Immunomodulatory drugs (IMiDs) could enhance both direct anti-tumour and graft-versus-tumour effects after allogeneic haematopoietic stem cell transplantation (AHSCT). However, clinical experience with IMiDs after AHSCT using adult peripheral blood (APB) as a stem-cell source has been limited by graft-versus-host disease. Characterization of the mechanisms by which IMIDs modulate alloresponses of T cells and identification of differential effects on T cells from different cell sources could facilitate more effective use of these drugs in the setting of AHSCT. Using in vitro modelling, multi-parameter flow cytometry and gene expression analysis, I have determined the impact of the widely used IMiD lenalidomide on alloresponses of APB and umbilical cord blood (UCB)-derived T cells. Lenalidomide-treatment potentiates net alloproliferation of APB-derived T cells by selectively enhancing proliferation of CD8+ T cells. These CD8+ T cells have enhanced effector memory differentiation, are enriched for polyfunctional effectors, have enhanced direct-cytotoxicity against heamatopoietic target-cells and have a distinct gene expression profile with altered expression of key immunoregulatory-genes and depletion of cellular ikaros. Importantly, while effects on CD8+ T cells derived from UCB are similar, lenalidomide has contrasting effects on allospecific proliferation of APB and UCB-derived CD4+ T cells. While lenalidomide-treatment has no effect on alloproliferation of APB-derived CD4+ T cells, it reduces alloproliferation of UCB-derived CD4+ T cells. The reduction in UCB-derived CD4+ T cell alloproliferation is accompanied by selective expansion of CD4+CD25+FOXP3+ regulatory T cells (Treg), resulting in an overall reduction in UCB-derived T cell alloproliferation. These findings demonstrate that lenalidomide has a differential impact on alloresponses of T cells from different cell sources; alloresponses of APB-derived T cells are increased via selective expansion of polyfunctional CD8+ effectors, while alloresponses of UCB-derived T cells are limited by expansion of tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHSCT.

Master of Science / Haematopoietic stem cell transplantation (HSCT) is a complicated and high-risk procedure used to cure disease or stop the spread of disease in a range of cancers. HSCT carries a high incidence of mortality and is associated with distressing short and long-term side effects. In addition, patients remain at risk of recurrence or mortality years after transplantation. Therefore, patients undergoing HSCT have been found to experience significant emotional and psychosocial distress because of the trauma associated with treatment. The literature suggests that about 50% of HSCT patients will experience clinical levels of distress. Carers and family members play an important role in caring for these ill patients in the short and long-term. Major role changes and financial stressors are experienced in many families, adding to the burden of care. However, very little is known about the rates of psychopathology in carers of HSCT patients. Due to the arduous nature of HSCT, psycho-educational programmes have been developed by major transplant centres and hospitals in order to provide HSCT patients and their families with information on the treatment process, side effects, risks, and long-term outcomes. Research on patient education in oncology has shown that providing patients and carers with information about their illness and treatment reduces anxiety and distress. To date, there have been no empirical evaluations to support the use of education programmes for HSCT patients. While it could be assumed that information would be helpful in reducing anxiety and depression in HSCT as it is in oncology generally, the information provided to these patients is usually more confronting and therefore, may be less reassuring. Thus, it is not known whether providing patients with education about HSCT reduces patient and carer distress or whether it might actually increase adverse outcomes. The aim of the present study was to evaluate the rates and correlates of distress in carers and patients and examine the effect of a psychoeducation programme for patients undergoing HSCT and their carers on knowledge, distress, information satisfaction, social support and caregiver burden. A randomised control trial was conducted to provide empirical data in relation to the latter aim. The following hypotheses were proposed. Firstly, it was hypothesised that patients and carers who received the education programme would have higher levels of knowledge, not evidenced in a group waiting to receive the programme. Secondly, it was hypothesised that the education program would not lead to increased anxiety and depressive symptoms. Thirdly, patients who know more about their condition would be the least distressed. As predicted, this study found high levels of distress, particularly in carers. Higher patient distress was related to having more concern about one’s illness and experiencing more symptoms. Education was effective in increasing patient and carer knowledge. Importantly there were no adverse effects of knowledge and greater patient knowledge following the education program was associated with less distress, although there was no direct effect of education on distress. Future research should aim to identify what aspects of the education program are helpful to patients. Finally, support interventions such as CBT are needed to help patients and carers, in particular, cope with the high levels of distress inherent in the HSCT experience.

