Dissertations / Theses on the topic 'Rejection antibody-mediated'

Although the short-term outcomes of solid allograft survival have improved substantially over the last few decades, there has been no significant improvement in long-term survival of solid allografts. This thesis presents the initial characterisation of alloantibody mediated rejection in a murine heart transplant model, with particular focus on the impact of the different phases of the humoral alloimmune response (follicular or germinal centre) on graft rejection.

Immune mediated injury is a major cause of late kidney graft loss. Donor specific HLA antibodies are widely believed to cause chronic rejection because their presence correlates strongly with worse graft outcome. However, some renal transplant recipients have circulating DSA for many years whilst maintaining stable graft function. This dichotomy has led to the hypothesis that the cause of the immune mediated damage observed in chronic rejection is likely to be multi-factorial. It has previously been demonstrated that donor specific CD4+ T cell activation can be observed in patients with chronic antibody mediated rejection using an IFNγ ELISpot assay. In some samples, this cellular activation was dependent on the presence of B cells, and in others the cellular activation appeared to be regulated by the presence of B cells. In this thesis, the same patterns of reactivity were observed in renal transplant recipients with chronic antibody mediated rejection recruited onto the RituxiCAN C4 clinical trial. HLA binding B cells were detected flow cytometrically and associations between their phenotype and the different patterns of reactivity were analysed. The majority of HLA binding B cells were found to be IgM+ and were underrepresented in the class switched memory B cell population (CD27+IgM-/CD45RBmem55+IgM-) relative to the global B cell population. B cell dependent IFNγ production in response to donor specific HLA was associated with a higher ratio of IgM+ memory to IgM+ naïve B cells in the HLA binding B cell population. CMV gB was used as a model protein to investigate the individual contribution that memory (CD27+) and naïve (CD27-) B cells make to antigen specific IFNγ production. The results suggest that memory B cells support IFNγ production and naïve B cells may be suppressing IFNγ production. Collectively the results in this thesis support a role for a cell-mediated component in chronic antibody mediated rejection. In order to develop more effective treatments for chronic rejection, further investigation into the contribution that T and B cells make to this process is warranted.

INTRODUCTION Heart transplant still represent the most important therapeutic strategy for end-stage cardiomyopathies. Humoral rejection was recognized as a distinct clinico-pathologic entity characterized by acute allograft rejection associated with the production of antidonor HLA antibodies and poor prognosis. For the first time on renal biopsy from patients with kidney function deterioration and absence of cellular rejection C4d was identified as an important diagnostic biomarker for antibody mediated rejection and was strongly correlated with the presence of circulating anti-HLA antibodies. In heart transplantation in the ISHLT working formulation of 2004 the diagnostic criteria of AMR were defined only as present or absent , recognizing grade 0 without histological and immunopathological signs of humoral rejection and grade 1 in the setting of both histological and immunopathological signs of humoral rejection. However the role of AMR remained controversial with different reported incidence between different centers. Even though we were able to obtain a better control of cellular rejection with a decreasing rate of positive Acute Cellular Rejection (ACR) requiring drug therapy modification, no concomitantly reduction in graft loss for cardiac allograft vasculopathy was observed. Recently the attention was mainly focused on humoral rejection and on our ability to detect it through immunohistopathological markers. The aims of the present thesis were summarized in three major objectives: 1) to study the pathophysiology of Antibody Mediated Rejection (AMR) in heart transplant recipients; 2) to assess the diagnostic ability of pathological features to identify AMR on monitoring post-transplant endomyocardial biopsies (EMBs); 3) to clarify the prognosis of AMR in the early and late post-transplant period. MATERIALS AND METHODS. As C4d staining has been performed on a routine basis in heart transplant recipients at the University of Padua's Heart Transplantation Center since 2005, 1.452 