Anti-DNA idiotype- and anti-idiotype-specific T cell responses in patients with systemic lupus erythematosus and their first-degree relatives.

We examined the proliferative responses of T cells of patients with systemic lupus erythematosus (SLE), their first-degree relatives, and healthy donors, to a human monoclonal antibody that bears a common anti-DNA idiotype, 16/6 Id, and to a murine, 16/6 Id-specific, monoclonal antibody. Both 16/6 Id+ and 16/6 Id-specific antibodies were previously shown to be involved in the induction of experimental SLE in mice. Here we show that T cells of fewer SLE patients, as compared with healthy donors, could proliferate to both antibodies. The difference between T cell responses of patients and controls to the 16/6 was found to be significant. The proliferative responses of T cells of first degree relatives of SLE patients to the anti-16/6 Id were found to be significantly lower compared with the responses detected in healthy donors and in SLE patients. The responses of T cells of SLE relatives to the 16/6 Id were found to be lower than those of healthy donors, but this difference was not significant. The present study suggests a possible involvement of T cells, and specifically of idiotype and anti-idiotype specific T cells, in SLE.     

Genetic idiotypic and tumor cell-based vaccine strategies for indolent non Hodgkin's lymphoma

B cell malignancies express a clear tumor-specific antigen (B cell immunoglobulin variable regions) known as idiotype (Id). It is now possible to immunize patients against autologous Id generating humoral and cellular immune responses that correlate with clinical and molecular remissions and the possibility of improved disease-free survival. In its present form, however, individual vaccine preparation by generating heterohybridomas is a technical and financial challenge. DNA vaccination provides a unique opportunity to streamline individual vaccine manufacture by circumventing the need for protein purification. DNA fusion vaccines have been developed in which genetic carriers promote adaptive immunity against the attached Id. Such carriers can specifically bind receptors on dendritic cells (DC) for targeted antigen delivery, or supply high levels of T cell help. Ideally, the carrier should be able to activate innate immunity to enhance the antigen-presenting capacity of DC. The correlates of immunity may vary depending upon the genetic carrier used. Translation to patients has begun with preliminary evidence of Id-specific immune responses. An alternative vaccination strategy that allows for the potential to vaccinate against multiple tumor antigens without the need to identify individual antigens is based on tumor cells themselves to be used as vaccine. To this purpose, however, each patient's tumor cells must be genetically modified to increase their immunogenicity. To overcome the technical limitations inherent with a fully autologous approach, strategies have been devised where a universal, genetically modified bystander cells is expected to provide the immunoenhancing cytokines to allow immune recognition of unmodified patients' tumor cells.     

Resting small B cells present endogenous immunoglobulin variable-region determinants to idiotope-specific CD4(+) T cells in vivo

Antigenic determinants localized within the highly diversified V-regions of Ig are called idiotopes (Id). Processed Id-peptides can be presented on MHC class II molecules to CD4(+) T cells. If B cells present their endogenous Id-peptides, T cell activation could occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. To test this idea, we used mice made transgenic for a lambda2 L-chain (Id(+) mice). Another transgenic mouse strain expresses TCR transgenes with specificity for the Id (lambda2), presented on MHC class II molecules. When highly purified sorted Id(+) B cells and Id-specific T cells were sequentially injected into MHC syngeneic SCID host, T cell became blastoid, CD69(+) and proliferated. To exclude any role of host APC, MHC incompatible Rag2(- / -) mice (H-2(b)) were used as recipients for the Id(+) B and Id-specific T cells, with similar results. Exposure to extracellular Id(+) immunoglobulin (Ig) was not sufficient for Id priming of B cells in vivo, highlighting the preferential presentation of Id peptides derived from endogenous Ig, by B cells. The results suggest that B cells presenting Id self-peptides generated by V(D)J recombinations or somatic mutations may directly stimulate T cell in vivo in the absence of conventional antigen.     

Idiotype specific T-cell lines inducing experimental systemic lupus erythematosus in mice.