Acute graft versus host disease (aGVHD) affects more than 40% of patients undergoing haematopoietic stem cell transplantation. aGVHD occurs after transplantation of donor haematopoietic cells into hosts incapable of rejecting the donor cells, when donor T cells attack host tissue. Despite extensive efforts, aGVHD remains problematic to prevent and difficult to control. Current therapies to prevent aGVHD induce profound immunosuppression, leaving patients at increased risk of infection and leukaemic relapse. Dendritic cells (DC) are professional antigen presenting cells of haematopoietic origin and are the primary stimulators of the immune system, uniquely being able to activate naïve T cells. A growing body of evidence suggests that DC are responsible for the stimulation of the donor T cells which cause aGVHD. I have used a model of aGVHD which utilizes conditioned severe combined immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMC). In this model human CD4+ T cells appear to be responsible for an aGVHD-like syndrome which results in death 15-30 days post transplant. I have shown, using in vitro depletion of individual populations, that other subpopulations of human PBMC did not affect the survival of the mice. I have also demonstrated that human DC are required for the induction of aGVHD in the majority of mice. This novel finding validated the use of this model to test the primary hypothesis; that antibody mediated depletion of DC would prevent aGVHD. The murine IgM monoclonal antibody (Mab), CMRF-44 Mab, is specific for an unknown molecule expressed on the surface of activated human DC. Previous work had shown that when mixed lymphocyte reaction stimulator cells were depleted of CMRF-44+ cells, there was a significant reduction in the proliferation of responder cells. Here I tested the efficacy of CMRF-44 as a therapy for the prevention of aGVHD in the model. CMRF-44 Mab did not improve survival of mice treated with human PBMC, despite recent data showing that CMRF-44 expression on DC was predictive of aGVHD in patients. In vitro depletion of CMRF-44+ cells from human PBMC prior to transplantation also did not reduce incidence of aGVHD. An alternate target for the depletion of human DC was CD83 which is also expressed on the surface of activated human DC. I generated a rabbit polyclonal antibody using a human CD83 fusion protein, which was then affinity purified in a multi-step process which yielded only antibody specific for human CD83. Treatment with this antibody greatly improved survival of transplanted mice. Further experiments showed that anti-CD83 treatment did not abrogate human leucocytes including CD8+ memory T cells suggesting that a therapy using an anti-CD83 antibody has the potential to prevent aGVHD without the immunosuppression associated with current anti-aGVHD therapies. The work described here has validated the use of a human mouse chimeric model as an in vivo assay of human DC function and shown that targeting CD83 has the potential to reduce the incidence of clinical aGVHD whilst preserving donor memory T cells.

