学位論文内容の要旨
博士の専攻分野の名称

博士（医 学）

氏 名

小杉 瑞葉

学 位 論 文 題 名Studies on inhibitory interaction between graft-derived and reconstituted T cells involves murine
chronic graft-versus-host disease（慢性移植片対宿主病における移植片由来および再構築由来 T 細胞の相互作用に関する研究）【Background and Objectives】
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative
therapy for a variety of hematopoietic disorders. However, allogeneic T cell responses inwhich donor T cells recognize the host tissues as non-self and attack them result ingraft-versus-host disease (GVHD). GVHD can be divided into two distinct syndromes,
acute and chronic GVHD. The pathogenesis of chronic graft-versus-host disease(cGVHD) remains elusive. Donor T cells in the cGVHD recipients are comprised of twodistinct T cell populations; graft-derived T cells (TG) and hematopoietic stem
cell-derived reconstituted T cells (THSC). Because cGVHD develops following thereconstitution of THSC, which underwent aberrant thymic negative selection thatoccurred during acute GVHD, THSC have been considered to be pathogenic. However,there is clinical evidence that older patients with poor thymopoiesis are at increased
risk of cGVHD, which suggests that THSC is not prerequisite for cGVHD but TG also
contribute to cGVHD. Consistent with this theory, previous reports have shown that TG
persist throughout the chronic phase in cGVHD mice with poor THSC reconstitution.
However, it remains unclear whether these persistent-TG sustain effector function andcontribute to cGVHD. Host-type histocompatibility antigens persist for a long time
following allo-HSCT, which may deprive allo-reactive persistent-TG of effector function
by mechanisms of exhaustion or replicative senescence, similar to the process that
occurs in the chronic viral infection or cancer. Thus, with the contributions of TG andTHSC being limited in supply or by a loss of function, respectively, the extent to which
each of these T cell populations is responsible for the pathogenesis of cGVHD has
remained unclear. One of the major hurdles to understanding the contribution of TG and
THSC is a lack of murine models that replicate the course of human cGVHD, specifically,models with autoimmune-like pathological features that meets clinical diagnostic
criteria and which develops as THSC numbers increase. Here, we established a clinically
relevant murine model of cGVHD in order to characterize the reciprocal regulation
between TG and THSC in cGVHD by selective T cell depletion.
【Material and Methods】
C3H.Sw (H2b) recipients received 9Gy total body irradiation (TBI) before transfer of T
cell depleted BM (TCD BM) with (“cGVHD group”) or without (“BMT group”) spleen
CD4+ and CD8+ T cells from MHC minor-mismatched B6 (H2b) donors. After 9 weeks,histological analysis was performed on cGVHD-affected organs (lung, liver, skin andsalivary gland) according to NIH criteria. The kinetics and function of TG and THSC inaffected-organs and secondary lymphoid organs (SLOs) were examined using congenicsystems. An anti-Thy1.2 monoclonal antibody (mAb) was used to selectively deplete TGor THSC during the chronic phase of disease.

【Results】
In the [B6 -> C3H.SW] cGVHD model, cGVHD mice developed pathology thatrecapitulates human cGVHD in the liver, lung, skin and salivary glands. To determinethe extent of THSC reconstitution, we examined the reconstitution of THSC by using
CD45.1 / CD45.2 congenic system. The number of CD4+ CD8+ double positive (DP)thymocytes of THSC in cGVHD group was significantly lower than in the BMT groupfrom day 35 to day 63. Delay and impairment of THSC reconstitution was observed. Total
T cell number in the liver and lung was higher in cGVHD group than in BMT group.
Time course analysis revealed that detectable numbers of THSC appeared in the liver,lung and spleen from day 21 after HSCT, TG outnumbered THSC for duration of ourexperiment. We next examined whether the persisted- TG in the affected organs at day
63 are exhausted or still functional. TG included a large proportion of PD-1+ exhausted
or KLRG-1+ replicative senescent T cells, but also included a functional population withthe potential to proliferate and produce inflammatory cytokines such as IFN and TNF .
To determine the contribution of persistent-TG and THSC to cGVHD pathogenesis, weperformed selective depletion of these cells in the chronic phase. Selective TG depletion
failed to block cGVHD development because of compensatory proliferation andactivation of THSC in affected organs. On the other hand, selective THSC depletion
resulted in activation of TG without increase leading to lethal exacerbation of cGVHD,
indicating that even a small number of THSC play a critical role in inhibiting TG
activation in the acute-to-chronic transition phase.
【Discussion】
Our cGVHD murine model recapitulates the important feature of human cGVHD suchas salivary gland and lung damage with concomitant immunodeficiency. In addition, thebiphasic development of GVHD symptoms recapitulated the acute-to-chronic transitionthat occurs in the course of human cGVHD course.
In our cGVHD model, a large number of TG persisted in cGVHD-affected organs up today 63 after allo-HSCT with an unexpected predominance of TG. The majority of TG
persisting in the affected organs had an effector phenotype, which is distinct from thememory stem cell population. PD-1- KLRG-1+ replicative senescent TG may be involvedin the cellular immunopathogenesis of cGVHD. Unlike the basal maintenance ofmemory T cells, a significant proportion of TG were actively proliferating in the liverand lung even at day 63. These results suggest that the number of TG is controlled by
active proliferation and cell death as well as by homeostatic cytokines. The extent of theinvolvement of microenvironment-derived allo-antigens and cytokines remains to beelucidated.
Reconstitution of THSC is markedly delayed and suppressed in the presence of GVHD.
TG may suppress THSC reconstitution by impairing primary lymphoid tissues. In thisstudy, depletion of TG resulted in a rapid increase of THSC in the liver and lung. Thisfinding points to the possible existence of a niche that antigenic signals and survival
factors to pathogenic T cells of TG or THSC origin. Such a “pathogenic T cell niche” might
have a fixed pool capacity, meaning that TG and THSC compete with each other for spacein the niche during cGVHD. Our observation that the depletion of a small number of
THSC did not influence the number of TG in cGVHD-affected organs is consistent withthe replicative senescent phenotype of TG and the hypothesis that the size of thepathogenic T cell niche is limited.
【Conclusion】
We have characterized the cellular mechanisms underlying the maintenance ofpathogenic T cells in a clinically relevant cGVHD model. Both TG and THSC with thepotential to proliferate and produce inflammatory cytokines infiltrated cGVHD-affectedorgans. However, a reciprocal regulatory interaction determined the balance between
the number and activity of TG and THSC, and thus maintains the pathogenic T cell pool.