Teresa M. Foy

Mice deficient for the CD40 ligand

To study the potential roles of CD40L in immune responses, we generated CD40L-deficient mice by gene targeting. Similar to the effects of CD40L mutations in humans (hyper-IgM syndrome), CD40L-deficient mice have a decreased IgM response to thymus-dependent antigens, fail altogether to produce an antigen-specific IgG1 response following immunization, yet respond normally to a T-independent antigen, TNP-Ficoll. Moreover, these mice do not develop germinal centers in response to thymus-dependent antigens, suggesting an inability to develop memory B cell responses. Although CD40L-deficient mice have low levels of most circulating immunoglobulin isotypes, they do not exhibit the spontaneous hyper-IgM syndrome seen in humans, at least up to 12 weeks of age. In summary, our study confirms the important role of CD40-CD40L interactions in thymus-dependent humoral immune responses and germinal center formation.

The role of CD40 in the regulation of humoral and cell-mediated immunity☆

The dynamic and reciprocal communication between T helper (Th) cells and B cells appears to rely on the provision of multiple signals. The first is antigen specific and is mediated by the interaction between the T-cell receptor (TCR) and antigen bound to the major histocompatibility complex (MHC). The subsequent signals are provided by the binding of accessory molecules such as CD28 and CD40 to their respective ligands. Here, Fiona Durie and colleagues discuss the co-stimulatory role of the interaction between CD40 on B cells and CD40 ligand (CD40L, gp39) on T cells, and review evidence that suggests blocking this interaction may induce T-cell tolerance.

In the absence of a CD40 signal, B cells are tolerogenic

When B cells are deprived of signaling through CD40, they exhibit the ability to induce T cell tolerance. The in vivo administration of anti-gp39 and allogeneic B cells diminished the ability of mice to mount an allogeneic response. Tolerance induction was specific for the haplotype expressed on the allogeneic B cells. Selective allospecific unresponsiveness was induced in the CD8 and CD4 compartments by the administration of anti-gp39 and class II-deficient B cells or class I-deficient B cells, respectively. As predicted by studies with anti-gp39 treatment, diminished allospecific responsiveness was induced by the administration of B cells to mice genetically deficient in gp39. Taken together, these data are consistent with the premise that deprivation of CD40 signaling engenders B cells with enhanced tolerogenicity. These studies provide insights into the tolerogenic capacity of resting B cells and outlines a practical approach to exploit this function.

Murine B1 B cells require IL-5 for optimal T cell-dependent activation.

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L−/− mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.

Vaccination with Her-2/neu DNA or protein subunits protects against growth of a Her-2/neu-expressing murine tumor

The present study utilizes an in vivo murine tumor expressing human Her-2/neu to evaluate potential Her-2/neu vaccines consisting of either full length or various subunits of Her-2/neu delivered in either protein or plasmid DNA form. Our results demonstrate that protective immunity against Her-2/neu-expressing tumor challenge can be achieved by vaccination with plasmid DNA encoding either full length or subunits of Her-2/neu. Partial protective immunity was also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of Her-2/neu. The mechanism of protection elicited by plasmid DNA vaccination appeared to be exclusively CD4 dependent, whereas the protection observed with ICD protein vaccination required both CD4 and CD8 T cells.

Characterization of a Proapoptotic Antiganglioside GM2 Monoclonal Antibody and Evaluation of Its Therapeutic Effect on Melanoma and Small Cell Lung Carcinoma Xenografts

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cells growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4s binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

A novel method for increasing the expression level of recombinant proteins.

Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.

Blockade of CD40-CD154 interferes with human T cell engraftment in scid mice

Antibodies to the ligand for CD40 (CD154) have been shown to exert profound effects on the development of cell-mediated immune responses in mice. The present study shows that an antibody to human CD154 (hCD40L) inhibits in vivo Tetanus toxoid (TT) specific secondary antibody responses in hu-PBL-scid mice, as well as the expansion of xenoreactive human T cells in the scid mice. A possible cause for the reduced expansion of xenoreactive, human T cells, was the decreased expression of murine B7.1 and B7.2 caused by the administration of anti-hCD40L. Therefore, it may be that defective maturation of murine antigen-presenting cells impeded the priming and expansion of human xenoreactive T cells.