Master of Science / Haematopoietic stem cell transplantation (HSCT) is a complicated and high-risk procedure used to cure disease or stop the spread of disease in a range of cancers. HSCT carries a high incidence of mortality and is associated with distressing short and long-term side effects. In addition, patients remain at risk of recurrence or mortality years after transplantation. Therefore, patients undergoing HSCT have been found to experience significant emotional and psychosocial distress because of the trauma associated with treatment. The literature suggests that about 50% of HSCT patients will experience clinical levels of distress. Carers and family members play an important role in caring for these ill patients in the short and long-term. Major role changes and financial stressors are experienced in many families, adding to the burden of care. However, very little is known about the rates of psychopathology in carers of HSCT patients. Due to the arduous nature of HSCT, psycho-educational programmes have been developed by major transplant centres and hospitals in order to provide HSCT patients and their families with information on the treatment process, side effects, risks, and long-term outcomes. Research on patient education in oncology has shown that providing patients and carers with information about their illness and treatment reduces anxiety and distress. To date, there have been no empirical evaluations to support the use of education programmes for HSCT patients. While it could be assumed that information would be helpful in reducing anxiety and depression in HSCT as it is in oncology generally, the information provided to these patients is usually more confronting and therefore, may be less reassuring. Thus, it is not known whether providing patients with education about HSCT reduces patient and carer distress or whether it might actually increase adverse outcomes. The aim of the present study was to evaluate the rates and correlates of distress in carers and patients and examine the effect of a psychoeducation programme for patients undergoing HSCT and their carers on knowledge, distress, information satisfaction, social support and caregiver burden. A randomised control trial was conducted to provide empirical data in relation to the latter aim. The following hypotheses were proposed. Firstly, it was hypothesised that patients and carers who received the education programme would have higher levels of knowledge, not evidenced in a group waiting to receive the programme. Secondly, it was hypothesised that the education program would not lead to increased anxiety and depressive symptoms. Thirdly, patients who know more about their condition would be the least distressed. As predicted, this study found high levels of distress, particularly in carers. Higher patient distress was related to having more concern about one’s illness and experiencing more symptoms. Education was effective in increasing patient and carer knowledge. Importantly there were no adverse effects of knowledge and greater patient knowledge following the education program was associated with less distress, although there was no direct effect of education on distress. Future research should aim to identify what aspects of the education program are helpful to patients. Finally, support interventions such as CBT are needed to help patients and carers, in particular, cope with the high levels of distress inherent in the HSCT experience.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23782号 / 医博第4828号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 波多野 悦朗, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an established therapy for many haematological disorders. Unfortunately, the new donor-derived immune system may damage host cells (graft-versus-host disease (GvHD)), causing significant morbidity and mortality. Since regulatory T cells (Tregs) can modulate immune responses, it was hypothesised that Treg numbers in the haematopoietic stem cell grafts and/or peripheral blood may influence the development of GvHD and other transplant-related complications. In this project, a prospective observational clinical study of putative Tregs in human alloHSCT was performed in Oxford. Flow cytometry and methylation-specific qPCR assays were developed to quantify putative Tregs and lymphocyte populations within the grafts and post-transplant blood samples. Although low CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell numbers were not associated with increased incidence of GvHD, low proportions of CD25(+)FOXP3(+)CD127(-/dim) cells in the graft (as a percentage of total CD4(+) T cells) were independently associated with poor engraftment, increased non-relapse mortality and inferior overall survival. Similarly, falling CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell counts over the first three months post-transplant were associated with higher non-relapse mortality and inferior overall survival. In view of these novel findings, strategies that increase CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T cells in alloHSCT may improve clinical outcomes. One possible route for increasing Tregs is through cellular therapy. This project therefore tested the hypothesis that CD4(+)CD25(+)FOXP3(+) Tregs can be produced in vitro from conventional CD4(+) T cells. In the presence of TGFβ and Azacitidine, FOXP3 was expressed in the majority of activated CD4(+) T cells. These cells also had a demethylated FOXP3 TSDR enhancer which is specific to natural Tregs. However, most of these cells produced pro-inflammatory cytokines, for example, TNFα. Therefore, under these conditions, FOXP3 expression was not sufficient to produce a Treg phenotype. It is proposed that current focus for generating Tregs for human clinical trials should be directed towards improving isolation and expansion of ex vivo isolated Tregs.

In patients after allogeneic haematopoietic stem cell transplantation (HSCT) is a human cytomegalovirus (CMV) one of the most important viral pathogens. Its detailed characteristic could provide information about the impact of each CMV genotype on overall survival of the patient, and some serious complications, such as graft versus host disease (GvHD). This thesis deals with retrospective genetic analysis of samples from 1877 patients transplanted at the Clinic of Pediatric Hematology and Oncology, University Hospital Motol and the Institute of Hematology and Blood Transfusion since 2002. DNA from biological samples (especially whole blood) was isolated kit Qiagen DNA Blood Mini or Qiagen DNA Mini and samples were prospectively detected presence of CMV DNA. Samples were subsequently stored at -20 řC. Genotyping was performed using real-time PCR technologies to the genes of 2 structural proteins glycoprotein B, glycoprotein H and using sequence specific primers and probes. In 1343 samples (71.6%) from 390 patients there was only one strain of CMV; in 256 (13.6%) samples from 113 patients have detected mixed infection caused by two or more strains of CMV. The most common genotype demonstrated in "single" infection was in pediatric and adult patients gB1/gH2 detected in 118 (28.4%) patients. Most...

