OBJETIVOS: Avaliar o perfil e o número absoluto e percentual de células NK verdadeiras (CD56+CD16+CD3-) e de células NK e NKT (CD56+) no sangue periférico de uma coorte de pacientes com artrite reumatóide (AR) antes e durante o tratamento com rituximabe (RTX). MÉTODOS: Foram analisados dez pacientes do grupo controle (doadores de sangue) e dez pacientes com AR que receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. As análises imunofenotípicas para avaliação do perfil e quantificação de células NK foram realizadas pré e após a infusão ou até a recaída clínica. Pacientes respondedores e não respondedores foram classificados de acordo com os critérios do Colégio Americano de Reumatologia (ACR) em 6 meses. RESULTADOS: A quantidade de células NK verdadeiras não demonstrou variação significativa pré e após o tratamento com RTX. Contudo, houve aumento percentual de células CD56+ entre o primeiro e o segundo mês após a infusão com RTX. Além disso, os pacientes respondedores apresentaram uma tendência de aumento do número absoluto de células NK verdadeiras após dois meses de tratamento. Já em relação ao grupo controle, observou-se um aumento significativo do número de células NK basais nos pacientes com AR (p<0,05). CONCLUSÕES: Foi identificada uma tendência de aumento nos valores absolutos de células NK verdadeiras entre os pacientes respondedores no segundo mês após a infusão com RTX. Não foi identificada uma variação significativa no perfil e quantidade de células NK nos pacientes com AR pré e após o tratamento com RTX. Contudo, foi observado que os pacientes com AR possuem uma quantidade maior de células NK do que os controles, sugerindo um possível envolvimento destas células na AR. / OBJECTIVES: To assess the profile as well as the absolute number and percentage of true NK cells (CD56+CD16+CD3-), and NK and NKT cells (CD56+) in the peripheral blood of a cohort of patients with rheumatoid arthritis (RA) before and during rituximab (RTX) therapy. METHODS: Ten control patients (blood donors) and ten patients with RA were assessed. The latter group received two intravenous infusions of 1g RTX, separated by a 14 day interval. Immunophenotypic analyses of NK cells were conducted before and after infusion, or until clinical relapse. After six months, respondents and nonrespondents were reassessed according to American Rheumatology Criteria (ARC). RESULTS: The number of true NK cells did not significantly change after treatment with RTX. However, an increase in the percentage of CD56+ cells was observed between the first and second month after RTX infusion. Respondents also displayed a tendency toward an increased number of true NK cells after two months of treatment. At baseline, the number of NK cells was also found to be significantly higher in patients with RA than in control individuals (p<0.05). CONCLUSIONS: Respondents displayed a tendency toward an increase in the absolute number of true NK cells in the second month after RTX infusion. No significant changes in the profile and frequency of NK cells were found between preand post-RTX treatment assessments of patients with RA. However, it was found that patients with RA have a higher number of NK cells than control partcipants, suggesting a possible role of these cells in RA.

No description available.

Dosis- und Zeitintensivierungen von Chemotherapie verbesserten das ereignisfreie Überleben bei Patienten mit aggressiven Non-Hodgkin-Lymphomen. Klinische Studien zeigten jedoch, dass zu starke Therapien in schlechteren Überlebensraten resultieren können. Rituximab ist ein monoklonaler Antikörper, der zu einem Durchbruch der Immuntherapie bei CD20-positiven B-Zell-Lymphomen geführt hat. Unterschiede bei den Überlebensraten zwischen einzelnen Therapievarianten werden durch Rituximab allerdings abgeschwächt. In dieser Promotionsarbeit wurde ein Modell entwickelt, welches diese Phänomene aus klinischen Studien durch die Annahme eines Anti-Tumor-Effekts des Immunsystems erklärt. Ein Differentialgleichungsmodell beschreibt die Dynamiken und Interaktionen zwischen Tumor- und Immunzellen unter Immunchemotherapie. Spezielle Parameter des Modells wurden durch Überlebenskurven aus klinischen Studien geschätzt. Dazu wurde ein Algorithmus entwickelt, der die Heterogenität der Überlebens- und Rezidivraten innerhalb eines Patientenkollektivs auf die Variabilität einiger weniger Parameter zurückführt. Das Modell wurde so an verschiedene Patientenkollektive angepasst. Schlechtere Ergebnisse bei zu intensiven Therapien werden im Modell durch eine zu starke Schädigung des Immunsystems erklärt, welches nicht mehr in der Lage ist, den residualen Tumor nach Therapieende zu bekämpfen. Ein weiterer Bestandteil des Modells ist die Vorhersage neuer Chemo- sowie Immuntherapievarianten, um vielversprechende Therapieszenarien zu ermitteln, die in die Konzeption neuer klinischen Studien einfließen können. Prognosen in Abhängigkeit von bestimmten Risikogruppen der Patienten können gestellt werden, indem Modellparameter mit messbaren Risikofaktoren assoziiert werden. Die wesentlichen Ergebnisse dieser Arbeit werden in zwei Publikationen vorgestellt.

