The success of the cord blood bank program in Japan was associated with the establishment of the Japanese Cord Blood Bank Network in 1999. Using the network system patients and their physicians were able to search for the most suitable cord blood units as their transplants. On the first screen of donor information, HLA mismatched loci, sex, blood type, and the number of nucleated cells in the unit was indicated. However, the number of CD34⁺ cells was not indicated until 2008. The main reason was that the comparison of these numbers between banks was difficult because the methods were different among the banks. The method of CD34⁺ cell enumeration was finally reached to the ISHAGE single platform method after improvement by changing dual platform to single. This was accomplished by using the control beads and by using 7-AAD reagents. The ISHAGE method has made it possible to accurately measure freeze-thawed samples that included dead cells. However, it took 5 years before all banks adopted this method. Keihan cord blood bank has used the CD34⁺ cell standard in addition to the nucleated cell standard for selection of cord blood to be processed since 2008. This contributed to the rise in quality of cord blood units and resulted in increase of their supply. This has made Keihan the largest contributor of cord blood in Japan. Due to fine constraints other banks have yet to adapt the CD34⁺ cell standard. Therefore, the development of the method to shorten the time for measurement is expected to make the CD34+ cell standard more feasible for other cord blood banks. This cell standard will also improve the quality of cord blood units.

当院では，1999 年から末梢血幹細胞採取（PBSCH；peripheral blood stem cells harvest）を開始し，CD34 の測定は，2002 年からベックマン・コールター社のStem-Kit を用いて開始した。今回2011 年1 月～4 月に自施設及び熊本医療センターのPBSCH 検体及び骨髄採取検体を用いて，自施設の当日，翌日のCD34 陽性率と検査センターで翌日測定したCD34 陽性率を比較検討した。CD34 陽性率はCD34 抗体の種類やクローンの違いにより，データに差異がでてくると思われるが，今回のように，クローン581 と8G12 のようなクラスIII の抗体であれば，陽性率は相関係数r=0.996 と全く問題を認めなかった。またフローサイトメトリーの機種の違いや，翌日測定することもCD34 陽性率にはほとんど影響与えなかった。

要旨 近年，移植技術の進歩に伴い，骨髄だけでなく，末梢血幹細胞や臍帯血が造血器悪性疾患の治療に用いられている。 2011 年末からは，非血縁者間の末梢幹細胞移植の導入が開始されるなど，造血器悪性疾患における造血幹細胞移植の適用拡大が期待されている。 これらの造血幹細胞移植において，CD34 陽性細胞数の正確な測定は，移植細胞の品質評価における重要な要素の一つと考えられている。フローサイトメトリー法を用いたCD34 陽性細胞数の測定は，国際血液療法・移植学会現国際細胞療法学会：ISCT）より，その標準化を目的としたガイドラインが策定されている^1）2）。しかしながら，信頼性の高い測定結果を得るためには，測定サンプルの調製やデータ解析における十分な経験が必要とされてきた。 本稿では，ISHAGE ガイドライン等の推奨項目に準じて新しく製品化された，BD Stem Cell Enumeration Kit を用いたCD34 陽性造血幹細胞の絶対数測定の有効性に関して紹介したい。

Invariant natural killer T cells (iNKT cells) are a subset of αβ T cell receptor (TCR) positive T cells and express Vα 14 (mouse) or Vα24 TCR (human). iNKT cells recognize lipid antigen, such as α-galactosylceramide (αGC) loaded on CD1d, which is non-classical major histocompatibility complex (MHC) class I-like molecule, and rapidly produce a huge amount of cytokines after stimulation. Although CD1d molecules were expressed on various cell types, dendritic cells (DCs) most efficiently presented αGC to iNKT cells both in vitro and in vivo. Through the antigen-presentation, DCs and iNKT cells reciprocally activated each other. Interestingly, although iNKT cells received both TCR and co-stimulatory stimuli from DCs, they became unresponsive to secondary stimulation (anergy). When restimulated in the prensene of exogenous IL-2, anergized iNKT cells produced IL-4 comparable to control cells but not IFN-γ. After primary stimulation, programmed death-1 (PD-1) molecules were expressed at high level on iNKT cells for certain period. However, αGCactivated iNKT cells in PD-1 deficient mice became anergic as in normal mice. Furthermore, anti-PD-1 blocking mAb was unable to restore their responsiveness. When iNKT cells were stimulated with immobilized anti-CD3 mAb in the presence or absence of anti-CD28 mAb, they produced cytokines in a dose-dependent manner. Unlike in conventional naïve CD4 T cells, the strong TCR-mediated signaling with co-stimulation rendered iNKT cells anergic to the subsequent stimulation with αGC and spleen DCs. Consistent with in vitro assay, the injection of αGC-pulsed DCs was more potent in inducing anergy than B cells. These results indicate that strong TCR-mediated activation with co-stimulation provides signals induces the anergic state in iNKT cells.

