Cutting edge: A/WySnJ transitional B cells overexpress the chromosome 15 proapoptotic Blk gene and succumb to premature apoptosis.

Better knowledge of peripheral B lymphocyte homeostasis is needed to address the human hypogammaglobulinemia diseases. A defect in the Bcmd gene shortens the B cell life span and causes B cell deficiency in A/WySnJ mice. Previous genetic mapping placed Bcmd near Srebf2 on chromosome 15. Inspection of the human chromosome 22 syntenic region identified the proapoptotic Bik gene as a candidate. Two mapping methods placed the homologous mouse gene, Blk, near Srebf2. The Blk genomic structure was highly homologous to BIK: Sequence analysis ruled out coding region mutations, but Blk transcripts were overly abundant in sorted A/WySnJ T1 B cells. Moreover, enriched transitional B cells showed a cell-autonomous defect leading to excessive apoptosis. Thus, Bcmd may be a direct mutation in Blk, or in a gene involved in Blk regulation, such that excess expression pushes the A/WySnJ transitional B cells past the apoptosis checkpoint to cell death.

Rag-1-dependent cells are necessary for 1,25-dihydroxyvitamin D(3) prevention of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a demyelinating disease involving genetic and environmental risk factors. Geographic, genetic, and biological evidence suggests that one environmental risk factor may be lack of vitamin D. Here, we investigated how 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) inhibits experimental autoimmune encephalomyelitis (EAE), an MS model. The experiments used adoptive transfer of TCR-transgenic (TCR1) cells specific for myelin basic protein (MBP) peptide into unprimed recipients. When unprimed TCR1 splenocytes were transferred, and the recipients were immunized with peptide, the mock-treated mice developed EAE, but the 1,25-(OH)(2)D(3)-treated recipients remained disease-free. Both groups had TCR1 T cells that proliferated in response to MBP Ac1-11 and produced IFN-gamma but not IL-4 in the lymph node. In the central nervous system (CNS), the mock-treated mice had activated TCR1 T cells that produced IFN-gamma but not IL-4, while the 1,25-(OH)(2)D(3)-treated mice had TCR1 T cells with a non-activated phenotype that did not produce IFN-gamma or IL-4. When activated TCR1 T cells producing IFN-gamma were transferred into unprimed mice, the mock-treated and the 1,25-(OH)(2)D(3)-treated recipients developed EAE. Likewise, the 1,25-(OH)(2)D(3) did not inhibit Th1 cell IFN-gamma production or promote Th2 cell genesis or IL-4 production in vitro. Finally, the 1,25-(OH)(2)D(3) inhibited EAE in MBP-specific TCR-transgenic mice that were Rag-1(+), but not in animals that were Rag-1-null. Together, these data refute the hypothesis that the hormone inhibits Th1 cell genesis or function directly or through an action on antigen-presenting cells, or promotes Th2 cell genesis or function. Instead, the evidence supports a model wherein the 1,25-(OH)(2)D(3) acts through a Rag-1-dependent cell to limit the occurrence of activated, autoreactive T cells in the CNS.

A quantitative-trait locus controlling peripheral B-cell deficiency maps to mouse Chromosome 15.

Peripheral B-lymphocyte homeostasis is determined through incompletely defined positive and negative regulatory processes. The A/WySnJ mouse, but not the related A/J strain, has disturbed homeostasis leading to peripheral B-lymphocyte deficiency. B lymphopoeisis is normal in A/WySnJ mice, but the B cells apoptose rapidly in the periphery. This B cell-intrinsic defect segregated as a single locus, Bcmd, in (A/WySnJxA/J)F2 mice. Here we mapped a quantitative-trait locus (QTL) that contributes to the A/WySnJ B-cell deficiency by examining the F2 progeny of a cross between strains A/WySnJ and CAST/Ei. In this cross, minimally 1.9 QTLs controlling peripheral B lymphocyte deficiency segregated. The (A/WySnJxCAST/Ei)F2 mice were phenotyped for splenic B-cell percentage and the DNA from progeny with extreme phenotypes was used to map the QTL by the simple-sequence length polymorphism method. A genome scan showed linkage between peripheral B-cell deficiency and Chromosome (Chr) 15 markers. When closely spaced Chr 15 markers were analyzed, the 99% confidence interval for the QTL map position extended along the entire chromosome length. The peak lod scores >17 occurred between 30 and 45 cM. We conclude that a significant QTL segregating in (A/WySnJxCAST/Ei)F2 mice resides in this middle region of Chr 15.

IL-12 and IFN-gamma are required for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C.B-17 mice.