Introduction: Rituximab is a monoclonal antibody used to treat hematological malignancies. The antibody depletes CD20+ B cells via cytotoxic immune mechanisms, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), which is mainly induced by natural killer (NK) cells. Rituximab is mostly well-tolerated but has been reported to induce the release of large amounts of cytokines in blood, thus causing systemic inflammatory response. Aim: To study rituximab-induced B cell depletion and cytokine release in blood from healthy volunteers and how this was affected by Fc modified versions of the antibody. Methods and materials: Fresh blood from healthy donors (n=3) was incubated with rituximab and Fc modified versions that influence the antibody’s target functions, namely ADCC and CDC, for 4 hours in a blood loop system. Results were measured using multicolor flow cytometry, except for cytokine release in plasma which was measured by enzyme-linked immunosorbent assay (ELISA). Results: Of all treatments, rituximab wild type (WT) showed superior B cell depletion than Fc mutant rituximab. The C1q knock-out variant (rituximab-P331S) and the variant with improved affinity to Fc receptor CD16 (rituximab-GASDALIE) did not differ in depletion. A cytokine release was not detected with the treatments, however, a cytokine stimulation in NK cells was observed. Rituximab-GASDALIE had the most prominent cytokine stimulation and CD107a (marker of NK cell functional activity) expression on NK cells. Rituximab-WT and rituximab-P331S had a minor and similar cytokine stimulation and CD107a expression between each other. Rituximab-IgG2 had minimal B cell depletion, CD107a expression and cytokine stimulation. Conclusions: Rituximab depleted B cells without inducing measurable cytokine release for healthy individuals. Among the treatments, Fc mutant rituximab seem to induce less B cell depletion. Moreover, rituximab-GASDALIE appear to elicit an enhanced NK cell activation. Further studies should include more donors as supplement and the results should be interpreted as complementary data to future data analyzed by performing the loop experiment using blood from patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20281号 / 医博第4240号 / 新制||医||1021(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 髙折 晃史, 教授 岩田 想, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le pemphigus est une maladie bulleuse auto-immune sévère causée par des auto- anticorps (Ac) ciblant la desmogléine (Dsg) 1 et/ou 3, principalement de la sous-classe IgG4. Certains Ac non spécifiques à la Dsg ont été décrits, comme la desmocolline 3 (Dsc3), mais leur pertinence est peu connue. Suite à l’étude RITUX3 en 2017, le traitement de première intention du pemphigus est le rituximab (RTX). Ce projet comprend trois volets qui s’inscrivent dans la caractérisation immunologique des patients inclus dans l’étude RITUX3, visant à mieux comprendre la pathogénèse et la prise en charge du pemphigus. Nous avons d’abord étudié la diversité isotypique des Ac anti-Dsg3. Nous avons démontré qu’un nombre d’isotypes plus élevé mène à un risque de rechute, particulièrement l’IgG3 anti-Dsg3 qui était détecté chez 71% des rechuteurs, comparativement à 12% des patients en rémission complète. Ensuite, nous avons étudié la prévalence et la pathogénicité in vitro des Ac anti-Dsc3. Ils étaient détectés chez 21% des patients, soit significativement plus qu’une population de donneurs sains. L’isotype principal était l’IgA, et leur pathogénicité in vitro a été démontrée à partir de sérums de patients et de souris immunisées. La présence de ces Ac permettait d’expliquer une bonne proportion des cas de discordance entre le profil sérologique d’anti-Dsg et le phénotype clinique des patients. Finalement, nous avons étudié la prévalence d’Ac anti-rituximab (ARA) chez les patients traités par RTX. Ils étaient détectés chez 31% des patients, mais n’affectaient pas l’atteinte d’une rémission complète et ne seraient pas une contre-indication à des perfusions subséquentes. Par contre, un petit groupe de patients qui présentaient des ARA fonctionnels étaient à risque de rechute. / Pemphigus is a severe auto-immune blistering disease caused by auto-antibodies (Abs) targeting desmoglein (Dsg) 1 and/or 3, mainly of the IgG4 subclass. Several Abs non-specific to the Dsg have been described, including desmocollin (Dsc) 3, but their relevance is not well known. Since the RITUX3 clinical trial in 2017, rituximab (RTX) is recommended as the first-line treatment for moderate-to-severe pemphigus. This project consists of three parts with the main goal of immunologically characterizing patients who were included in the RITUX3 trial, to allow a better understanding of the pathogenesis and treatment of pemphigus. First, we studied the diversity of IgG anti-Dsg3 subclasses. A higher number of subclasses was associated with a significant risk of relapse, especially with IgG3 anti-Dsg3 detected in 71% of relapsing patients, compared to 12% of patients in complete remission. Then, we studied the prevalence and pathogenicity of anti-Dsc3 Abs. They were detected in 21% of patients, significantly more than healthy donors. The main isotype was IgA, and their in vitro pathogenicity was demonstrated with sera from patients and immunized mice. Their presence explained a good proportion of cases who presented discrepancies between the clinical phenotype and the serological profile of anti-Dsg Abs. Finally, we studied the prevalence of anti-RTX Abs (ARA) in patients treated with RTX. They were detected in 31% of patients but did not affect the rate of complete remission and are not a contra-indication to receive subsequent perfusions. However, a small group of patients who presented functional ARA were at risk of relapse.

