Akemi Sakamoto

CD25+CD4+ T cells contribute to the control of memory CD8+ T cells

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. In animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.

Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells

Naïve T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8+ T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8+ T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8+ T cells

Central memory CD8+ T cells (TCM) are considered to be more efficient than effector ones (TEM) for mediating protective immunity. The molecular mechanism involved in the generation of these cells remains elusive. Because Bcl6 plays a role in the generation and maintenance of memory CD8+ T cells, we further examined this role in the process in relation to TCM and TEM subsets. In this study, we show that TCM and TEM were functionally identified in CD62L+ and CD62L− memory (CD44+Ly6C+) CD8+ T cell subsets, respectively. Although TCM produced similar amounts of IFN-γ and IL-2 to TEM after anti-CD3 stimulation, the cell proliferation capacity after stimulation and tissue distribution profiles of TCM differed from those of TEM. Numbers of TCM were greatly reduced and elevated in spleens of Bcl6-deficient and lck-Bcl6 transgenic mice, respectively, and those of TEM were constant in nonlymphoid organs of these same mice. The majority of Ag-specific memory CD8+ T cells in spleens of these mice 10 wk after immunization were TCM, and the number correlated with Bcl6 expression in T cells. The proliferation of Ag-specific memory CD8+ T cells upon secondary stimulation was dramatically up-regulated in lck-Bcl6 transgenic mice, and the adoptive transfer experiments with Ag-specific naive CD8+ T cells demonstrated that some of the up-regulation was due to the intrinsic effect of Bcl6 in the T cells. Thus, Bcl6 is apparently a crucial factor for the generation and secondary expansion of TCM.

The Blimp1–Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte–induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets ‘osteoclastic’ molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1–Bcl6–osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.

Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

The role of transforming growth factor (TGF)-&bgr; and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-&bgr;, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-&bgr; caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-&bgr; in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-&bgr;. TGF-&bgr; increased transcript level of &agr;2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-&bgr; were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

A Role for c-fos/Activator Protein 1 in B Lymphocyte Terminal Differentiation

Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) transcription factor is essential for promoting B cell differentiation into plasma cells. However, a critical transcription factor for Blimp-1 expression in activated B cells is unclear. When splenic B cells were stimulated with CD40 ligand (CD40L) and IL-4, terminal differentiation was induced in the B cells from c-fos transgenic (H2-c-fos) mice but barely in those from control littermates and from c-fos-deficient mice. AP-1 family and Blimp-1 mRNAs were transiently induced in the control B cells, and overexpression of c-Fos induced a sufficient amount of Blimp-1 for terminal differentiation in the H2-c-fos B cells. When normal and c-fos-deficient B cells were stimulated with LPS, a sufficient amount of Blimp-1 for terminal differentiation was induced in those B cells. However, expression of c-fos/AP-1 family mRNAs in LPS-stimulated normal B cells was similar to that of normal B cells stimulated with CD40L and IL-4. EMSA and chromatin immunoprecipitation assays using the AP-1-binding DNA sequence in the murine Blimp-1 promoter region demonstrated that AP-1-binding activity in nuclear protein of LPS-stimulated normal B cells was prolonged more than that in normal B cells stimulated with CD40L and IL-4. Furthermore, the percentage of CD138+ B cells within germinal center B cells in the spleen and the number of Ab-forming cells in the bone marrow of H2-c-fos mice was larger than that of control mice 12 days after immunization. Thus, although c-Fos is not essential for Blimp-1 expression, c-Fos/AP-1 positively regulates Blimp-1 expression and terminal differentiation of activated B cells.