Immunization of mice with either antibodies bearing the 16/6 idiotype (16/6 Id) or anti-idiotypic antibodies against the 16/6 Id induces experimental systemic lupus erythematosus (SLE). We report here the establishment and characterization of 16/6 Id-specific T-cell lines from C3H.SW (H-2b) and BALB/c (H-2d) mice. Both lines proliferate specifically in response to the 16/6 Id in an H-2-restricted manner. The injection of 16/6 Id-specific T cells into syngeneic mice led to the development of experimental SLE. Furthermore, inoculation of the 16/6 Id-specific T-cell line derived from C3H.SW mice into the H-2 compatible C57BL/6 mice, which are non-responders to the 16/6 Id, induced experimental SLE. This report provides direct evidence for the role of idiotype-specific T cells in the induction of experimental SLE.     

Hepatitis B vaccine-specific CD4(+) T cells can be detected and characterised at the single cell level: limited usefulness of dendritic cells as signal enhancers

Hepatitis B surface antigen (HBsAg)-specific T cells were analysed in 16 healthy individuals vaccinated against hepatitis B, using optimised protocols. HBsAg-specific IFN-γ producing CD4⁺ T cells were found at a similar low frequency (0.01%) within the naive T cell subset, the central and the effector memory T cell subset. Overall, HBsAg-specific total CD4⁺ T cells were detected in approximately 81% of the HBV vaccinees. The use of dendritic cells substantially increased the otherwise low proliferative responses but not the percentage of IFN-γ producing cells. The analysis of readily detectable CMV-specific CD4⁺ T cell responses was used as a positive control and confirmed the adequacy of our assays. T cell responses associated with (in-) adequate hepatitis B vaccination can now be analysed in more detail.     

Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses

Vaccination with in vitro-generated dendritic cells (DC) that present tumor-associated antigens is a promising approach for immunotherapy of malignant tumors. For optimization of DC-based vaccination protocols, preclinical tumor models that mimic the clinical situation closely are highly desirable. Strong non-specific T cell activation was observed in experimental immunization of mice with syngeneic DC generated in standard FCS-supplemented culture medium. To avoid deviation of the immune response to FCS-derived antigens, a serum-free culture protocol for in vitro generation of murine DC from bone marrow progenitor cells was developed. In comparison to DC differentiated with FCS supplementation, DC generated under serum-free conditions (sfDC) have a more homogeneous phenotype with higher expression of IL-12 and the differentiation and activation markers CD11c, CD40, CD80, CD83, CD86, DEC-205, and MHC class II. Demonstration of strong uptake of protein and carbohydrate antigens and analysis of the in vivo migration behaviour of sfDC also indicated excellent APC function. Vaccination of mice with peptide-pulsed sfDC efficiently induced an antigen-specific T cell response as assessed by MHC tetramer staining, IFN-γ ELISPOT and in vivo cytotoxicity assay. sfDC may therefore represent a valuable tool to improve active tumor immunotherapy in animal models.     

The J558 V(H) CDR3 region contributes little to antibody avidity; however, it is the recognition element for cognate T cell control of the alpha(1-->3) dextran-specific antibody response [In Process Citation]

Analysis of the humoral immune response of BALB/c mice to alpha(1-->3) dextran (Dex) reveals novel aspects of T cell-mediated control of 'type 2 thymus-independent' responses against polysaccharide antigens. The IgM and IgG antibody response, dominated by the J558 idiotype (Id), is controlled by Id-specific T cells. These regulatory T cells, for which the T cell clone 178-4 Ts with characterized TCR alpha and beta chain sequences is the prototype, expand in all BALB/c mice upon immunization with Dex. They suppress in a cognate interaction the expansion of J558 Id-bearing B cells, committed for production of IgG antibodies. Furthermore they provide a gate which precludes variability in the VH CDR3 region of IgG antibodies appearing occasionally in the periphery. The VH CDR3 region is the recognition element of 178-4 Ts analogous T cells but contributes little to affinity for the antigen. For recognition by 178-4 Ts cells not even minimal sequence deviations of the J558 Id peptide are allowed. The tight germline programmed complementarity between J558 Id-bearing Dex-specific B and J558 Id- specific 178-4 Ts analogous T cells leaves little room on both sides for ontogenetic variability.      

Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma.

The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.     

A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses

The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.     

