M. not induce immunity to IOE. Antisera from both wild-type and MHC-II-deficient mice provided at least partial resistance to challenge infection, and protection could also be achieved following transfer of total, but not B-cell-depleted, splenocytes obtained from can induce class switch recombination in the absence of classical T-cell-mediated help. These studies highlight a major protective role for classical T-cell-independent humoral immunity during an intracellular bacterial infection. Despite increasing evidence that humoral immunity can play a role in protection against intracellular bacterial pathogens, it is often generally accepted that cellular immunity is the dominant form of protection during intracellular bacterial infections. We have drawn similar conclusions on the basis of our previous studies OTX008 of protective immunity during ehrlichial infections, where we have shown that CD4 T-cell production of gamma interferon (IFN-) is essential (4). However, work from several laboratories, using a variety of infection models, has demonstrated that antibodies can also play an important role in immunity (6, 8, 10, 26, 33, 35, 44, 51). In our own studies, for example, we found that immune serum or outer membrane protein (OMP)-specific monoclonal antibodies could protect susceptible SCID mice from fatal monocytotropic ehrlichia infection (24, 26). Although these and other studies indicated that antibodies could control ehrlichia infections in immunodeficient mice, they did not reveal the extent to which antibodies mediate protection in immunocompetent mice. More recent studies of immunity to highly pathogenic bacteria isolated from (IOE) revealed that B cells were essential for protection in immunocompetent mice following a low-dose sublethal infection (51). However, low-dose IOE-infected wild-type mice generated relatively poor antibody responses and were not protected from a subsequent fatal high-dose IOE challenge infection (5, 51). In contrast, infection with a closely related low-pathogenicity ehrlichia, (19), was shown to generate effective immunity to IOE challenge (17). In these latter studies, infection was associated with production of IFN- by CD4 T cells, although the requirement(s) for CD4 T cells, B cells, and inflammatory cytokines in protective immunity was not fully resolved. Here we have addressed the underlying mechanisms of protective immunity induced by infection. In contrast to our expectations that CD4 Th1 cells would play an important and essential role in immunity, we found instead that B cells and antibodies were required for protection. Moreover, B-cell-dependent protective immunity was generated in the absence of CD4 T cells. These findings indicate that B cells and antibodies can play not only auxiliary but also central roles in host defense during an intracellular bacterial infection in immunocompetent mice. MATERIALS AND METHODS Mice. The mice used in these OTX008 studies were obtained OTX008 from Jackson Laboratories, Bar Harbor, ME, or were bred in the Animal Care Facility at the Wadsworth Center under microisolator conditions, in accordance with institutional guidelines for animal welfare. The inbred strains were C57BL/6 and C57BL/6-(B6.CB17-immunization. infection was performed by intraperitoneal inoculation of 7.2 104 CFU. CFU were determined on blood agar. CD4 T-cell purification. CD4 T cells were purified from mouse spleen cell homogenates using a CD4 T-cell isolation kit (BD Biosciences) following the instructions of the manufacturer. For further CD4 T-cell enrichment, the samples were sorted by flow cytometry using a FACSVantage flow cytometric cell sorter (BD Biosciences), which yielded cells of a purity of 99%. For T-cell adoptive transfers, CD4 T cells were purified by negative magnetic bead selection (Miltenyi Biotec) and were resuspended in Hanks balanced salt solution at a concentration of 2 106/ml prior to transfer (0.5 ml) to recipient mice by tail vein injection. B-cell depletion. B cells were depleted from whole splenocyte suspensions using goat anti-mouse polyclonal IgG microbeads (Polysciences Inc.). The beads were washed and mixed with the splenocytes (4 ml/spleen), and the suspension was incubated at 4C on a rocker for 30 min prior to binding to the magnet. The supernatant containing unbound cells was used in cell transfer experiments. Fluorescence-activated cell sorter (FACS) analysis revealed that the depleted cell suspensions contained fewer than 2% B220-positive cells. Cell IL1A and cytokine neutralization. For neutralization of CD4 T cells, mice were administered.
