称号及び氏名

博士（学術）Viskam Wijewardana

学位授与の日付

平成 1９年９月３０日

論「Basic study of canine dendritic cells：In vitro generation,

文

名characterization and regulatory factors（イヌ樹状細胞の基礎研究： In vitro における分化誘導、特性の解析および制御因子）」論文審査委員

主査

松尾三郎

副査

稲葉俊夫

副査

馬場栄一郎

副査

杉浦喜久弥

論文要旨
Introduction
Dendritic cells (DCs) are specialized to take up, process and present antigens, andhave the capacity to stimulate naïve T cells and initiate the primary immune response.
Since patients with growing tumor are impaired to recognize the abnormal cells as
non-self, these properties of DC can be utilized to recall immune responses against
tumor cells.

Furthermore, since the highly specific immune responses against tumor

cells are persistently memorized and take place wherever the immune cells can arrive
at, the DC based immunotherapy is expected to cope with tumor relapses andmetastasis.
For approaching to the immune therapy, much effort has been done to establish theoptimal method to induce DCs in vitro from monocytes and bone marrow precursors
using purified cytokines such as granulocyte-macrophage colony stimulating factor(GM-CSF) and interleukin (IL)-4, to overcome the scarcity of DCs in blood. In human,
using the in vitro developed DCs, many clinical trials have been carried out against
variety of tumors. However, in most of the cases the DC based therapy has not beenenough succeeded, and many of patients has showed progress of the disease. Recently it
has been found that the in vitro developed DCs were inactivated along with reverse
maturation in the in vivo environment where the stimulation for DC activation andmaturation was not available. Furthermore, it has also found that some tumors produce
factors to suppress the DC activation. Therefore, it is necessary for the success of theDC based therapy to constitute the in vivo environment to support the development andactivation of DCs.
Suffering from common types of cancer with humans, success in DC therapy against
canine cancers promises not only advancement in cancer treatment in veterinary
medicine, but also provides effective information that advances DC therapy in humans.
However, the development and characteristics of DCs in the dog has not been subjected
to a thorough study. Therefore, a series of experiments were conducted in order tounderstand in vitro differentiation, characterization of canine DCs and regulatory

factors for optimizing their activity in the tumor therapy. First, the method to effectively
induce fully matured DC from canine peripheral blood monocytes (PBMOs) wasestablished. Second, using biophysical phenotypes, hematopoietic progenitor cells fromcanine bone marrow (BM) was isolated to be used ultimately as sources of DC. Third,
genes of canine cytokine were cloned and expression vector including them wereconstructed to constitute the in vivo environment.

Chapter 1: Generation of canine DCs from peripheral blood monocytes without using
purified cytokines
Since the DC population in blood and tissues is very small, recent work has aimed togenerate DCs in vitro from PBMOs or BM precursors, using granulocyte-macrophage(GM)-CSF, IL-4 and maturing factors in the human and mouse. But this generation
method is not applicable in animal species for which recombinant cytokines are notavailable. The differentiation and maturation of canine DCs from PBMOs also have notyet been well characterized, since appropriate conditions for the in vitro differentiation
were not known. However, it has been reported that monocytes differentiate into DCs invivo in the T cell area of lymph nodes, and that maturation of DCs takes place in thelymphoid tissues through their interaction with T cells. Therefore, it is suggested thatthe conditioned media produced by activated T cells provides suitable conditions fordifferentiation and maturation of DCs. To verify this hypothesis and to cope withlimitations in purified cytokines of the dog, canine T cell conditioned medium (TCCM)
was developed by culturing peripheral T cells with immobilized anti-canine CD3 mAb,
and the differentiation of DCs from PBMOs (isolated CD14+ cells) was examined bymorphology, phenotype and functional aspects.
There has been tremendous progress in characterizing DCs in both mice and humans.
In all these studies, mature DCs have been identified based on their morphological andfunctional characteristics: 1) Large cells with long dendritic processes; 2) significant
expression of molecules for antigen presentation, such as MHC (particularly MHC II)
and CD1a; 3) potent ability to stimulate allogeneic T cells in MLR; and 4) poor
phagocytotic activity. In addition, DCs produce cytokines such as IFN-γ, TNF-α andIL-18, to augment immune responses. In the present study morphological andfunctional characteristics of cells derived from the TCCM-culture of PBMOs wasinvestigated, and found that the day 12 cells from the TCCM culture provided featurecompletely according with the DC-characters described above. Furthermore, the effectsof TCCM are reproducible over different batches. It follows from these results that
TCCM provide optimal condition for the in vitro differentiation of DCs from PBMOs. Inthis study, a model for DC generation without using pure cytokines is exhibited.

