The existence of a hyaluronic acid-rich node and duct system (HAR-NDS) within the lymphatic and blood vessels was demonstrated previously. The HAR-NDS was enriched with small (3.0-5.0 μm in diameter), adult stem cells with properties similar to those of the very small embryonic-like stem cells (VSELs). Sca-1(+)Lin(-)CD45(-) cells were enriched approximately 100-fold in the intravascular HAR-NDS compared with the bone marrow. We named these adult stem cells "node and duct stem cells (NDSCs)." NDSCs formed colonies on C2C12 feeder layers, were positive for fetal alkaline phosphatase, and could be subcultured on the feeder layers. NDSCs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(+), while VSELs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(-). NDSCs had higher sphere-forming efficiency and proliferative potential than VSELs, and they were found to differentiate into neuronal cells in vitro. Injection of NDSCs into mice partially repaired ischemic brain damage. Thus, we report the discovery of potential adult stem cells that may be involved in tissue regeneration. The intravascular HAR-NDS may serve as a route that delivers these stem cells to their target tissues.

4-1BB (CD137) is expressed on dendritic cells (DCs) and its biological function has remained largely unresolved. By comparing 4-1BB-intact (4-1BB(+/+)) and 4-1BB-deficient (4-1BB(-/-)) DCs, we found that 4-1BB was strongly induced on DCs during the maturation and that DC maturation was normal in the absence of 4-1BB. However, DC survival rate was low in the absence of 4-1BB, which was due to the decreased Bcl-2 and Bel-x(L) in 4-1BB(-/-) DCs compared with 4-1BB(+/+) DCs after DC maturation. Consistent with these results, 4-1BB(-/-) DCs showed an increased turnover rate in steady state and more severely decreased in spleen by injecting LPS compared with 4-1BB(+/+) DCs. When OVA-pulsed DCs were adoptively transferred to recipient mice along with OVA-specific CD4(+) T cells, 4-1BB(-/-) DCs did not properly migrate to the T cell zone in lymph nodes and poorly induced proliferation of CD4+ T cells, although both DCs comparably expressed functional CCR7. Eventually, 4-1BB(-/-) DCs generated a reduced number of OVA-specific memory CD4(+) T cells compared with 4-1BB(+/+) DCs. To further assess the role of 4-1BB on DC longevity in vivo, 4-1BB(+/+) and 4-1BB(-/-) C57BL/6 were administrated with Propionibacterium acnes that develop liver granuloma by recruiting DCs. Number and size of granuloma were reduced in the absence of 4-1BB, but the inflammatory cytokine level was comparable between the mice, which implied that the granuloma might be reduced due to the decreased longevity of DCs. These results demonstrate that 4-1BB on DCs controls the duration, DC-T interaction, and, therefore, immunogenicity. The Journal of Immunology, 2009, 182: 4107-4115.

4-1BB (CD137) is expressed on dendritic cells (DCs) and its biological function has remained largely unresolved. By comparing 4-1BB-intact (4-1BB+/+) and 4-1BB-deficient (4-1BB−/−) DCs, we found that 4-1BB was strongly induced on DCs during the maturation and that DC maturation was normal in the absence of 4-1BB. However, DC survival rate was low in the absence of 4-1BB, which was due to the decreased Bcl-2 and Bcl-xL in 4-1BB−/− DCs compared with 4-1BB+/+ DCs after DC maturation. Consistent with these results, 4-1BB−/− DCs showed an increased turnover rate in steady state and more severely decreased in spleen by injecting LPS compared with 4-1BB+/+ DCs. When OVA-pulsed DCs were adoptively transferred to recipient mice along with OVA-specific CD4+ T cells, 4-1BB−/− DCs did not properly migrate to the T cell zone in lymph nodes and poorly induced proliferation of CD4+ T cells, although both DCs comparably expressed functional CCR7. Eventually, 4-1BB−/− DCs generated a reduced number of OVA-specific memory CD4+ T cells compared with 4-1BB+/+ DCs. To further assess the role of 4-1BB on DC longevity in vivo, 4-1BB+/+ and 4-1BB−/− C57BL/6 were administrated with Propionibacterium acnes that develop liver granuloma by recruiting DCs. Number and size of granuloma were reduced in the absence of 4-1BB, but the inflammatory cytokine level was comparable between the mice, which implied that the granuloma might be reduced due to the decreased longevity of DCs. These results demonstrate that 4-1BB on DCs controls the duration, DC-T interaction, and, therefore, immunogenicity.

4-1BB (CD137) triggering typically induces Th1 response by increasing IFN-gamma from T cells upon TCR ligation. We found recently that 4-1BB costimulation increased the expression of IL-13 from CD4(+) T cells, as well as CD8(+) T cells. The enhanced IL-13 expression by agonistic anti-4-1BB treatment was mediated via MAPK1/2, PI-3K, JNK, mammalian target of rapamycin, NF-AT, and NF-kappaB signaling pathways. The signaling for IL-13 induction was similar to that of IFN-gamma production by anti-4-1BB treatment in T cells. When the anti-4-1BB-mediated IL-13 expression was tested in an in vivo viral infection model such as HSV-1 and vesicular stomatitis virus, 4-1BB stimulation enhanced IL-13 expression of CD4(+) T, rather than CD8(+) T cells. Although IL-13 was enhanced by anti-4-1BB treatment, the increased IL-13 did not significantly alter the anti-4-1BB-induced Th1 polarization of T cells--increase of T-bet and decrease of GATA-3. Nevertheless, anti-4-1BB treatment polarized T cells excessively in the absence of IL-13 and even became detrimental to the mice by causing liver inflammation. Therefore, we concluded that IL-13 was coinduced following 4-1BB triggering to maintain the Th1/2 balance of immune response.

To explore the roles of 4-1BB (CD137) and CD28 in corneal transplantation, we examined the effect of 4-1BB/4-1BB ligand (4-1BBL) and/or CD28/CD80/CD86 blockade on corneal allograft survival in mice. Allogeneic corneal transplantation was performed between two strains of wild-type (WT) mice, BALB/c and C57BL/6 (B6), and between BALB/c and B6 WT donors and various gene knockout (KO) recipients. Some of the WT graft recipients were treated intraperitoneally with agonistic anti-4-1BB or blocking anti-4-1BBL monoclonal antibody (mAb) on days 0, 2, 4 and 6 after transplantation. Transplanted eyes were observed over a 13-week period. Allogeneic grafts in control WT B6 and BALB/c mice treated with rat immunoglobulin G showed median survival times (MST) of 12 and 14 days, respectively. Allogeneic grafts in B6 WT recipients treated with anti-4-1BB mAb showed accelerated rejection, with an MST of 8 days. In contrast, allogeneic grafts in BALB/c 4-1BB/CD28 KO and B6 CD80/CD86 KO recipients had significantly prolonged graft survival times (MST, 52.5 days and 36 days, respectively). Treatment of WT recipients with anti-4-1BB mAb resulted in enhanced cellular proliferation in the mixed lymphocyte reaction and increased the numbers of CD4(+) CD8(+) T cells, and macrophages in the grafts, which correlated with decreased graft survival time, whereas transplant recipients with costimulatory receptor deletion showed longer graft survival times. These results suggest that the absence of receptors for the 4-1BB/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival, whereas triggering 4-1BB with an agonistic mAb enhances the rejection of corneal allografts.