consecutive EMBs from 131 patients (mean age 48.9±18.3) were reviewed. Donor mean age was 41,6±15,2 years and 15% had more than 60 year old. Adult population. To study survival in patients with C4d positivity on EMBs we evaluated 985 consecutive biopsies from a total of 107 adult patients (n=85, 79% of these were males) with a median age at the time of transplantation of 55 years (range 17-73 years). Our study population was divided into 4 groups on the basis of the patients’ C4d, DSA, and graft function profile: - C4d positive, DSA negative, without graft dysfunction (asymptomatic AMR) = group 1; - C4d positive, DSA positive, without graft dysfunction (asymptomatic AMR) = group 2; - C4d positive, DSA positive, presence of graft dysfunction (symptomatic AMR) = group 3 - C4d negative, DSA negative, without graft dysfunction, control group = group 0. Pediatric population. We evaluated 226 consecutive biopsies from 24 transplant patients (mean age 8,1±6,3 years). The immunoperoxidase staining was applied routinely both in paediatric and adult patients. The staining for C4d was performed retrospectively using affinity-purified antihuman C4d rabbit polyclonal antibody. The grading system was standardized and was defined in four grades: grade 0 = negative; grade 1 = weak staining with focal distribution, grade 2 = moderate staining with multifocal distribution; grade 3 = strong staining with diffuse pattern. We considered positive grades 2 and 3.The cut off value between grade 1 and grade 2 was 50% of intramyocardial capillaries involved. Immunostaining for C3d, was performed on paraffin embedded section and using a polyclonal antibody C3d.The grading system for C3d immunostaining was the same as for C4d. Histological features of AMR on EMBs. Of the 1452 EMBs, 87 were positive for C4d staining (6% 87/1452) from 61 patients, 26 EMBs from 13 patients had repeated C4d positivity. Sixty-six patients staining negative for C4d were matched for pre-transplant diagnosis, time after transplantation age, and acute cellular rejection (ACR) grading (case-control study). Among the 61 C4d positive patients 9 were diagnosed as AMR. In this case-control study we also applied C3d staining. Two pathologists reviewed blindly all morphological features associated to humoral damage: endothelial swelling, interstitial edema, intracapillary mononuclear cells, intracapillary neutrophils, myocite damage, myocite necrosis, hemorrhage, intravascular microthrombi according to ISHLT 2005 classification. Donor specific Antibodies (DSA) Assessment. Since the study of C4d has been mainly retrospective only in a subgroup of patients circulating anti HLA antibodies could be performed. In thirty-seven patient circulating Anti HLA antibodies were assessed and resulted 26 positive and 9 negative. IgG anti-HLA reactivity in the sera, obtain before transplantation and at the time of C4d positive detection on EMBs, was analyzed using bead-based screening assays, referred to as Luminex methodology. All sera tested positive at screening, were retested with Single Antigen beads in order to determine the antibody specificity. Contrast-enhanced transthoracic Doppler echocardiography. We selected 19 patients who at the time of biopsy stained positive for C4d and in whom echocardiography was performed for Coronary Flow Reserve (CFR) evaluation using CE-TTE before and after adenosine infusion. CFR was calculated as ratio of peak diastolic velocity(hyperemia) and peak diastolic velocity (basal). Statistical analysis. . The comparison between C4d groups was conducted with the Fisher’s exact test in case of categorical variables and the Kruskal-Wallis test for quantitative variables. The Kaplan-Meier method was applied to estimate the C4d groups’ survival functions and the log–rank test was used to compare survival between groups. The Cox’s regression model was used to estimate unadjusted and sex and age adjusted hazard ratios (HR). Sensitivity and specificity, were reported with 95% confidence interval calculated with the exact method RESULTS Clinic-pathological profile of adult population The 22 patients in group 1 (61% of total C4d+ve) showed a 18 fold higher mortality risk compared to the C4d negative patients (95% CI 1.960 to 160.022). The 6 patients in group 2 had a 61 fold higher mortality risk (95% CI 3.399 to 1110.360). The 8 patients in group 3 had a 32 fold higher mortality risk (95% CI 5.884 to 179.432), overall p<0.0001 Overall the C4d positive patients showed a statistically significant reduction in survival compared to the C4d negative patients and this observation was maintained in the three different groups When the asymptomatic (groups 1 and 