No description available.

No description available.

No description available.

La thrombopénie immunologique ou purpura thrombopénique immunologique (PTI) est une maladie auto-immune rare responsable d’une destruction périphérique immunologique des plaquettes associée à une production médullaire inadaptée. Dans la première partie de ce travail, nous exposons les connaissances actuelles de sa physiopathologie ainsi que certaines données concernant la réponse immunitaire T, le rôle des lymphocytes T régulateurs (Treg), l’implication de la rate dans la réponse immunitaire ainsi que les modes d’action d’une thérapeutique anti-lymphocytaire B, le rituximab. Dans une seconde partie, nous rapportons les résultats obtenus chez 40 patients atteints de PTI. Nous avons montré que le taux des Treg circulants CD4+CD25HighFoxp3+ est similaire chez les patients et les témoins, avec une élévation de leur taux chez les sujets répondeurs aux traitements. A l’inverse, il existe un déficit quantitatif en Treg au sein de la rate des patients. L’analyse des sous-populations lymphocytaires B spléniques a montré une augmentation du taux de lymphocytes B de la zone marginale chez les patients. Concernant les mécanismes d’action du rituximab, nous avons montré qu’une déplétion lymphocytaire B sanguine et splénique n’est pas suffisante pour obtenir une rémission, et que les plasmocytes ne sont pas sensibles à cette thérapeutique. Par ailleurs, nous proposons un mécanisme d’échappement à ce traitement. En effet, nous avons montré que les patients résistants au RTX présentent une élévation du ratio Th1/Treg spléniques. Chez ces sujets non répondeurs, nous avons également observé une élévation du ratio lymphocytes T CD8+/CD4+, au sein de la rate, suggérant une participation des lymphocytes T cytotoxiques dans la physiopathologie du PTI. Ces résultats ouvrent donc de nouvelles perspectives dans la compréhension de la physiopathologie du PTI, notamment la possible implication des lymphocytes B de la zone marginale et le défaut de contrôle de la réponse immunitaire splénique par les Treg. Concernant le rituximab, son action sur la réponse immunitaire ne semble pas se limiter à une déplétion lymphocytaire B qui n’est pas suffisante pour obtenir une rémission. Un mécanisme d’échappement ou de résistance à cette thérapeutique passe par une orientation Th1 et une probable implication des lymphocytes T CD8+. / Immune thrombocytopenia (ITP) is an autoimmune disease responsible for a peripheral immune destruction of platelets associated with an inappropriate bone marrow production. In this work, we first review the mechanisms involved in the pathogenesis of ITP. We also focus on the T cell immune response, highlighting the key role of regulatory T cells (Treg) in peripheral tolerance. The implication of the spleen in the immune response and the effects of rituximab, a B cell depleting therapy, are discussed. Then, our results obtained from 40 ITP patients are reported. Despite the fact that CD4+CD25HighFoxp3+ circulating Treg levels are similar between patients and controls, a significant increase is observed in responder patients. In the spleen, the rate of Treg is lower in ITP patients. Analyses of the spleens also reveal an increase in the level of marginal zone B cells in ITP. Rituximab is responsible for a complete depletion of both circulating and splenic B cells, which is not sufficient to achieve a response. Moreover, plasma cells are still observed after treatment. An increase in the Th1/Treg ratio in the spleen of non responder patients after rituximab infusion could trigger an escape to this therapy. The involvement of CD8+ T cells in the pathogenesis of ITP is highlighted by the increase in the CD8+/CD4+ ratio in the spleen after rituximab. New fields in the understanding of the pathogenesis of ITP are opened with these results, particularly by showing a quantitative deficiency in splenic Treg and the possible involvement of marginal zone B cells. Regarding rituximab effect on the immune response, we demonstrate on the one hand that complete circulating and splenic B cell depletion is not sufficient to achieve remission, and on the other hand that Th1 response and increase in CD8+ T cells level may represent an escape to this treatment.