L'immunosuppression générale est utilisée pour traiter les patients transplantés ou greffés avec des cellules souches. Ils ont des effets secondaires graves et sont peu efficaces à long terme. li faut donc développer de nouvelles stratégies thérapeutiques, plus ciblées, pour limiter ces effets secondaires et améliorer la qualité de vie des patients. Le niveau d'expression de la molécule CD45RC permet de distinguer, les cellules CD45RChigh associées au rejet de greffe et à la GVHD, et les cellules CD45RClow qui ont des fonctions régulatrices importantes et inhibent ces maladies. Nous avons étudié le potentiel thérapeutique d'un anti--CD45RC mAb injecté à court terme dans des modèles de greffe et de GVHD. Dans le modèle de greffe cardiaque chez le rat, l'anticorps prévient le rejet de greffe dans 60 à 80% des cas. Dans les modèles de GVHD chez le rat et la souris NSG, l'anti-CD45RC mAb associé à la rapamycine permet de prévenir la GVHD dans 50% des rats el la totalité des souris. Nous avons montré que cet anticorps induit la déplétion transitoire des L Ts CD45RChigh par apoptose. Le nombre de LTs CD45RClow est augmenté au cours du traitement el leur potentiel suppresseur est accru à long terme in vitro et in vivo. En effet le transfert adoptif des cellules, de rats tolérants leur greffon à long terme, à de nouveaux rats greffés cardiaque (même configuration génétique}. prévient le rejet de greffe. Nous avons également montré que les réponses immunitaires naïves et mémoires ne sont pas affectées. Le traitement court avec un anti-CD45RC mAb a donc un potentiel thérapeutique important dans la prévention du rejet de greffe et de la GVHD. / General immunosuppressants serve lo treat solid organ or CSH transplanted patients. But they have many side effects and their efficacy is limited at long term. So its necessary lo develop new therapeutic strategies, more specific, to limit these side effects and lo improve patient quality life. The leval of expression of CD45RC distinguishes Iwo different cells types. Cells expressing CD45RChigh are associated with allograft rejection and GVHD, while cells that dont express CD45RC haveregulatory properties and inhibit these diseases. We studied the therapeutic potential of anti-CD45RC mAb as a short treatment in allograft and GVHD models. ln the heart allograft rat model, the antibody prevents rejection in 60 to 80% of the case. ln the GVHD model (rat of NSG mice), association of anti-CD45RC mAb and rapamycin prevents 50% of GVHD in the rat and all in NSG mice. We showed that the antibody induces a transitory depletion of CD45RChigh T cells throughapopolosis, while the number of CD45RClow T cells is increased and their regulatory properties are improved in vitro and in vivo. Indeed, adoptive transfer of tolerant rat lo new irradiated heart grafted rat prevents allograft rejection. We also describe that naive and memory immune responses are not impaired by the treatment Short course treatment with anti--CD45RC mAb hes a great potential lo prevent allograft rejection and GVHD.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22147号 / 医博第4538号 / 新制||医||1039(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 濵﨑 洋子, 教授 竹内 理, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Etude des Treg dans la physiopathologie du syndrome de Wiskott-Aldrich et étude de la reconstitution des Treg dans les greffes de cellules souches hématopoïétiques après un conditionnement nonmyéloablateur.

No description available.

Graft versus Host Disease (GvHD) is one of the main complications patients face after receiving a bone marrow transplant. Between 40-60% of bone marrow transplant recipients develop GvHD, with consequent systemic inflammation/fibrosis, reduced quality of life, graft failure, and mortality. We have previously demonstrated that donor-derived C5 is involved in the initiation and propagation of GvHD. Current approaches to inhibition of C5 share a serious flaw of indiscriminately blocking production of a mediator that is crucial for host defense. Targeted therapies to block C5 in specific cells, or anatomical sites, are the only way in which to achieve therapeutic benefit without compromising host defenses. Three lentiCRISPR v2-dCas9 gene editing viral constructs were created to selectively cleave the complement C5 gene, at three different sites. Our objective was to knockout complement C5 function in infected donor BM cells in a GVHD mouse model. Each of the three lentiCRISPR plasmids was separately co-cultured with PMDG2 and PSPAX2, in human embryonic kidney (HEK) 293T cells. Resultant viral particles were able to transfer the Cas-9 endonuclease gene into donor BM cells in vitro with a transduction efficiency of 52%. Treated donor BM cells were then retro-orbital injected into irradiated recipient mice. Control mice were transplanted following the same protocol excluding the lentiCRISPR treatment of BM. The lentiCRISPR treatment group demonstrated significantly lower total airway resistance (p = 0.05) and higher lung compliance (p = 0.014) when compared to the control group. When compared to the saline treated group however the lentiCRISPR group showed significantly higher total airway resistance (p = 0.004) and significantly lower lung compliance (p = 0.014). These results taken together suggest a possible reduction in GvHD severity in mice that received the lentiCRISPR treatment. This study can serve as a starting point for the development of this novel treatment of GvHD. / Thesis / Master of Science (MSc)

Graft-versus-host disease (GvHD) of the skin is a severe allo-immune reaction and complication following allogeneic stem cell transplantation. Over the past years, intensive pre-clinical research has led to an improved understanding of the pathophysiology of acute and to a lesser extend chronic GvHD. This has translated into the approval of several new agents for the treatment of both forms of GvHD. This review summarizes the most recent advances in underlying pathomechanisms, clinical trials and newly approved agents for GvHD, with a special focus on skin involvement.

