The Role of CD40 in the Immune Response

A successful humoral immune response requires reciprocal signaling between T helper cells and B cells. The first signal is antigen specific and is mediated by the interaction between the T cell receptor and antigen in association with major histocompatibility complex on B cells. The subsequent signals are provided by the costimulatory molecules CD40 and CD28 and their respective ligands. CD40L which is expressed on activated T helper cells interacts with CD40 on B cells providing the essential signal for the induction of B cell activation and immunoglobulin production. The importance of the CD40-CD40L interaction has been shown in patients suffering from X-linked immunodeficiency with hyper-IgM (HIGM1). The disease is characterized by the inability of B cells to undergo immunoglobulin isotype switching. Affected males experience recurrent infections and most infections are of bacterial origin, but HIGM1 patients are also unusually susceptible to infections with opportunistic pathogens and often suffer from Pneumocystis carinii pneumonia and Cryptosporidium intestinal infection. Mutations in CD40L molecules are responsible for hyper IgM syndrome in humans. To study the role of the CD40-CD40L interaction in vivo and to derive an animal model for HIGM1, I generated CD40 deficient mice using homologous recombination in embryonic stem (ES) cells. A targeting vector was constructed using an 8 kb CD40 genomic fragment. The G418 resistance gene (NEO) was inserted into the 3rd exon to disrupt the CD40 gene and to allow selection. The targeting vector was transfected into ES cells and G418 resistant clones were isolated - and screened for homologous recombination by Southern blot analysis. Chimaeric mice were generated by injection of targeted ES cell clones into blastocysts. Germline transmission was obtained and heterozygous mutant mice bred to generate CD40 deficient mice. Flow cytometric analysis of lymphocytes in CD40 deficient mice revealed normal development of B and T lymphocytes. CD40 deficient mice were immunized with KLH and assessed for germinal centre formation. CD40 deficient mice did not generate germinal centres. Analysis of serum immunoglobulin levels showed that CD40 deficient mice displayed reduced levels of isotype switched immunoglobulins compared to wild-type mice. These results confirm the crucial role of the CD40-CD40L interaction in humoral immunity. CD40 is also expressed on other antigen presenting cells and the involvement of the CD40-CD40L interaction in cell-mediated immunity was determined in CD40 deficient mice infected with Mycobacterium Bovis (BCG). Although CD40 deficient mice survived mycobacterial infection, the increased numbers of bacilli in spleen and lungs and the reduced production of IFN-ɣ in response to mycobacterial infection indicates that CD40 deficient mice are more susceptible to infection with BCG than control mice