2) and symptomatic patients (group 3) were compared with the control group an 18 and 26 fold increase mortality risk was observed, respectively. Clinic-pathological profile of pediatric population. Seven patients (33%) showed a C4d +ve intra-graft capillary deposition. Of these 4 were positive for circulating donor specific anti HLA antibodies; none were positive in the C4d negative group. One patient presented also graft dysfunction (14% of C4d+ve) within 3 months after transplantation. One patient with C4d positivity died for heart failure after 2 years since C4d positivity detection. The other five patients with C4d positivity are still alive at 2, 3, 4 , 14 years respectively. In the C4d positive group the rejection score was higher compare to C4d negative patients. (2.2 vs 0.2, p<0,05) Morphological parameters on Endomyocardial biopsy (EMB) to detect Asymptomatic AMR (AsAMR) and Symptomatic AMR (AMR). Of the 8 histological characteristic evaluated on Hematoxilyn Eosin , only two (endothelial swelling and interstitial edema) could be considered fair predictors of C4d capillary positivity in the light of their sensitivity: endothelial swelling with a 78.7 % sensitivity (its specificity was very poor 28.8%) and interstitial edema with a 77.1% sensitivity (its specificity was 31.8%). Intracapillary macrophages had a sensitivity of 39.3% only and a specificity of 68.2%. The sensitivity and specificity of the histological parameters in EMBs of patients with C4d positivity (both asymptomatic and symptomatic) were similar to those observed in EMBs of patients affected by symptomatic AMR ROC curve combining endothelial swelling and intra-capillary aggregates of macrophages did not improve the capacity to predict presence of circulating anti HLA antibodies. Immunostaining for C3d in the diagnosis of AMR. In the asymptomatic AMR group (52 EMBs of 52 patients) 31 were also positive for C3d (59,6%, 31/52). In the symptomatic AMR group (9 EMBs of 9 patients) 5 were also positive for C3d (55% 5/9). The sensitivity of C3d to predicted DSA was 42.3% and the specificity of C3d was 56% for DSA. Combining C4d and C3d to predicting circulating DSA did not increase the sensitivity and specificity of C4d. Alloantibody profile in symptomatic AMR. Of the 9 symptomatic AMR patients, 5 had been followed up for C4d positivity on EMBs and circulating DSA. The results showed that MFI for class I and class II changed according to the treatment. High level of MFI (>10000) strongly correlated with diffuse and strong intensity of C4d staining; with MFI ranging between 1000-9000 C4d was variably detected; with MFI < 1000 C4d was always negative. Microvasculopathy and C4d. Seven out of 19 patients, in whom we performed echostress(or where ECHO stress was reduced) had C4d positive immunostaining while 12 were negative (average age 50.1±8.7 years). In the group of patients with C4d positivity 4 were late symptomatic AMR (mean time after Htx 13.9±4.9 years) and three of them died after few months since the diagnosis of AMR. Three patients had asymptomatic AMR (mean time after HTx 5.3±3.4 years). Coronary flow reserve (CFR) of patients with C4d+ was statistically significant reduced compared to C4d- group (1.28±0.48 vs 3.28±1.03 respectively, p=0.0297) suggesting a relation between humoral rejection and microvascular damage. Even in pts without CAV at angiography C4d positivity identified a low value of CFR in the C4d+ group (p=0.0502). CONCLUSIONS. Our findings indicate that C4d+ve is an important marker for diagnosis of AMR , thus identifying asymptomatic AMR . C4d predicts worse prognosis and DSA and graft dysfunction further improve risk stratification. C4d+ve and DSA can be used as early mortality predictors in patients without signs of graft dysfunction. AMR is a complex and ongoing phenomenon with different phenotypic features. Morphological parameters alone, are not adequately sensitive and carry low positive likelihood ratio as screening tools for early asymptomatic AMR detection. Screening recommendations has been recently modified to include more sensitive tests such as C4d staining in the routine protocol to improve patient risk stratification. The assessment of anti HLA antibodies, class I and II and their relation with C4d (complement activation)showed : 1) a difference in type of anti HLA-DSA in the early and late period post-transplant with class I anti HLA early appearance and class II anti HLA late onset after transplant 2) high levels of MFI correlate with C4d deposition on EMBs. Endothelial damage and complement deposition produce microvascular remodeling leading to microvasculopathy with increased graft loss.