BACKGROUND: Graft versus host disease (GVHD) is a significant cause of morbidity and mortality following stem cell transplantation. Donor T cells are thought to be the main mediators of this disease, although we have recently identified that host T cells are present and active during acute GVHD suggesting contributions from both donor and host T cells. Whether both donor and host T cells can survive GVHD and coexist harmoniously after disease resolves is unknown. OBJECTIVE: The goals of this thesis are two-fold: (i) to study T cell chimerism in post-GVHD skin and (ii) to understand what effect, if any, treatment has on T cell chimerism in skin. METHODS: Acute GVHD and post-GVHD skin samples were obtained from male patients that had been transplanted with female donor cells. Chimerism was assessed using fluorescence in situ hybridization for the X and Y chromosomes concurrently with immunofluorescence staining for CD3, a T cell marker. Regulatory T cells were stained by immunofluorescence for CD3, CD4 and Foxp3. Medical record data was collected for all patients. RESULTS: We found that the percent of host T cells decreased significantly after resolution of acute skin GVHD compared to during active acute skin GVHD in skin samples obtained from five male patients that had been transplanted with female donor cells. The T cell composition in these patients in post-GVHD skin was primarily donor. We identified chimerism shifted toward donor T cells in patients treated with systemic steroids and this correlated with an increased number of donor T cells infiltrating into skin rather than a decrease in the number of host T cells in skin. With regard to frequency of Tregs, there was no significant difference between the group that had been treated with systemic steroids prior to biopsy and the group that had not. CONCLUSIONS: We discovered that donor chimerism predominates in post-GVHD skin and in active skin GVHD of patients who received systemic steroids, suggesting a role of donor cells in acute GVHD resolution. We were not able to identify a higher frequency of regulatory T cells in the treatment group. It is possible that the Treg recruited to skin by steroid treatment is Foxp3 negative, and therefore missed by our staining approach. The use of another marker is required for future studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble serum stimulation-2 (sST2) are elevated during human and murine aGVHD and are correlated to a type 1 T cell response. Membrane-bound ST2 (ST2) on donor T cells has been shown to be protective against aGVHD. ST2 signals through the adapter protein myeloid differentiation primary response 88 (MyD88). The role of MyD88 signaling in donor T cells during aGVHD remains unknown. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of interleukin 1 receptor (IL-1R) and toll-like receptor 4 (TLR4) signaling, both of which also signal through MyD88, in two murine HCT models. This protection was entirely driven by MyD88-/- CD4 T cells, leading to a decreased type 1 response without affecting T cell proliferation, apoptosis, or migration. In our aGVHD models, loss of intrinsic MyD88 signaling is not responsible for the observed protection. However, transplanting donor MyD88-/- T conventional cells (Tcons) with wild type (WT) or MyD88-/- T regulatory cells (Tregs) ameliorated aGVHD severity and lowered aGVHD mortality. Transcriptome analysis of sorted MyD88-/- CD4 T cells from the intestine ten days post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, and Jak2, among others. Decreased sST2 was confirmed at the protein level with less secretion of sST2 and more expression of ST2 compared to WT T cells. Transplanting donor ST2-/- Tcons with WT or ST2-/- Tregs mirrored observations when using donor MyD88-/- Tcons. This suggests that Treg suppression from lack of MyD88 signaling in Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways. The results of our study confirm that ST2 represents an aGVHD therapeutic target that spares Treg function.

