of lipid rafts were highly expressed and unusually partitioned in T-reg thymic precursors as compared with the conventional T cell precursors, 2) the CD28 receptor density on cell membrane is proportional with the content of cholesterol in lipid rafts and with the level of Foxp3 mRNA expression in T-reg precursors, and 3) the CD28-mediated increase of Foxp3 mRNA life span was paralleled by an increased proliferative and suppressogenic capacity of terminally differentiated CD4(+)25(high)Foxp3(+) T-reg precursors. Thus, the functional integrity of CD28 receptor p56(lck) and plasma membrane lipid rafts are all prerequisites for up-regulation and long-term expression of Foxp3 mRNA transcripts in CD4(+)25(high)Foxp3(+) T-reg precursors.

Despite the expression of non-self or neo-epitopes, many tumors such as lymphoid malignancies or cancers induced by oncogenic viruses are able to gradually overcome the immune defense mechanisms and spread. Using a preclinical model of hematological malignancy, we show that Ig-associated idiotypic determinants are recognized by the immune system in a fashion that results in immune deviation, allowing tumor progression and establishment of metastases. Using gene-targeted mice, we show that anti-idiotypic MHC class I-restricted immunity is promoted by ITAM motif (ITAM+) FcgammaR, but kept in check by ITIM motif (ITIM+) FcgammaRIIB-mediated mechanisms. In addition to interfering with the functionality of ITIM+ FcgammaR, effective anti-idiotypic and antitumoral immunity can be achieved by FcgammaR-targeted delivery of epitope in conjunction with administration of stimulatory motifs such as dsRNA, correcting the ineffective response to idiotypic epitopes. The immune process initiated by FcgammaR-mediated targeting of epitope together with dsRNA, resulted in control of tumor growth, establishment of immune memory and protection against tumors bearing antigenic variants. In summary, targeted delivery of MHC class I-restricted epitopes via ITAM+ FcgammaR, in conjunction with use of TLR-binding immune stimulatory motifs such as dsRNA, overcomes suboptimal responses to idiotypic determinants and may constitute a novel approach for the treatment of a broad range of malignancies. Finally, the results shed light on the mechanisms regulating the idiotypic network and managing the diversity associated with immune receptors.

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.

We had previously shown that a genetically engineered Ig expressing an immunodominant CD4+ T epitope corresponding to the 110-120 amino acids of influenza virus hemagglutinin (HA), activated T cells more efficiently than the synthetic peptide. Taking advantage of a T cell hybridoma specific for HA 110-120 and transfected with a reporter gene under the IL-2 promoter, we studied the kinetics of generation and persistence on surface MHC-II of the HA 110-120 peptide derived from various carriers. Our results show that the generation rate of immunogenic MHC II-peptide complex is dependent on the nature of the carrier. Abs specific for HA 110-120 peptide or for other epitopes on HA affect through various mechanisms the presentation of HA 110-120 peptide to T cells. The persistence of immunogenic MHC II-peptide complex on the surface of APC is not dependent on the nature of the carrier and correlates with the half-life of class II molecules, suggesting an irreversible binding of peptides to MHC-II molecules in 2PK3 murine B lymphoma cells.

Serum samples from 147 patients with different systemic autoimmune diseases (SLE, Sjögren's syndrome, and progressive systemic sclerosis) were tested for anti-Fc gamma R activity using mouse rFc gamma RII in an ELISA. High reactivity compared to normal individuals was found for patients with all three diseases. The anti-Fc gamma R antibody was purified from several serum samples by affinity chromatography on a Sepharose column coupled with denatured, murine rFc gamma RII. Both IgM and IgG antibodies were found. To analyze the specificity of the affinity-purified autoantibody, cells (human neutrophils, IFN-gamma-stimulated neutrophils, monocytes, and the THP-1 monocytic cell line) that express different combinations of Fc gamma R (CD64, CD32, CD16) were stained with the affinity purified Ig. Ig directed against all three types of Fc gamma R were found. The results may reflect on the role of Fc gamma R-specific antibodies in the pathology of autoimmune diseases.