Chapter 2: Isolation of hematopoietic progenitor cells in canine bone marrow
The in vitro differentiation of DC was first found from BM progenitor cells. From theresults of the study described in Chapter 1, it is suggested that efficiency of DC
generation is affected by the purity of precursor. Therefore, as the first step ofinvestigating the canine DC-differentiation in the BM cell route, hematopoietic
progenitor cells (HPCs) in canine bone marrow were isolated using phenotypes
concerned with physiological properties: Cell density, surface glycoproteins which bindto wheat germ protein (WGA) and capability for efflux of vital dye, Rhodamine-123 (Rh)which is mediated by multidrug resistance genes such as P-glycoprotein.
Canine BM cells were separated by equilibrium-discontinued centrifugation, andHPCs, detected by in vitro colony formation, were significantly enriched in therelatively low density (LD) fraction. In flow cytometry, many CD34, a marker of human
HPCs or MHC class II expressing cells were detected in the LD fraction, but these werenot significantly enriched. When the LD cells were separated, using a cell-sorting
method, into cells with high affinity of WGA (WGAhigh) and cells with WGAlow, almost all
multipotent HPCs (MHPCs) and HPCs committed to myeloid lineage were found in theWGAhigh population. However, forty percent of CD34 positive cells were found asWGAlow. Therefore, the WGA separation is superior to that by CD34 for isolation of DCprogenitors. When the WGAhigh population was further stained for Rh, almost allMHPCs were included in the dull population (Rhlow). Morphologically, most Rhlow cells
were round blastic cells containing a large nucleus with nucleoli and narrow cytoplasm.
Based on these results, it is suggested that all of the MHPCs in canine BM show theRhlow WGAhigh LD phenotype, and may contain hematopoietic stem cells, which are theprimitive HPCs.

Chapter 3: Cloning and production of canine cytokine genes for generation andactivation of dendritic cells in vivo
IL-12 shows potent ability to activate immune cells concerning in cellular immunity,
such as cytotoxic T lymphocyte (CTL) against tumors. While IL-12 is produced by
activated mature DCs, the cytokine in turn takes a role in maintenance of DC activity.
On the other hand a combination of GM-CSF and IL-4 exert an effect to induce
differentiation of PBMOs into DCs. Therefore, if an environment producing GM-CSF,
IL-4 and IL-12 exists near or around tumor tissues, DCs will be continuously recruitedby the differentiation from infiltrated PBMOs, and maintain the activity to primespecific immunity against the tumor. The long term existence of such environment will
be achieved by transfection of gene-expression vectors containing the cytokine genes. In
this study, as the first step of the project, the molecular cloning of canine IL-4, GM-CSF
and IL-12 gene was carried out, and the expression of functional cytokine by the gene
was examined.
Full length of canine IL-4, GM-CSF, IL-12p35 and IL-12p40 cDNAs were amplified by
PCR with specific primers, inserted into blunt-end vector and cloned in E. coli clone
DH5α. After the certification of correct sequence, cDNA of IL-4, GM-CSF or IL-12 p35(p35) was inserted into expression vector, pcDNA3.1, which contains neomycin-resistantgene and cDNA IL-12 p40 (p40) into pSVL, which does not contain neomycin-resistantgene. The vectors were transfected into CHO cells and the transfected cells wereselected with neomycin.
For expression of IL-12, the p35-pcDNA3.1 and the p40-pSVL vectors wereco-transfected into CHO cells. After the selection with neomycin, the expression of IL-12
was examined in immunofluorescence assay using anti-canine p40 antibody. About 50%of the CHO cells was successfully transfected with both the p35-pcDNA3.1 and thep40-pSVL vectors. Ninety to100% of CHO cells transfected with IL-4-gene or
GM-CSF-gene was detected to express IL-4 or GM-CSF cytokine in flow cytometry.

Supernatants were collected from cultures of IL-4-expressing CHO (IL-4-CHO),
GM-CSF-CHO and IL-12-CHO, and examined for functions of DC induction andmaturation. When PBMOs were incubated with supernatants of GM-CSF-CHO andIL-4-CHO, cell morphology changed to that of DCs, and expression of MHC class II,
CD1a, CD80 and CD86 on the cells significantly elevated. The expressions of CD80 andCD1a were significantly enhanced by addition of the IL-12-CHO supernatant. Theseresults suggest that cloned canine IL-4 and GM-CSF genes express functional cytokinesfor DC induction, and IL-12 gene for DC maturation. In addition, the IL-12-CHO
supernatant activated T cells and induced secretion of IFN-γ. Taken together, it issuggested that these cytokine gene vectors can be utilized to constitute the in vivoenvironment for recruiting DCs and maintaining DC activity against tumors.

