We demonstrate in this study that the size of T cell zone and the number of resident CD4+
T cells in the spleen were markedly reduced in the spleen of SIRPα MT mice. In addition, the mRNA expression of CCL19, CCL21, and IL-7 or the protein expression of the former two chemokines was also decreased in the spleen of SIRPα MT mice. Given that CCL19 and CCL21 as well as IL-7 are thought to be important for organization and maintenance of the T cell zone (1
), the reduction in the size of the T cell zone and CD4+
T cell number in the spleen of SIRPα MT mice is likely, at least in part, attributable to the decreased expression of CCL19, CCL21, or IL-7. Both CCL19 and CCL21, as well as IL-7, are produced by stromal cell FRCs in the PALS of spleen. Indeed, the immunoreactivity of gp38, a marker for FRCs, was markedly decreased in the spleen of SIRPα MT mice, suggesting that the decrease of the cell population of FRCs might be a cause for the reduced expression of CCL19, CCL21, or IL-7 in the spleen of SIRPα MT mice.
The expression of SIRPα is minimal in T cells as shown in Supplemental Fig. 1
, suggesting that SIRPα is unlikely required in a cell-autonomous manner for homeostatic regulation of T cells in the spleen. However, the reduction of the T cell zone as well as the decreased mRNA expression of CCL19 or CCL21 and IL-7 in the spleen were observed in hematopoietic BM chimeras. We indeed found that the mRNA expression levels of LTα, LTβ, and LIGHT were markedly decreased in the spleen of SIRPα MT mice. LTα, LTβ, and LIGHT are thought to be important for ho-meostatic regulation of T cells by producing of CCL19 or CCL21 from FRCs. Indeed, the spleens of adult LTα−/ −
, LTβ −/ −
, and LTβR−/ −
mice showed marked defects in the development of white pulps in the spleen (2
). LTα and LTβ are predominantly expressed on B cells or T cells (11
), whereas LIGHT is expressed in T cells, DCs, or NK cells (39
). Thus, these results suggest that hematopoietic SIRPα is, at least in part, important for homeostatic regulation of T cells or expression of CCL19 or CCL21 in the spleen through the action of LTαβ or LIGHT. Of interest is that the expression level of either LTα or LTβ in isolated T cells or B cells from SIRPα MT mice did not differ from that of WT mice. Thus, the reduction of LTα and LTβ in the whole spleen of SIRPα MT mice is, at least in part, attributable to the reduction of T cell population. However, LTα and LTβ are also expressed in lymphoid tissue inducer (LTi) cells (40
) or NK cells (38
). Moreover, expression of CCL21 or of CCL19 was reduced in the secondary lymphoid organs of LTβ KO mice but not in B cell-specific LTβ KO or T and B cell-specific LTβ KO mice, suggesting that expression of CCL21 or CCL19 is largely independent of LT produced by T or B lymphocytes (41
). Thus, SIRPα is required for expression of LTα or LTβ that is produced by yet-unidentified hematopoietic cells such as LTi cells, which might express SIRPα. By the use of BM chimera, we found that nonhematopoietic SIRPα is also required for expression of CCL21 or CCL19 in the spleen. Given the expression of SIRPα in non-hematopoietic cells such as fibroblasts and endothelial cells (30
), SIRPα is potentially expressed in FRCs in the spleen and thus required for the homeostatic regulation of FRCs.
In addition to the reduction of LTα or LTβ mRNA expression in the spleen, we showed that the effect of the agonistic mAb to LTβR on mRNA expression of CCL21 or p100 was markedly reduced in the spleen of SIRPα MT mice. Such reduced response is presumably attributable to the reduction of LTβR mRNA expression in the spleen of SIRPα MT mice. Furthermore, SIRPα might be important for the signaling pathway downstream of LTβR, and impairment of LTβR signaling thus participates in the reduced expression of homeostatic chemokines CCL21 or CCL19 in the spleen of SIRPα MT mice.
SIRPα MT mice manifest mild splenomegaly that is likely attributable to the increased number of RBCs in the spleen (15
). Thus, a decrease of the white pulp of SIRPα MT mice might be a secondary effect of the splenomegaly. However, we demonstrated in this study that the number of CD4+
T cells, but not that of B cells, was indeed reduced in the spleen of SIRPα MT mice. Moreover, CD47 KO mice also manifested the reduction of T cell zone and the decreased cell number of CD4+
T cells, whereas they did not manifest splenomegaly (M. Sato-Hashimoto, Y. Saito, and T. Matozaki, unpublished observations). Thus, the reduced size of white pulp is unlikely attributable to a secondary effect of the splenomegaly in SIRPα MT mice. By contrast, the number of CD11chigh
DCs (cDCs) is markedly reduced in the spleen of SIRPα MT mice (25
). Given that interaction of self-Ag–presenting MHC molecules of DCs with TCR promotes the survival of naive T cells (6
), the reduction of cDCs in the spleen of SIRPα MT mice might participate in the decrease in CD4+
The reason why the phenotypes of MT mice are specific to the spleen is currently unknown. However, such tissue-specific difference was noted in some gene-KO mice. BCR-deficient mice manifested a marked decrease of T cells as well as of CCL21 in the spleen but not in pLNs (42
). In addition, CD30-deficient mice showed impaired B/T segregation in the spleen but not in pLNs (43
), and the authors suggested the importance of LTi cells in regulation by CD30 of B/T segregation. We indeed measured the mRNA expression of CCL21, CCL19, CXCL13, and IL-7 in the pLNs. Expression levels of mRNA for these chemokines or a cy-tokine in the pLNs did not differ between WT and SIRPα MT mice, again suggesting that the phenotypes of SIRPα MT mice are specific to the spleen. Thus, SIRPα is likely an element that regulates T cell homeostasis differentially in the spleen and pLNs.
Similar to SIRPα MT mice, CD47 KO mice manifested the marked reduction of the T cell zone and the decreased cell number of CD4+
T cells, as well as reduced expression of CCL19 and CCL21, CXCL13, or IL-7 in the spleen. Given that CD47 is a li-gand for SIRPα, the similarity of these CD47 KO phenotypes to those of SIRPα MT mice suggests that CD47 and SIRPα form a pathway that regulates splenic T cell microenvironment. In contrast to SIRPα, however, we have not determined yet whether hematopoietic or nonhematopoietic CD47 is important for such regulation. With regard to the role of hematopoietic CD47, trans
interaction of CD47 on T cells with hematopoietic SIRPα might be important. With regard to the role of nonhematopoietic CD47, trans
interaction of CD47 on nonhematopoietic cells, such as stromal cells or endothelial cells, with hematopoietic SIRPα might be important for the homeostatic regulation of T cells in the spleen. Indeed, CD47 is expressed in splenic and BM stromal cells (44
) (Y. Saito and T. Matozaki, unpublished observations) as well as in endothelial cells (18
Overall, our present study provides a new insight into the molecular basis for regulation of T cell homeostasis in the spleen by membrane-bound molecules. Further study is clearly necessary to understand the detailed mechanism by which SIRPα that is not expressed in T cells regulates T cell homeostasis by interacting CD47.