The epitope corresponding to amino acid residues 147-161 of the nucleoprotein (NP) of influenza A virus is recognized by CTL in association with H-2Kd class I Ag. Herein, we engineered an Ig molecule carrying this CTL epitope by replacing the diversity gene segment of the H chain V region of an anti-arsonate antibody with an oligonucleotide that encodes the CTL epitope. The chimeric H chain gene was expressed either alone or together with the parental L chain in the nonsecreting BALB/c myeloma B cell line, SP2/0. The Ig produced by cells transfected with both the chimeric H chain and parental L chains genes expressed the NP epitope but lost the original arsonate binding activity. In addition, SP2/0 cells expressing the chimeric H chain either alone or together with the parental L chain were lysed by class I restricted NP-epitope specific CTL. By contrast, SP2/0 cells pulsed with soluble chimeric Ig molecules were not lysed by the specific CTL. These observations indicate that: 1) this particular CTL epitope can be expressed on Ig molecules without altering the H and L chain pairing; 2) this CTL epitope can be generated from this chimeric Ig in which it is surrounded by flanking regions distinct from those of the viral NP; and 3) the generation of this CTL epitope from the Ig molecule requires the endogenous pathway as do viral proteins.

We have identified a new murine V kappa family that contains five to seven members, one member of which encodes the L chain V region of an anti-dsDNA antibody produced by a BALB/c hybridoma, C8.5. The cloned C8.5 V kappa gene exhibits highest homology with a human V kappa gene that was cloned from a nonproductive rearrangement but has never been seen in an expressed repertoire. Because this family was first identified in an autoantibody, we studied its expression in an autoimmune mouse strain. This V kappa family is expressed in 20% of hybridomas from NZB mice.

The BALB/c myeloma protein ABPC48 binds beta(2-6)-linked fructosans and expresses genes derived from the VHX24 and V kappa 10 gene families. We have selected 30 hybridomas expressing the VHX24 gene family derived from mitogen-stimulated spleen cells of naive BALB/c mice and mice injected at birth with the syngeneic monoclonal anti-ABPC48Id, IDA10. The majority of mAb with kappa L chains uses V kappa 1. Antibodies reacting with IDA10 use both V kappa 10 and V kappa 1. Most of these VHX24+ mAb reacted with one or more members of a limited panel of predominantly polysaccharide Ag that have been previously observed to interact with antibodies expressing the VHX24 gene family. Nucleotide sequencing of selected VH and V kappa genes shows a very low frequency of somatic mutation. The effect of neonatal anti-Id injection on VHX24-V kappa pairing and Id expression is discussed.

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.

Two purified peptides extracted from E. coli or B. megaterium strongly stimulated the spleen lymphocytes of rabbits and of normal or nude mice. Both preparations can substitute for Mycobacteria in Freund's complete adjuvant. The peptidoglycan extracted from M. lysodeikticus by a similar procedure which lacks adjuvant activity, did not induce blast transformation. However, the monomere of the E. coli peptidoglycan was devoid of mitogenicity although it has also a marked adjuvant activity.

Nocardia water soluble mitogen (NWSM), a mitogen specific for mouse and rabbit B cells, activates blood, spleen, and tonsil lymphocytes from all the humans tested. Evidence is presented that it acts selectively on human B lymphocytes. B-enriched cells were recovered from anti-Ig-coated Sephadex columns or after elimination of E rosette-forming cells: they were able to incorporate tritiated thymidine when they were cultivated in the presence of NWSM whereas they were not activated by PWM. Conversely T-enriched suspensions did not respond to NWSM although they were able to be stimulated by PHA and PWM. Moreover, the response to NWSM remained unchanged after T cells had been killed by anti-HTLA serum and complement and disappeared after B cells had been killed by anti-HBLMA serum. After stimulation by NWSM, the number of direct plaque-forming cells to HRBC, LPS, and TNP was significantly increased, showing a polyclonal activation of human B lymphocytes. Since no evidence of T cell help for B cell activation has been found, NWSM thus appears to be a T-independent human B lymphocyte mitogen.

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.