INTRODUZIONE Il trapianto cardiaco rappresenta ancora la più importante strategia terapeutica nei pezienti con scompenso cardiaco refrattario alla terapia medica. Nell’ultimo decennio la attenzione clinica è stata rivolta al rigetto umorale in seguito al dato che la percentuale di perdita del garft per rigetto cronico non è cambiata, nonostante la terapia immunosoppressiva controlli in modo ottimale il rigetto cellulo-mediato. Il rigetto umorale viene definito come una entità clinico-patologica caratterizzata dalla presenza di anticorpi anti HLA (complesso maggiore di istocompatibilità) circolanti che provocano danno endoteliale sull’organo trapiantato con conseguente impatto sulla mortalità. Per la prima volta in biopsie renali di pazienti con disfunzione del graft in assenza di rigetto cellulo-mediato, è stato evidenziato deposito di C4d sulla superficie endoteliale, sinonimo di attivazione del complemento , strettamente correlato con la presenza di anticorpi anti HLA circolanti nel siero. Le linee guida internazionali che definiscono i criteri patologici per la diagnostica del rigetto nel trapianto di cuore, solo nel 2004 per la prima volta definiscono il rigetto umorale come entità patologica distinta indicandone i criteri diagnostici istologici e immunopatologici maggiori, riconoscendo come grado 0 assenza di segni istologici e immunopatologici e grado 1 la presenza di segni istologici e immunopatologici di rigetto umorale. Nell’ultima decade la letteratura internazione ha rivolto la propria attenzione alla ricerca del significato prognostico del rigetto umorale in relazione al rigetto cronico e alla ricerca di nuovi biomarcatori per la diagnosi precoce del rigetto umorale. Gli obiettivi della presente tesi di dottorato possono essere riassunti in tre principali puntii: 1) studio della fisiopatologia del rigetto umorale o rigetto anticorpo mediato nel trapianto di cuore; 2) valutazione della capacità diagnostica dei criteri istopatologici definiti dalle linee guida internazioni del 2004, su biopsie endomiocardiche di monitoraggio post-trapainto; 3) definizione del significato prognostico del rigetto anticorpo mediato sia nella fase precoce che tardiva post-trapianto. MATERIALI E METODI. Dal 2005, nel nostro centro di riferimento per il trapianto cardiaco dell’Università di Padova, abbiamo studiato routinariamente il biomarcatore di attivazione del complemento (C4d) con tecnica immunoistotchimica su tessuto (biopsie endomiocardiche di monitoraggio). Abbiamo valutato 1452 biopsie endomiocardiche (BEM) da 131 pazienti (età media 48.9±18.3). Popolazione adulta. Per studiare la sopravvivenza di pazienti adulti che presentano una positività al C4d nel loro follow up, abbiamo studiato 985 BEM di 107 pazienti (79% maschi) con età al trapianto di 55 anni (mediana) range 17-73 anni. La nostra popolazione adulta è stata suddivisa il quattro gruppi sulla base del profilo del C4d nelle BEM, degli anticorpi circolanti anti HLA donatore specifici (DSA), e della disfunzione del graft (riduzione della frazione di eiezione, anomalie elettrocardiogarfiche): • C4d positivo, DSA negativi, assenza di disfunzione del garft (rigetto anticorpo-mediato asintomatico)= gruppo 1 • C4d positivo, DSA positivi, assenza di disfunzione del graft (rigetto anticorpo-mediato asintomatico)= gruppo 2 • C4d positivo, DSA positivi, presenza di segni clinici di disfunzione del graft (rigetto anticorpo-mediato sintomatico)= gruppo 3. • C4d negativo, DSA negativi, assenza di disfunzione clinica del graft (gruppo controllo )= gruppo 0. Popolazione pediatrica. Abbiamo valutato 226 biopsie provenienti da 24 pazienti trapiantati (età media 8,1±6,3 anni). Tecnica immunoistochimica è stata applicata routinariamente sia nella popolazione adulta sia nella popolazione pediatrica. La tecnica immunoistochimica è stata applicata sia retrospetticamente che prospetticamente usando anticorpo policlonale per C4d. Il grading per la valutazione della