Conclusions
1. Mature DCs can be successfully generated using TCCM from PBMO and thiscould be used as a model to develop DCs in any species especially whererecombinant cytokines have not developed.
2. HPCs including DC progenitors were significantly enriched in the LD WGAhigh
Rhlow cells of the dog BM.

3. IL-12 not only activates T cells but facilitates the DC maturation also.
4. All results in the series of experiments together will provide new strategy for theDC based tumor immunotherapy not only in dogs but also in human.

審査結果の要旨
樹状細胞は、抗原提示に特化した能力を持つ血液細胞で、抗原特異的免疫反応を惹起できる唯一の細胞である。他方、がん患者の体内では、がん細胞に対する免疫機能が著しく障害されて、本来排除されるべきがん細胞が排除されずに増殖している。そこで、生体外（in vitro）において骨髄中の前駆細胞や末梢血単球から樹状細胞を分化誘導させて育て、がん抗原を取り込ませて提示させた後に生体内（in vivo）に戻せば、がん細胞に対する免疫反応が賦活され、がんを退縮に導くことが期待される。現在、この考えに基づき、ヒトにおいて in vitro で育てた患者の樹状細胞を用いてがんの治療が試みられているが、invitro で育てた樹状細胞を in vivo に戻すと樹状細胞の免疫活性化能が急激に低下するため、これまではかばかしい成果が得られていない。
これまでに、いくつかの研究グループによってイヌ樹状細胞の in vitro における分化誘導と特性の解析が試みられているが、樹状細胞の分化誘導に必要な精製イヌサイトカインが、入手困難なため、未だ分化誘導の至適条件が確立されていない。また、骨髄中の樹状細胞の前駆細胞を分離するための方法も確立されていない。
そこで本研究では、１）精製サイトカインを用いることなくイヌ樹状細胞を in vitro で分化誘導する方法の確立、２）樹状細胞の前駆細胞を含んだイヌ骨髄中の造血前駆細胞の分離方法の確立、および３）In vivo において樹状細胞の成熟、活性化を維持する環境を構築するためのイヌサイトカイン遺伝子のクローニングを試み、以下の成果を得た。
１．In vivo では、免疫細胞が分泌する炎症性サイトカイン群によって樹状細胞の分化誘導が起こる。本研究では、培養中でイヌ T 細胞を活性化刺激することで、炎症性サイトカインの溶液（T cell conditioned medium：TCCM）を取得し、それを用いて単球から樹状細胞への分化誘導を試みたところ、12 日間の TCCM 培養によって、ほとんどの単球が①形態、②細胞表面マーカー、③免疫反応活性化能、④貪食能のすべてに樹状細胞の特徴を表し、成熟樹状細胞への分化誘導が証明された。
２．骨髄細胞を比重、細胞表面糖鎖への小麦胚芽凝集素（WGA）の親和性および生体染料であるRhodamine-123（Rh）の染色性（細胞外への排出性）によって分別したところ、樹状細胞の前駆細胞を含む多能性造血前駆細胞は、低比重で、WGAの親和性が高く（WGAhigh）、Rhの染色性が低い（Rhlow）細胞集団中に有意に多く含まれることが判明した。
３．樹状細胞の分化誘導に必須とされる顆粒球・マクロファージ‐コロニー刺激因子（GM-CSF）とインターロイキン‐4（IL-4）のイヌ遺伝子、および腫瘍免疫の活性化亢進に重要である IL-12 のイヌ遺伝子をクローニングした。これらを適切なプラスミドに組み込んで、哺乳類細胞発現ベクターを作製し、CHO 株細胞に導入したところ、それぞれのサイトカインタンパク質の細胞からの産生が認められた。イヌ末梢血単球を GM-CSF および IL-4
遺伝子導入細胞から産生されたサイトカインと培養すると、樹状細胞へ分化誘導され、その培養に IL-12 遺伝子導入細胞から産生されたサイトカインをさらに加えると樹状細胞の成熟、活性化が促進した。また、この IL-12 遺伝子導入細胞産生サイトカインは、腫瘍免疫を活性化するインターフェロンγのイヌ T 細胞からの産生を誘導した。これらの結果か
らこれらのサイトカイン遺伝子は、in vivo において樹状細胞の成熟、活性化を維持する環境を構築できることが明らかとなった。

以上のように本研究では、イヌにおける腫瘍免疫治療法の開発をめざして、in vitro における樹状細胞の分化誘導、特性の解析およびその制御因子を明らかにした。イヌは、ヒトと同じ生活空間を持ち、ヒトと同様な腫瘍の発生がみられる。したがって、本研究の成果に基づくイヌにおける新しいがん治療法の開発は、獣医学の発展のみならず、ヒトの臨床応用にもつながるものであり、本論文の審査ならびに最終試験の結果と併せて、博士（学術）の学位を授与することを適当と認める。