Bcl6 controls granzyme B expression in effector CD8+ T cells

Bcl6, a sequence‐specific transcriptional repressor, is important for generation and maintenance of memory CD8+ T cells. Although memory CD8+ T cells are generated from effector CD8+ T cells, a role for Bcl6 in effector CD8+ T cells is largely unknown. We show here that Bcl6 expression was transiently induced in activated CD8+ T cells and continuously up‐regulated in effector CD8+ T cells. The amount of granzyme B mRNA among effector molecules produced by effector CD8+ T cells inversely correlated with the amount of Bcl6 mRNA in CD8+ T cells. Overexpression of Bcl6 in CD8+ T cells resulted in lower killing activity at their effector phase, supporting the reduction of granzyme B expression in effector CD8+ T cells by Bcl6. We identified a putative Bcl6‐binding DNA sequence in the promoter region of the granzyme B gene. Binding of Bcl6 to the Bcl6‐binding sequence was detected in naive CD8+ T cells but not in activated CD8+ T cells by chromatin immunoprecipitation assay. Furthermore, the Bcl6‐binding sequence was required for Bcl6 to repress the luciferase reporter gene expression controlled by the granzyme B promoter. Thus, the granzyme B gene is a molecular target of Bcl6 in effector CD8+ T cells.

A Lysosomal Protein Negatively Regulates Surface T Cell Antigen Receptor Expression by Promoting CD3ζ-Chain Degradation

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

Bcl6 Is a Transcriptional Repressor for the IL-18 Gene

Bcl6 functions as a sequence-specific transcriptional repressor, and Bcl6-deficient (Bcl6−/−) mice have been reported to display Th2-type inflammatory diseases in multiple organs. Since IL-18 is a potent stimulator of Th2 cells, we examined the expression of IL-18 mRNA in bone marrow-derived macrophages from Bcl6−/− mice after LPS stimulation. Here we show that the expression was strikingly up-regulated after stimulation. The expression was also up-regulated in RAW264 cells, a murine macrophage cell line, by transfection with the dominant negative type of Bcl6 gene. We identified a putative Bcl6-binding DNA sequence (IL-18BS) upstream of exon 1 of the murine IL-18 gene and three IL-18BSs in the promoter region of human IL-18 gene. Binding of Bcl6 in nuclear protein from resting RAW264 cells to murine IL-18BS was detected by gel retardation assay and chromatin immunoprecipitation assay. The binding activity was diminished gradually in RAW264 cells after LPS stimulation. However, the amount of Bcl6 protein in these cells was constant over the period examined, suggesting the functional modification of Bcl6 protein after stimulation. Furthermore, murine IL-18BS was required for Bcl6 to repress the expression of the luciferase reporter gene under control of the IL-18 promoter. Taken together, Bcl6 is a key regulator of IL-18 production by macrophages.

Impaired lymphocyte development and function in Clast5/Stra13/DEC1‐transgenic mice

Clast5/Stra13/DEC1 is a member of the helix‐loop‐helix family of transcriptional repressors. We have previously shown that Clast5 is rapidly down‐regulated upon B cell activation and its overexpression inhibits cell cycle progression in B lymphoma cells. In the present study, we show that Clast5 expression is developmentally regulated during B cell differentiation, being expressed at theprogenitor B cells, down‐regulated at the precursor B cells, elevated in immature and mature resting B lymphocytes, and down‐regulated again in germinal center B ells. To investigate the function of Clast5 in regulating lymphocyte development, we have generated transgenic mice expressing Clast5 in B‐ and T‐lineage cells (Clast5‐Tg). Clast5‐Tg mice grew and bred normally but their spleen and thymus cellularity was reduced compared with control littermates. The development of B cells in the bone marrow and T cells in the thymus was impaired, with the expansion of progenitor B and T cells most strongly affected. The frequency of IL‐7‐responsive cells in the bone marrow of Clast5‐Tg mice was reduced by >80% and their proliferative response to IL‐7 was also compromised. Mature B cells from Clast5‐Tg mice were hyporesponsive to antigen receptor cross‐linking and exhibited mild reduction in the proliferative response to CD40 ligation or lipopolysaccharide stimulation. Moreover, thedevelopment of germinal center B cells and antibody production against a T‐dependent antigen were reduced in Clast5‐Tg mice. These results reveal a critical role for Clast5/Stra13/DEC1 in negatively regulating lymphocyte development and function in vivo.