colorazione immunoistochimica è stata standardizzato in quattro gradi: grado 0= negativo; grado 1= debole intensità, focale coinvolgimento dei capillari; grado 2= moderata intensità e coinvolgimento multifocale dei capillari e grado 3= colorazione intensa e coinvolgimento diffuso dei capillari. Si considera positivo grado 2 e grado 3. E’ stata inoltre eseguita l’immunoistochimica per C3d su materiale paraffinato applicando lo stesso schema di valutazione del C4d. Valutazione dei criteri diagnostici morfologici di rigetto anticorpo mediato. Delle 1452 BEM valutate, 87 erano positive per il C4d (6% 87/1452) in 61 pazienti, 26 BEM di 13 pazienti hanno mostrato un C4d positivo in più di un biopsia. Sessantasei pazienti negativi per il C4d sono stati selezionati e confrontati secondo i seguenti criteri: diagnosi pre-trapianto, età dopo il trapianto, grading del rigetto cellulo-mediato (studio caso-controllo). Tra i 61 pazienti positivi per il C4d 9 avevano rigetto anticorpo-mediato sintomatico, In questo studio caso-controllo abbiamo studiato anche il C3d. Due Patologi hanno rivisto “blindly” tutti i criteri istologici associati al rigetto umorale: edema endoteliale, edema interstiziale, cellule mononucleari intracapillari, neutrofili intracapillari, danno miocitario, necrosi miocitaria, emorragia, microtrombi intravascolari in accordo con le linee guida internazionali “ISHLT working formulation “ del 2004. Determinazione degli anticorpi anti HLA donatore specifici. La valutazione degli anticorpi circolanti è stata eseguita su 37 casi, in virtù del fatto che il nostro studio è stata condotto anche retrospetticamente. E’ stata utilizzata la tecnica Luminex per testare la reattività degli anticorpi anti HLA tipo IgG con tecnica “bead-based screening assay” da sieri ottenuti pre-trapianto e post-trapianto, quest’ultimo in concomitanza con la positività del C4d nelle BEM. Inoltre in una sottopopolazione di pazienti con rigetto umorale sintomatico gli anticorpi circolanti sono stati quantificati misurando la media di intensità di fluorescenza (MFI). Valutazione della riserva coronarica tramite tecnica ecocardiografica non invasiva. Abbiamo selezionato 19 pazienti in cui al momento della biopsia endomiocardica di monitoraggio post-trapianto è stata eseguita ecocardiograficamente.La Riserva Coronarica valutata prima e dopo l’infusione di adenosina La riserva coronarica (CFR) è stata calcolata con rapporto tra picco di velocità diastolica (iperemia) e picco di velocità diastolica (basale). Analisi statistica. Il confronto tra i vari gruppi C4d è stato calcolato con test Fisher nei caso di variabili categoriali e con test di Kruskal-Wallis per variabili quantitative. E’ stato applicato il metodo di Kaplan-Meyer per studiare la sopravvivenza dei vari gruppi positivi per C4d. Test di regressione di Cox per stimare Hazard ratio. Sono state calcolate sensibilità e specificità dei vari parametri morfologici e riportate con intervallo di confidenza del 95%. RISULTATI. Profilo clinico-patologico della popolazione adulta. Ventidue pazienti del gruppo 1 (61% del totale dei C4d positivi) mostano un rischio di mortalità 18 volte maggiore rispetto al gruppo di controllo (95% CI 1,960 – 160,022).. I sei pazienti del gruppo 2 hanno un rischio di mortalità 61 volte maggiore rispetto al gruppo di controllo (95% CI 3.399-1110.360). Gli 8 pazienti del gruppo 3 hanno un rischio di mortalità 32 volte maggiore rispetto al gruppo di controllo con una p<0,0001. Se si considerano nella totalità i pazienti C4d positivi mostrano una riduzione statisticamente significativa della sopravvivenza rispetto al gruppo C4d negativo. Si osserva un aumento del rischio di mortalità sia nei gruppi 1 e 2 (asintomatici) che nel gruppo 3 sintomatico Profilo clinico-patologico della popolazione pediatrica. Sette pazienti (33%) mostrano una positività al C4d nelle BEM di monitoraggio. Di questi 4 presentavano anticorpi anti HLA circolanti; nessuno era positivo nel