A murine model was used to assess the role of cytokines in initiating protective T-cell-mediated immunity in the lung. A pulmonary infection was initiated by intratracheal inoculation of Cryptococcus neoformans (Cne). Previously, we had established that Cne lung clearance was mouse-strain-specific: C.B-17 mice were resistant and developed a Th1-like response, whereas C57BL/6 mice were susceptible and did not develop a Th1 response. In the present study we showed that monoclonal anti-interferon-gamma (IFN-gamma) and anti-interleukin-12 (IL-12) antibody administration prior to infection of resistant C.B-17 mice inhibited lung clearance of Cne. Cytokine profiles of lung and lung-associated lymph nodes (LALN) from monoclonal antibody (mAb)-treated C.B-17 mice were switched from Th1-like to Th2-like, and mAb-treated C.B-17 mice exhibited lung eosinophilia, which was absent in control C.B-17 mice. Additionally, C.B-17 mice treated with anti-IFN-gamma and anti-IL-12 mAb demonstrated a significantly lower percentage of lung macrophages expressing inducible nitric oxide synthase (iNOS) than did control mice. These studies clearly demonstrate that both IFN-gamma and IL-12 are required for initiation of a Th1 response in resistant C.B-17 mice.

Early cytokine production in pulmonary Cryptococcus neoformans infections distinguishes susceptible and resistant mice.

A murine pulmonary infection model utilizing intratracheal inoculation of Cryptococcus neoformans was used to analyze cytokines produced in response to opportunistic pathogens acquired via the respiratory tract. The specific question asked was whether early cytokine secretion in lung-associated lymph nodes (LALN) would predict whether this organism would be cleared from the lung. Lung colony-forming units (CFU) were analyzed in two strains of mice over 12 wk, and lung clearance was found to be strain dependent. C.B-17 mice reduced their lung CFU burden between day 7 and day 14 of infection, had significantly higher in lung CFU than C.B-17 mice. The capacity of cells from lungs and LALN to secrete cytokines was significantly different between the strains when assessed at day 7 and day 14 after inoculation. When compared with sensitive C57BL/6 mice 7 days after infection, resistant C.B-17 mice demonstrated (1) increased interferon-gamma secretion by LALN cells in vitro in response to media alone, heat-killed cryptococci, and the T cell mitogen concanavalin A and (2) increased interleukin (IL)-2 secretion by both LALN and lung cells in response to concanavalin A. IL-4 and IL-10 were comparable or undetectable in both mouse strains, whereas IL-5 was significantly higher in all lung cell cultures of C57BL/6 mice. Thus, an early regional Th1 immune response in C.B-17 mice correlated with resistance to the organism, whereas the absence of this response in C57BL/6 mice correlated with susceptibility.

The role of CD4+ and CD8+ T cells in the protective inflammatory response to a pulmonary cryptococcal infection.

Moderately virulent strains of Cryptococcus neoformans, inoculated via the trachea, cause a pulmonary infection in BALB/c mice that was gradually resolved by T lymphocyte-dependent mechanisms. The current studies, using monoclonal antibodies to deplete T cell subsets, demonstrated that CD4+ and CD8+ T cells combined to mediate a prominent pulmonary inflammatory infiltrate that included lymphocytes, macrophages, neutrophils, and eosinophils. The inflammatory response peaked 2 weeks after infection and coincided with the beginning of gradual pulmonary clearance of the infection. CD4/CD8 double deficiency (4-8-) markedly reduced the influx of all cells into the lungs. A CD4 deficiency had a more profound effect on the total number of inflammatory cells recruited to the lungs than a CD8 deficiency. Depletion of either CD8+ or CD4+ T cells significantly decreased pulmonary macrophages and neutrophils, but only a CD4 deficiency prevented the influx of eosinophils. Recruitment of CD8+ T cells occurred independently of CD4+ T cells, but CD4+ T cell recruitment to the lungs was significantly reduced in CD8-deficient mice. Mitogen-stimulated infiltrating lung lymphocytes from infected 4+8+ mice secreted both T helper cell type 1 (Th1) [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] and Th2 (IL-4, IL-5, and IL-10) cytokines. CD4 deficiency resulted in loss of T cells secreting IL-4, IL-5, and IL-10. However, residual CD8+ T cells still secreted IL-2 and IFN-gamma. Lung T cells from CD8-deficient mice secreted similar levels of IL-4, IL-5, and IL-10 on a per lung basis compared with 4+8+ mice despite decreased numbers of CD4+ T cells, but secreted reduced levels of IFN-gamma. These experiments indicate that (1) CD4+ T cells play a dominant role in recruiting macrophages and granulocytes to the lung and (2) CD8+ T cells also mediate cellular recruitment, increase the magnitude of CD4+ T cell numbers in the infiltrate, and contribute to the local secretion of IFN-gamma. Thus, these studies demonstrate that CD8+ T cells can independently mediate an inflammatory response to a large, particulate, extracellular antigen, a role heretofore attributed almost solely to CD4+ T cells.