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23794号 / 医博第4840号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 小川 誠司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A mucosite oral (MO) constitui uma condição dolorosa que se desenvolve entre 47% e 100% dos pacientes submetidos a transplante de células-tronco hematopoiéticas (TCTH), impactando enormemente em sua qualidade de vida. Investigar fatores preditivos para MO por meio de exames não invasivos faz-se necessário, visando melhorar a qualidade de vida dos pacientes. O objetivo do estudo foi investigar a relação dos regimes de condicionamento e dos níveis salivares de Interleucina 6 (IL- 6) e Fator de Necrose Tumoral alfa (TNF-?) com a MO, bem como investigar o impacto destes na qualidade de vida. Foram selecionados 82 pacientes submetidos a TCTH, que foram avaliados em 4 momentos diferentes: no início do condicionamento para o TCTH (M1), no dia da infusão das células (M2), após 12/20 dias do início do condicionamento para transplante autólogo e alogênico, respectivamente (M3), e após 30 dias ou na alta hospitalar para ambos (M4). Nestes momentos, foi avaliado clinicamente o grau de MO segundo critérios da Organização Mundial da Saúde (OMS), coletada saliva total e aplicados 3 questionários de avaliação de qualidade de vida em relação à MO e à saúde bucal: PROMS, OHIP-14 e OMQoL. As informações clínicas e laboratoriais foram correlacionadas através do STATA 13.0 com 5% de nível de significância. Verificou-se que a maior incidência e intensidade de MO, os piores índices de qualidade de vida e os maiores níveis de IL- 6 e TNF-? foram registrados no M3, porém não houve correlação entre as citocinas e graus de MO. Houve associação entre altos níveis salivares de IL-6 e maiores pontuações no PROMS. O regime de condicionamento mieloablativo (ML) foi relacionado à MO intensa (graus 3 e 4) e à maiores pontuações nos 3 questionários de qualidade de vida, e os escores dos questionários foram maiores conforme maior foi intensidade da MO (p<0,05). / Oral mucositis (OM) is a painful condition that develops between 47% and 100% of patients undergoing hematopoietic stem cell transplantation (HSCT), impacting greatly on their quality of life. Investigation of predictive factors for OM through noninvasive exams is necessary, in order to improve the quality of life of patients. The aim of the study was to investigate the relationship of conditioning regimens and salivary levels of Interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-?) with OM, and to investigate their impact on quality of life. We selected 82 patients undergoing HSCT, which were assessed at four different moments: at the start of conditioning for HSCT (M1), on the cell infusion day (M2), after 12/20 days of the start of conditioning for autologous and allogeneic transplantation, respectively (M3), and after 30 days or at hospital discharge for both (M4). In these moments, it was clinically evaluated the degree of OM according to criteria of the World Health Organization (WHO), collected whole saliva and applied 3 questionnaires of assessment of quality of life related to OM and oral health: PROMS, OHIP-14 and OMQoL. Clinical and laboratorial data were correlated using STATA 13.0 in a 5% significance level. It was found that the highest incidence and intensity of OM, the worst indices of quality of life and higher IL-6 and TNF-? levels were found in M3, but there was no correlation between cytokines and levels of OM. There was an association between high levels of salivary IL-6 and higher scores in PROMS. The myeloablative conditioning regimen (ML) was related to intense OM (grades 3 and 4) and to highest scores in the 3 questionnaires of quality of life, and the scores of questionnaires were higher as higher was the intensity of OM (p<0,05).

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.