Bei Leukämien, Lymphomen und dem Multiplen Myelom stellt die allogene hämatopoetische Stammzelltransplantation (allo-HCT) oft die letzte kurative Therapieoption dar. Spender-T-Zellen (v.a. CD8+-T-Zellen), die im Transplantat enthalten sind, erkennen nach Chemo-/Strahlentherapie verbliebene Reste des entarteten Empfängergewebes, eradizieren dieses und verhindern somit ein Tumorrezidiv (Graft-versus-Leukämie Reaktion/GvL). Häufig attackieren Spender-T-Zellen (v.a. CD4+-Th1-Zellen) aber auch nicht-malignes Gewebe (z.B. Haut, Leber und Darm), was bis zum Tod des Patienten führen kann (Graft-versus-Host Disease/GvHD). Calcineurin-Inhibitoren wie Cyclosporin A (CsA) und Tacrolimus, die oft schon prophylaktisch verabreicht werden, verhindern über eine unselektive Inhibition aller Mitglieder der NFAT-Transkriptionsfaktorfamilie (Nuclear factor of activated T-cells) die Aktivierung der Spender-T-Zellen. Es folgt eine klinische Besserung der GvHD-Symptomatik, während jedoch der GvL-Effekt ebenfalls supprimiert wird. Bisherige Untersuchungen unserer Arbeitsgruppe am Mausmodell hatten gezeigt, dass die selektive Inhibition eines NFAT-Familienmitgliedes (NFATc1 oder NFATc2) in den Donor-T-Zellen zu einer signifikanten Besserung der aGvHD bei jedoch erhaltener GvL führt. Es wurde nun der Einfluss des dritten, in Lymphozyten exprimierten NFAT-Mitglieds NFATc3 im Kontext der aGvHD untersucht. Zur Basisanalyse der neu kreierten Nfatc3fl/fl.Cd4cre- und Nfatc1fl/fl.Nfatc3fl/fl.Cd4cre-Mauslinien erfolgten durchflusszytometrische und Western-Blot-Analysen. Anschließend wurden In-vivo-Untersuchungen unter Verwendung eines etablierten major-mismatch-aGvHD-Modells (H-2b→H-2d) durchgeführt. Es konnte gezeigt werden, dass durch eine NFATc3- (+/- NFATc1-) Defizienz direkt ex vivo die CD4+/CD8+-Ratio durch Abnahme der CD4+- hin zu den CD8+-T-Zellen verschoben wird. Auch zeigte sich in den entsprechenden Genotypen eine Abnahme der naiven- und dafür vice versa eine Zunahme der Effektor-T-Zellen. In den wiederholt durchgeführten aGvHD-Versuchen zeigte sich in vivo als Korrelat der (ebenfalls erneut nachgewiesenen) Abnahme des CD4+/CD8+-Quotienten in den Zielorganen eine geringere Expansion der NFAT-defizienten als der wildtypischen T-Zellen. Leider spiegelte sich dies nicht in dem clinical score zur Quantifizierung der aGvHD-Symptomatik wider. Auch das Körpergewicht der Versuchsgruppe nahm rapide ab. Ursächlich hierfür ist – als Korrelat zur direkt ex vivo nachgewiesenen Aktivierungsneigung – ein vermehrter Th1-Shift der NFATc3 (+/-NFATc1-) defizienten T-Zellen. Eine Inhibierung von NFATc3 – im Gegensatz zu NFATc1 und NFATc2 – ist demzufolge kein sinnvoller Ansatzpunkt für eine mögliche, zielgerichtetere aGvHD-Therapie. Der positive Effekt der reduzierten Proliferationsneigung der NFATc3-defizienten Lymphozyten wird durch deren vermehrte Aktivierungsneigung mit erhöhter Sekretion von pro-inflammatorischen Zytokinen zunichte gemacht. / In malignant diseases such as multiple myeloma, leukemia and lymphoma the allogenic hematopoietic stem cell transplantation (allo-HCT) often represents the final curative treatment option. Donor T cells (esp. CD8+ T cells) within the graft recognize and eradicate tumor cells which have remained after chemo- and radiotherapy. This graft-versus-leukemia (GvL) effect can prevent tumor relapses. However, donor T cells (esp. CD4+ Th1 cells) often attack non-malignant tissue (e.g. skin, liver, colon) with potentially life-threatening consequences for the host. (Graft-versus-host disease = GvHD). To prevent the development of aGvHD, calcineurin-inhibitors (CNI) like cyclosporin A (CsA) and tacrolimus are often administered prophylactically. By means of an unselective suppression of the nuclear factor of activated T cells (NFAT) transcription factors, both drugs inhibit the activation of donor T cells. While leading to a clinical improvement of the GvHD-symptoms, coevally, the GvL effect is also suppressed. Previous research of our study group showed that a selective inhibition of one NFAT family member (NFATc1 oder NFATc2) in donor T cells leads to a significant decline of aGvHD symptoms while maintaining GvL. We have now analysed the iκluence of NFATc3, the third NFAT member expressed in lymphocytes, in context of aGvHD. Initially we analysed the new created Nfatc3fl/fl.Cd4cre- and Nfatc1fl/fl.Nfatc3fl/fl.Cd4cre-mouse strains by western blot and flow cytometry. Subsequently, these were followed by in vivo studies, using an already established major-mismatch-aGvHD-model (H-2b→H-2d). It could be shown that directly ex vivo a NFATc3- (+/- NFATc1-) deficiency leads to a reduction in the CD4+/CD8+ ratio. This is mainly caused by a diminution of CD4+ T cell population, while the CD8+ population remains unaffected. Furthermore, a lower number of naive but an increased number of effector T cells has been observed. This effect (which was also present in the aGvHD-experiments) correlated in vivo with a decreased expansion of NFAT-deficient T cells – compared to wild type T cells – in target organs. Unfortunately, the expected clinical improvement could not be demonstrated. The clinical score which objectifies the aGvHD-symptoms, as well as the body weight of the mice in the experimental group declined rapidly, comparable with or even worse than in the control group due to an increased Th1-shift of the NFATc3- (+/- NFATc1) deficient T cells. This also correlates with the increased effector function which has been observed in the previous ex vivo experiments. In conclusion an inhibition of NFATc3 – in contrast to NFATc1 or NFATc2 – is not a useful target point for a more specific aGvHD-therapy. The positive effect of a reduced proliferation in NFATc3-deficient lymphocytes is over-compensated by their augmented activation.

No description available.