Dendritic cells (DC) are specialized antigen presenting cells that play critical roles to link innate and adaptive immunity. DCs are highly heterogeneous populations and can be divided into several subsets according to their physiological functions and expression patterns of cell surface molecules. We have found that XC chemokine receptor 1 (Xcr1) is exclusively expressed in one DC subset, CD8α⁺ DCs which have abilities to uptake apoptotic cells and cross-present soluble/cells-associated antigens on MHC class I to cytotoxic CD8 T cells. Xcr1-expressing DCs showed chemotactic migration to an Xcr1 ligand, Xcl1. We also showed that Xcl1 was constitutively expressed by NK cells and its expression was enhanced upon IL-2 stimulation. Furthermore, Xcl1 was also induced in CD8, but not CD4, T cells after crosslinking of anti-CD3ε plus anti-CD28 antibody, suggesting that a CD8α⁺ DC subset might migrate to NK cells or activated CD8 T cells via Xcr1 during immune responses.

Plasmacytoid dendritic cells (pDCs) play not only a central role in antiviral immune response in innate host defense but also a pathogenic role in the development of the autoimmune responses by vicious spiral based on the dysregulated type I IFN production through sensing immune complexes containing self-nucleic acids. Thus, type I IFNs and pDCs represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Accordingly, control of the dysregulated pDC-derived type I IFN production provides an alternative treatment strategy for SLE. We focused on two agents, IκB kinase inhibitor (BAY11-7082) and HMG-CoA reductase inhibitors (statins), and investigated their immunomodulatory effects in targeting the IFN response on pDCs. Our study identified that both BAY11 and statins have the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. We also found that these agents inhibited both in vitro IFN-α production induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with TLR ligand. These findings suggest that BAY11 and statins have the therapeutic potential to attenuate the IFN environment by regulating the pDC function and thus provide the novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.

We conducted two clinical trials of dendritic cell (DC)-based immunotherapy for elderly patients with acute myeloid leukemia. In both trials, autologous DCs were generated by culturing monocytes which were obtained from patients after recovery of normal hematopoiesis by chemotherapy, in the presence of GM-CSF and IL-4. In the first trial, DCs pulsed with autologous apoptotic leukemic cells were administered intradermally together with OK-432 to induce maturation of DCs in vivo. Injection sites were pretreated with OK-432 to facilitate DCs to migrate to regional lymph nodes by local skin inflammation. In the second trial, DCs were pulsed with HLA-A^＊24:02-restricted WT1235-243 modified peptide and zoledronate to activate Vγ9Vδ2 T cells, which exert the enhancing effect on the T cell-activating capacity of DCs. DCs were matured with TNF-α and PGE₂ ex vivo, and administered intradermally and intravenously. In the first trial, two of four patients showed induction of anti-leukemic immune response. In one patient with HLA-A^＊24:02, CD8 T cell responses against HLA-A^＊24:02-restricted WT1 and hTERT peptides were detected by IFN-γ ELISPOT assay and HLA tetramer staining. These results demonstrate cross-presentation of leukemia antigens in vivo by DCs pulsed with apoptotic leukemic cells. In the second trial, two of three patients showed induction of WT1 modified peptide-specific immune response. Notably, a small fraction of WT1 modified peptide-specific T cells was cross-reactive to the WT1 natural peptide. Moreover, whereas most of the modified peptide-specific T cells disappeared shortly after completion of DC vaccination, T cells cross-reactive to the natural peptide persisted longer presumably owing to presentation of the natural peptide by leukemic cells in vivo. In both trials, inductions of anti-leukemic immune response were associated with longer periods of stable disease. DC-based immunotherapy for elderly patients with acute myeloid leukemia is feasible and safe, and has the potential to improve clinical outcome of these patients.