Peripheral T cell tolerance as a tumor escape mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a plasmacytoma

Tumors could escape an immune attack by inducing peripheral T cell tolerance. To test this, T cell receptor (TCR)-transgenic mice were injected with plasmacytoma cells secreting a highly tumor-specific antigen, a monoclonal immunglobulin (Ig), for which the transgene-encoded TCR is specific. The TCR recognizes a third hypervariable region idiotypic (Id) peptide of the Ig, presented by a class II molecule on host antigen-presenting cells. The TCR-transgenic mice have previously been shown to be protected against an Id⁺ plasmacytoma challenge. In the present experiments, the protection was deliberately overwhelmed by subcutaneous injection of large numbers of plasmacytoma cells. Such tumor mice, chronically exposed to increasing amounts of monoclonal Ig, delete Id-specific CD4⁺ T cells in their peripheral lymphoid organs and in the tumor. The residual CD4⁺ cells express endogenous, rather than transgene-encoded TCR α chains. Peripheral deletion, functional T cell unresponsiveness, and thymocyte deletion are all first detected at the same serum concentration of monoclonal Ig, ∼50 μg/ml (0.3 μM), and become more and more profound as the tumor burden increases. The results suggest that peripheral T cell tolerance to Id could be a tumor escape mechanism in patients with B cell malignancies. In addition, the findings have implications for T cell tolerance to Ig V regions in normal individuals.     

Characterization of a carcinogen-induced murine B lymphocyte cell line of C3H/eB origin.

A carcinogen-induced lymphoid tumor, denoted 38C-13, obtained in a T cell-depleted mouse of C3H/eB strain, was adapted to continuous culture in vitro and characterized with respect to its cell surface components. The cells possess IgM class immunoglobulins on their surface but do not secrete it. This membrane IgM is composed of mu and L-chains that are similar in apparent molecular weight to those of an IgM myeloma protein. It is also homogeneous as revealed by isoelectric focusing. The cells possess Fc receptors but lack complement receptors as well as Thy-1 and Ia alloantigens. These characteristics indicate that 38C-13 cells are transformed counterparts of small B lymphocytes at an early stage of differentiation.     

An IgM-producing tumor with biochemical characteristics of a small B lymphocyte.

A lymphocytic tumor, 38C-13, induced by the chemical carcinogen 7, 12-dimethylbenz(a)anthracene in C3H/eB mice and adapted to tissue culture, produces 7-8 S IgM with "core" carbohydrates (N-acetylglucosamines, mannoses), but not "branch" carbohydrates (neuraminic acids, fucoses, galactoses) attached to the mu heavy, but not to the light chains. Turnover of the 7-8 S 38C-13 IgM is slow (half disappearance time = 10-15 h). The IgM is released from the cells as 7-8 S IgM. The ratio of IgM synthesis to the synthesis of all cellular glycoproteins is 0.005-0.01. After comparison of these data with data obtained with normal B lymphocytes before and after mitogenic stimulation, we conclude that 38C-13 tumor cells are transformed counterparts very near or within the population of small, mitogen-sensitive, resting B lymphocytes.     

CD4 T cells inhibit in vivo the CD8-mediated immune response against murine colon carcinoma cells transduced with interleukin-12 genes

Retroviral-mediated cytokine gene transfer into tumor cells is a highly effective way of inducing tumor inhibition and immunity. We analyzed the tumorigenicity of C-26 murine colon carcinoma cells transduced with genes encoding the two subunits of murine interleukin-12 (IL-12) in a polycistronic retroviral vector and selected for resistance to G418 and for IL-12 production (30–80 pg/ml). BALB/c mice injected s.c., i.v. and intrasplenically with C-26/IL-12 cells from three different IL-12-producing clones showed delayed tumor onset as compared with mice injected with control Neo^R-transduced or parental tumor cells. Although C-26/IL-12 tumor-bearing mice eventually died of lung metastasis, their survival time was twice as long as that of mice injected with control cells. In experiments with mice selectively depleted of natural killer (NK) cells before tumor cell injection, the time of tumor onset and survival of mice injected with C-26/IL-12 s.c. and i.v., respectively, was reduced. CD8⁺ T cell depletion had no effect on latency or survival, whereas removal of CD4⁺ T cells led to C-26/IL-12 tumor regression in about 40% of mice. Histological and immunocytochemical characterization of leukocytes infiltrating C-26/IL-12 tumors showed only slight infiltration with few T cells in non-depleted mice but abundant infiltration by CD8⁺ T cells and asialo-GM1+ NK cells in tumors of mice depleted of CD4⁺ T cells. The lack of CD8⁺ T cell infiltration is not due to a CD4-mediated suppression of their activation because irradiated C-26/IL-12 cells primed for the induction of a strong cytotoxic T lymphocyte response against C-26 parental cells and induced CD8⁺ effector cells that protected against C-26/IL-12 in a Winn assay. Rather, the results suggest that, although C-26/IL-12 cells injected in vivo stimulate both NK and CD8⁺ T cells, tumor infiltration by the latter is inhibited by CD4⁺ T cells.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4⁺ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0×10⁷ cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-γ, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4⁺ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c×C57BL/6 F₁ mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-γ than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-γ-producing ability but rather derived from their poor responsiveness to IFN-γ.     