gruppo di controllo. Un paziente con positività al C4d ha presentato disfunzione del graft (14% dei C4d+) entro i 3 mesi dal trapianto. Un paziente C4d positivo è morto per scompenso cardiaco dopo circa 2 anni dalla comparsa della positività del C4d nelle BEM. Gli altri cinque pazienti con C4d + sono attualmente vivi a rispettivamente 2,3,4,14 anni di follow up. Nel gruppo C4d+ il “rejection score” per rigetto cellulare è maggiore rispetto al gruppo di controllo (2,2 vs 0,2, p<0,05). Parametri morfologici per la diagnosi di rigetto anticorpo-mediato sintomatico e asintomatico nelle biopsie endomiocardiche di monitoraggio. Degli 8 criteri morfologici valutati all’ematosillina ed eosina, solo due (edema endoteliale ed interstiziale) possono essere considerati predittivi di positività capillare al C4d. la sensibilità dell’edema endoteliale è del 78,8% e la sensibilità dell’edema interstiziale è del 77,1%. Accumulo intracapillare di cellule mononucleari hanno una sensibilità del 39,3% nel predire la positività al C4d. La curva ROC mostra come i due criteri valutati assieme e riconosciuti essere i più importanti associati al danno anticorpo-mediato non aumentano la capacità di predite la presenza di anticorpi circolanti anti HLA. Immunoistotchimica per C3d nella diagnostica del rigetto umorale. Nel gruppo degli asintomatici (52 BEM di 52 pazienti) 31 BEM sono positive anche per C3d (59,6%, 31/54). Nel gruppo dei sintomatici (9 BEM per 9 pazienti) 5 BEM erano positivi al C3d (55% 5/9). La sensibilità del C3d nel predire la presenza di anticorpi anti HLA circolanti è del 42,3% e la specificità del 56%. Combinando C4d e C3d la sensibilità e specificità nel predire la presenza di anticorpi circolanti non aumenta. Profilo anticorpale nei pazienti con rigetto anticorpo mediato sitomatico. Dei 9 pazienti sintomatici della nostra popolazione studiata, 5 sono stati seguiti in follow up sia per C4d sia per anticorpi circolanti. I risultati mostrano che la quantità di anticorpo circolante (MFI) cambia in relazione al trattamento. Inoltre alti livelli di MFI >10000 correlano strettamente con grado 3 del C4d (diffuso coinvolgimento capillare con colorazione intesa), con MFI tra 1000-9000 il C4d ha una intensità ed una distribuzione variabile.; con MFI <1000 C4d è sempre negativo. C4d e microvasculopatia. La riserva coronarica di pazienti con C4d + è ridotta rispetto al gruppo di controllo rappresentato da pazienti con C4d- (1,28±0,48 vs 3,28±1,03 rispettivamente, p=0,0297) suggerendo una relazione tra il rigetto anticorpo mediato e il danno microvascolare. Anche nei pazienti senza coronaropatia del graft i pazienti con C4d+ mostrano una riserva coronarica inferiore rispetto al gruppo di controllo (C4d-) (p=0,0502). CONCLUSIONI I nostri risultati dimostrano come il C4d sia un indicatore oltre che diagnostico anche prognostico per rigetto umorale e la sua positività è sinonimo di prognosi negativa anche in assenza di sintomi clinici . La valutazione di C4d, anticorpi circolanti e disfunzione del graft ci permette di stratificare il rischio di mortalità. I parametri morfologici da soli non sono sufficientemente sensibili e specifici per la diagnostica del rigetto umorale e dunque non sono adeguati per una valutazione di screening. Alla luce dei nostri risultati raccomandiamo il C4d come test di screening per individuare pazienti asintomatici. Lo studio degli anticorpi circolanti hanno mostrato che alti livelli di anticorpi correlano con la positività capillare del C4d . Il danno endoteliale e il deposito di complemento determinano rimodellamento vascolare che porta a lungo termine

