Kiyoshi Takatsu

The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4⁺ T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4⁺ T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4⁺ T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs.

Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4+ T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4+ T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection.

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4⁺ T cells induces transient T-bet expression, interleukin (IL)-12 receptor β2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A^b-transfected Chinese hamster ovary cells in the absence of interferon-γ (IFN-γ) and IL-12. Sustained IFN-γ and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet^–/– naive CD4⁺ T cells are stimulated through TCR in the absence of IFN-γ or IL-12. Stimulation of naive CD4⁺ T cells in the absence of IFN-γ or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4⁺ T cells to Th1 cells.

Summary: Interleukin (IL)‐5 induces CD38‐activated splenic B cells to differentiate into immunoglobulin M‐secreting cells and undergo µ to γ1 class switch recombination (CSR) at the DNA level, resulting in immunoglobulin G1 (IgG1) production. Interestingly, IL‐4, a well‐known IgG1‐inducing factor does not induce immunoglobulin production or µ to γ1 CSR in CD38‐activated B cells. In the present study, we implemented complementary DNA microarrays to investigate the contribution of IL‐5‐induced gene expression in CD38‐stimulated B cells to immunoglobulin‐secreting cell differentiation and µ to γ1 CSR. IL‐5 and IL‐4 stimulation of CD38‐activated B cells induced the expression of 418 and 289 genes, respectively, that consisted of several clusters. Surprisingly, IL‐5‐inducible 78 genes were redundantly regulated by IL‐4. IL‐5 and IL‐4 also suppressed the gene expression of 319 and 325 genes, respectively, 97 of which were overlapped. Genes critically regulated by IL‐5 include immunoglobulin‐related genes such as J chain and immunoglobulinκ, and genes involved in B‐cell maturation such as BCL6, activation‐induced cytidine deaminase (Aid) and B lymphocyte‐induced maturation protein‐1 (Blimp‐1) and tend to be induced slowly after IL‐5 stimulation. Intriguingly, among genes, the retroviral induction of Blimp‐1 and Aid in CD38‐activated B cells could induce IL‐4‐dependent maturation to Syndecan‐1⁺ antibody‐secreting cells and µ to γ1 CSR, respectively, in CD38‐activated B cells. Taken together, preferential Aid and Blimp‐1 expression plays a critical role in IL‐5‐induced immunoglobulin‐secreting cell differentiation and µ to γ1 CSR in CD38‐activated B cells.

CD4⁺ Th1 cells play a critical role in the induction of cell-mediated immune responses that are important for the eradication of intracellular pathogens. Peptide-25 is the major Th1 epitope for Ag85B of Mycobacterium tuberculosis and is immunogenic in I-A^b mice. To elucidate the role of the TCR and IFN-γ/IL-12 signals in Th1 induction, we generated TCR transgenic mice (P25 TCR-Tg) expressing TCR α- and β-chains of Peptide-25-reactive cloned T cells and analyzed Th1 development of CD4⁺ T cells from P25 TCR-Tg. Naive CD4⁺ T cells from P25 TCR-Tg differentiate into both Th1 and Th2 cells upon stimulation with anti-CD3. Naive CD4⁺ T cells from P25 TCR-Tg preferentially develop Th1 cells upon Peptide-25 stimulation in the presence of I-A^b splenic antigen-presenting cells under neutral conditions. In contrast, a mutant of Peptide-25 can induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation is observed even in the presence of anti-IFN-γ and anti-IL-12. Furthermore, naive CD4⁺ T cells from STAT1 deficient P25 TCR-Tg also differentiate into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-A^b-transfected Chinese hamster ovary cells induce Th1 differentiation of naive CD4⁺ T cells from P25 TCR-Tg in the absence of IFN-γ or IL-12. These results imply that interaction between Peptide-25/I-A^b and TCR may primarily influence determination of the fate of naive CD4⁺ T cells in their differentiation towards the Th1 subset.

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R α-chain (IL-5Rα) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Rα-deficient (IL-5Rα^−/−) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Rα. IL-5 stimulation of wild-type B-1 cells, but not IL-5Rα^−/− B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Rα^−/− mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.

Bruton’s tyrosine kinase (Btk) is required for B cell development and signal transduction through cell‐surface molecules such as BCR and IL‐5 receptor. We have identified a Btk‐associated molecule, BAM11 (hereafter referred to as BAM) that binds to the pleckstrin homology (PH) domain of Btk, and inhibits Btk activity both in vivo and in vitro. In this study, we demonstrate BAM’s transcriptional co‐activation activity and its functional interaction with Btk. By using transient transcription assays, we demonstrate that the enforced expression of BAM enhances transcriptional activity of the synthetic reporter gene. The C‐terminus of BAM is essential for the transcriptional co‐activation activity. The ectopic expression of Btk together with BAM enhances BAM’s transcriptional co‐activation activity. BAM’s transcriptional co‐activation activity is enhanced through interaction with Btk, and requires both its intact PH domain and functional kinase activity. We also show that enforced expression of TFII‐I, another Btk‐binding protein with transcriptional activity, together with BAM and Btk, further augments BAM‐ and Btk‐dependent transcriptional co‐activation. Furthermore, BAM can be co‐immunoprecipitated with the INI1/SNF5 protein, a member of the SWI/SNF complex that remodels chromatin and activates transcription. We propose a model in which Btk regulates gene transcription in B cells by activating BAM and the SWI/SNF transcriptional complex via TFII‐I activation.