Monoclonal anti-idiotype antibodies against the murine B cell lymphoma 38C13: characterization and use as probes for the biology of the tumor in vivo and in vitro

To establish a murine model for the monoclonal anti-idiotype immunotherapy of B cell lymphoma, a panel of rat and murine monoclonal anti-idiotype antibodies of several different isotypes was generated against the surface immunoglobulin of the murine B cell tumor 38C13 (38C). Xenogeneic antibodies were made from fusions of rat spleen cells immunized with the 38C idiotype. Syngeneic monoclonal anti-idiotypes were generated from mice immunized with the idiotype conjugated to the protein carrier KLH. Small differences were noted in the ability of the antibodies to cross-block one another, but all appeared to be directed against the same or closely spaced idiotopes on the immunoglobulin molecule. The antibodies selectively precipitated surface Ig from 38C tumor cells and not from normal mouse spleen cells. They were used to selectively stain 38C tumor cells in cell suspensions for FACS analysis or immunohistochemical staining of tissue sections from mice bearing the tumor. As the malignancy progressed, the number of tumor cells found in all tissues examined increased. Thus, the anti-Id antibodies provided a specific probe for tumor cell detection. The antibodies had no detectable effect on cell growth in vitro; however, they did cause the rapid transient loss of the expression of cell surface Ig. This modulation was concentration and time dependent but not 100% complete. Re-expression of the Id occurred by 24 h following removal of the anti-Id antibodies. When these antibodies were used in sensitive radioisotope and enzyme linked immunoassays, the tumor cells were found to secrete small amounts of idiotype in vitro and in vivo. The level of idiotype detected in vivo correlated with tumor growth and inversely with survival. This work is an attempt to develop further an animal model system in which to test the diagnostic and therapeutic effects of monoclonal anti-idiotype antibodies.     

Aberrant T helper cell response in tumor-bearing mice limits the efficacy of dendritic cell vaccine

Dendritic cell (DC) vaccine is a promising immunotherapy for malignancies, but its clinical efficacy has been questioned. Here we examined the mechanisms of treatment failure with DC vaccine in a murine colon cancer model. DC vaccination of naive mice prevents tumor implantation, but it is ineffective in tumor-bearing hosts despite the induction of tumor-specific CTL activity. Analyses of tumor-specific T helper cell type 1 (Th1)/T helper cell type 2 (Th2) responses showed that DC vaccine induced a mixed Th1/Th2 response in naive mice. Interestingly, CD4+ T cells from tumor-bearing mice showed a Th1-predominant response before DC vaccination but Th2 after DC vaccination. Furthermore, interleukin-10 production was higher in CD4+ T cells from vaccinated tumor-bearing mice than in CD4+ T cells from unvaccinated tumor-bearing mice. CD4+ T cells from mice treated with lipopolysaccharide (LPS)-matured DC fusion vaccine had lower production of interleukin-10 than CD4+ T cells from mice treated with non-LPS-treated DC vaccine. However, similar to the non-LPS-treated DC vaccine, the LPS-matured DC vaccine failed to suppress tumor growth and induced a Th2 predominant tumor-specific response in tumor-bearing mice. These results suggest that the presence of tumor in the host induces an aberrant CD4+ T cell response to DC vaccine, which may contribute to the failure of the vaccine to eradicate established tumors.     