This thesis tests the hypothesis that Tregs with indirect allospecificity can prevent humoral rejection. Anti-CD4 monoclonal antibody and PVG.R8 donor-specific blood transfusion (DST) given pre-transplant led to long-term PVG.R8 heart graft survival in two-thirds of PVG.RT1^u recipients. Alloantibody production was abrogated in these animals and tolerance was allospecific. Histology of long-term surviving grafts showed no evidence of chronic rejection. Naïve syngeneic spenocytes injected into tolerant PVG.RT1^u animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4+ T cell-reconstituted congenitally athymic PVG.RT1^u rats confirmed that regulation in tolerant animals was mediated by spenic CD4+ T cells. These CD4+ Tregs were allospecific. Other mechanisms of tolerance in this model were explored by breaking regulation in tolerant rats, either by immunising with immunodominant linear allopeptide, or by depleting tolerant CD4+ T cells with anti-CD4 monoclonal antibody. Surprisingly, this resulted in neither alloantibody generation nor graft rejection, suggesting that tolerance also resides within alloantigen-specific B cells. These results demonstrated that anti-CD4 + DST treatments results in the development of CD4+ Tregs that recognise alloantigen via the indirect pathway and act in an antigen-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell component that persists after CD4+ T cell help has been made available. These findings may be of use in designing clinically-applicable tolerogenic strategies in the future.

We determined in a rat model (1) the presence and dynamics of alloantibodies recognizing MHC complexes on quiescent Brown-Norway (BN) splenic cells in the sera of Lewis (LEW) recipients of Brown-Norway iliolumbar vein grafts under tacrolimus immunosuppression; and (2) the presence of immunoglobulins in the wall of acute rejected vein allografts.

En transplantation d’organes solides, la principale cause de perte de greffons à long terme est le rejet humoral. Le rejet humoral est une maladie de la vasculature du greffon, caractérisé par une inflammation et une forte concentration sérique des anticorps dirigés contre le greffon. Les études récentes ont souligné la forte prévalence des anticorps spécifiques à HLA-DQ parmi des anticorps spécifiques du donneur formés post-transplantation. Cette thèse explore l’importance de l’expression de HLA-DQ par les cellules endothéliales dans la pathogenèse du rejet humoral. Afin d’étudier l’expression de HLA-DQ, des cellules endothéliales microvasculaires ont été stimulées avec des cytokines pro-inflammatoires. L’inflammation induite altère l’immunogénicité des cellules endothéliales et donc modifie la qualité des synapses immunologiques avec les lymphocytes T CD4+ alloréactifs. Ainsi, l’inflammation chronique a diminué la capacité des cellules endothéliales à induire l’expansion des lymphocytes T CD4+ régulateurs FoxP3high (Treg). De plus, la fixation des anticorps anti-HLA-DQ aux cellules endothéliales, en synergie avec l’inflammation, réduit davantage l’expansion des lymphocytes T CD4+ régulateurs. Cette baisse des cellules anti-inflammatoires peut altérer la tolérance des allogreffes, promouvoir les réponses pro-inflammatoires et aggraver le rejet
In solid organ transplantation, antibody-mediated rejection is currently the major cause of allograft failure. Antibody-mediated rejection is a disease of the allograft vasculature, characterised by inflammation and circulating donor-specific antibodies. Recent studies have exposed the particularly high prevalence of antibodies targeting HLA-DQ amongst de novo donor-specific antibodies produced post-transplantation. This thesis explores the significance of endothelial HLA-DQ expression in the pathogenesis of antibody-mediated rejection. HLA-DQ expression in in vitro microvascular endothelial cell cultures required sustained exposure to inflammatory cytokines. Such inflammation modified endothelial immunogenicity and subsequent synapses with alloreactive CD4+ T lymphocytes. As a consequence, sustained inflammation compromised the capacity of endothelial cells to expand anti-inflammatory regulatory T cells. Moreover, the binding of HLA-DQ alloantibodies to the endothelial cells synergised with inflammation to further diminish regulatory T cell expansion. This reduction in anti-inflammatory cells may impair allograft tolerance, promote proinflammatory responses and exacerbate rejection