Several idiotypic (Id) specificities have been identified by immunofluorescence on the membranes of B cells in the spleens of nonimmune mice. These determinants are displayed at frequencies varying from 0.22 to 1.83% of B cells and are entirely immunoglobulin (Ig) in nature. No Id determinants were observed on the membranes of four Slg- Abelson virus-transformed pre-B cell lines that are sensitive to LPS. Under our experimental conditions, we did not observe a significant increase in 3H-thymidine incorporation subsequent to the in vitro incubation of splenic lymphocytes from normal mice with various amounts of anti-Id antibodies specific for several cross-reactive (IdX) or individual Id. Similarly, in utero exposure to anti-Id antibodies against myeloma proteins specific for T-independent antigens displaying B cell mitogenic properties did not alter the proliferation of lymphocytes induced by these mitogens. In contrast, exposure to anti-Id antibodies in vitro as well as in utero had a profound and specific effect on the corresponding antibody responses and maturation of small lymphocytes into plasma cells. When normal B cells were cultured with the B cell mitogen LPS in the presence of anti-Id antibodies directed against the J558IdX, specific suppression of the maturation of IdX+ plasma cells was observed. In mice exposed to maternal anti-Id in utero, we observed a severe inability to mount an immune response to any compound that elicited antibody molecules bearing the Id that was complementary to the maternal anti-Id. These same maternally Id-suppressed mice, however, gave a normal response when the same compound was presented as a mitogen. Our results reinforce the concept that Id on membranes of B lymphocytes are associated only with Ig receptors and do not support the possibility of mitogen-like poly-clonal stimulatory properties of anti-Id antibodies.

Syngeneic anti-(anti-Id) antibodies were prepared against BALB/c anti-A48Id antibodies, BALB/c anti-460Id monoclonal antibodies, and A/J anti-J558 IdI monoclonal antibodies. With these anti-(anti-Id) antibodies we identified cross-reactive idiotypes on syngeneic and homologous anti-A48Id and anti-460Id antibodies. By contrast, tbe idiotypic determinants of A/J anti-J558 IdI monoclonal antibodies were not shared by other syngeneic, homologous, or xenogenic anti-J558 IdI or IdX antibodies. These results suggest that idiotype-antiidiotype reactions that serve as regulatory controls within the immune system are characteristic for each particular antigen system, strain, or species and that such interactions make the system self-limited with respect to the whole antild repertoire.

Pretreatment of BALB/c mice with antisera to a cross-reactive idiotype (E109IdX) expressed on many anti-bacterial levan (BL) and anti-inulin (Inu) antibodies leads to a prolonged suppression in production of IdX-bearing molecules in response to BL immunization. There is a comparable suppression in numbers of plaque-forming cells secreting IdX-bearing anti-BL and anti-Inu molecules. Furthermore, spleen cells from anti-E109IdX pretreated mice are unable to transfer to irradiated recipients the ability to produce IdX-bearing anti-BL and anti-Inu antibodies. These results indicate that the suppressive effect is at the precursor level and not simply a clearance of antibodies bearing the IdX. Suppression of IdX production can be achieved by pretreating nu/nu BALB/c mice with anti-E109IdX antibodies. Furthermore, spleen cells from pretreated mice do not inhibit the capacity of spleen cells from normal mice transferred to irradiated recipients to produce E109IdX in response to BL. This indicates that the suppression of IdX production in the anti-BL system is T independent and probably represents direct inhibition of precursors by anti-IdX.

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

We have dissected the human influenza-specific B cell repertoire by performing Epstein-Barr virus (EBV) limiting dilution analysis of lymphocytes obtained from donors before and after immunization with a commercially available influenza vaccine. In addition to an analysis of precursor frequency and light chain diversity, we studied sera and culture supernatants containing human anti-influenza antibodies with a panel of murine monoclonal antibodies specific for idiotopes identified on murine anti-PR8 and anti-X-31 antibodies. An idiotypic specificity present on the X-31-specific murine monoclonal PY206 has previously been shown to be shared by murine antibodies specific for PR8, X-31, and other influenza viruses. We observed little correlation among the following parameters: anti-viral titer, serum idiotope content, precursor frequency and immune status. More interestingly, there was a striking predominance of human influenza-specific antibodies that utilized lambda light chains. In addition, 12 of 26 human anti-influenza monoclonals strongly inhibited the binding of one of the murine anti-idiotopes to the labeled murine antibody, PY206. This is the same idiotope that is shared among murine antiinfluenza antibodies and all six individuals studied contained clones reactive with this anti-idiotope. Seven of these 12 idiotope-positive human antibodies gave partial cross-reactivity in a second anti-idiotypic system. These observations imply that a significant level of homology exists between the binding sites of human and murine influenza-specific antibodies and suggest that idiotypic manipulation of the human immune response to influenza virus may have important therapeutic implications.