Idiotype vaccination following ABMT can stimulate specific anti-idiotype immune responses in patients with B-cell lymphoma.

Vaccination with the idiotype (Id) protein derived from B-cell malignancies can produce Id-specific immune responses that correlate with improved remission duration and survival rates in patients with follicular non-Hodgkin's lymphoma (NHL). A state of minimal or no residual disease correlates strongly with the laboratory detection of a cellular or humoral immune response. High-dose cytotoxic therapy (HDCT) with autologous stem cell support (autologous bone marrow transplantation [ABMT]) can provide profound cytoreduction of B-cell NHL, but the potential immune suppression associated with myeloablative therapy may compromise a patient's ability to mount a specific immune response. To determine whether patients with NHL could mount detectable immuneresponses following ABMT, Id vaccines were administered at 2 to 12 months following myeloablative therapy to a series of patients with relapsed or resistant B-cell NHL. Two different vaccination strategies produced robust immune responses against KLH in all patients, supporting the capacity of the reconstituted immune system following HDCT to react against a strong antigen. Combining the results from both vaccination strategies, 10 of 12 patients mounted Id-specific humoral or cellular responses. Vaccinations were consistently well tolerated. Of the 12 patients, 7 have experienced prolonged remissions with a follow-up from HDCT ranging from 3 to more than 11 years. Our experience serves to document the ability of the recovering immune system to react against both self and xenotypic antigens and supports the feasibility and safety of antigen-specific vaccination following myeloablative therapy in patients with B-cell NHL.     

Immunoglobulin secretion by B1 cells: differential intensity and IRF4-dependence of spontaneous IgM secretion by peritoneal and splenic B1 cells

Peritoneal B1 cells are typified by spontaneous, constitutive secretion of IgM natural antibody, detected by ELISPOT assay, among other means. Recently, this key characteristic has been called into question, a reason for which we evaluated the integrity of IgM(+) ELISPOT spots. We found that fixed B1 cells fail to produce ELISPOT spots, that interference with Golgi function inhibits ELISPOT spot formation, and that B1 cell-derived immunoglobulin in supernatant samples is EndoH-resistant. These findings indicate that spots produced by B1 cells on ELISPOT assay reflect secretory IgM actively exported by viable B1 cells. Current paradigms propose that interferon response factor 4 (IRF4) is required for plasma cell differentiation and immunoglobulin secretion. However, we found that IgM secretion by peritoneal B1 cells is not altered in IRF4-null mice. In contrast, spontaneous IgM secretion by splenic B1 cells, which amounts to much more IgM secreted per cell, is dramatically reduced in the absence of IRF4. These results indicate that peritoneal B1 cells spontaneously secrete low levels of IgM via an IRF4-independent non-classical pathway, and, considering the low level of serum IgM in IRF-null mice, further suggest that accumulation of serum immunoglobulin depends on IRF4-dependent secretion by splenic B1 cells.     

Identification of capsid epitopes of Theiler's virus recognized by CNS-infiltrating CD4+ T cells from virus-infected C57BL/6 mice

Intracerebral infection of Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in some mouse strains but not in others. We report here for the first time two new predominant capsid epitopes (VP4(21-40) and VP2(201-220)) recognized by CD4+ T cells from virus-infected resistant C57BL/6 mice based on IFNgamma ELISPOT assay utilizing a 20-mer peptide library covering the entire capsid proteins. Further experiments by IFNgamma ELISPOT and flow cytometry for intracellular IFNgamma production using truncated peptides indicated that the epitope regions recognized by CNS-infiltrating CD4+ T cells are VP4(25-38) and VP2(206-220), respectively. No apparent reduction in the T cell response to these viral epitopes is seen in the CNS of IL-12- and ICAM-1-deficient C57BL/6 mice compared to those in control C57BL/6 mice, suggesting that T cell response to TMEV in the CNS is largely insensitive to the absence of these proinflammatory cytokine and adhesion molecules. Therefore, these newly defined CD4+ T cell epitopes are likely to provide an important tool to investigate the role of CD4+ T cell responses in H-2b-bearing congenic strains.