Background: Rejection is considered as a major barrier to achieve successful transplantation. Non self-human leucocyte antigen (HLA) is a well-known antigenic target for antibodies binding that can result in antibody-mediated rejection (AMR). To reduce risk of rejection in kidney transplant, preventive measures are undertaken, which include HLA-matching between donor and recipient, and in-vitro pre-transplant crossmatch with potential donor cells and recipient sera, furthermore, periodic HLA antibodies monitoring for donor-specific antibodies (DSA) is carried out before and after transplant. Nevertheless, allograft may still fail despite the above measures, which suggests other antigens besides HLA can also contribute to renal rejection. In fact, polymorphic major histocompatibility complex (MHC) class I–related chain A (MICA) antigens and Angiotensin II type 1 receptor (AT1R) antigens have been reported as likely targets in AMR. However, the effect of non-HLA antibodies such as anti-MICA and anti-AT1R antibodies in rejection are not fully defined. This implies there is an imminent need to elucidate the role of non-HLA antibodies in allograft AMR cases which are not mediated by HLA antibodies. Aim: To retrospectively evaluate the occurrence of MICA and AT1R antibodies in 21 clinical AMR cases without detectable HLA antibodies or HLA antibodies that were not target against donor HLA. Methods: Twenty-one cases with suspected non-HLA mediated post-transplant rejection were retrieved. Eplet analysis was utilized to confirm that the detectable HLA-DR antibodies in one of the samples were not cross-reactive towards a donor’s antigen. Sera from 21 non-AMR cases were used as controls. All sera were subjected to MICA antibody and AT1R antibody screening. Identified positive cases were further examined with their pre-transplant sera to assess whether the AT1R and/or MICA antibodies were already pre-formed before transplantation. The sensitization histories of transfusion, pregnancy and previous transplantation were recorded. Results: Nine of twenty-one cases were detected with MICA and/or AT1R antibodies. 7 samples were detected with MICA antibodies while 3 samples were detected with AT1R antibodies. A sample was detected with both MICA and AT1R antibodies. Importantly, the presence of MICA/AT1R antibodies appeared to be strongly associated with rejection caused by non-HLA antigens (p=0.0007). All controlled cases were found to be negative for MICA and AT1R antibodies. Pre-transplant sera of the positive cases were further screened and pre-formed antibodies were detected in 3 of the positive MICA cases, and 1 of the positive AT1R cases. Since no AT1R and MICA genotyping of the donor was carried out previously, it was uncertain that the allograft rejection was induced by the donor specific pre-formed antibodies generated in the pre-transplant sensitization events. Nonetheless, AT1R and MICA antibodies appeared to be induced by the allograft in the remaining 5 cases. Conclusion: Presence of MICA/AT1R antibodies appeared to be associated with the investigated AMR cases without detectable HLA antibodies. Some evidence suggested the production of these non-HLA antibodies could be induced by transfusion sensitization or allograft upon transplantation.
published_or_final_version
Pathology
Master
Master of Medical Sciences

Cardiac transplantation continues to be the main treatment pathway for end stage heart failure. The process holds many challenges for the recipients' immune system. Modern immunosuppression has improved survival to one year greatly, but the main limitation to survival following this remains chronic rejection, taking the form of coronary artery vasculopathy (CA V). Recent studies have found strong associations with antibody mediated rejection and CA V. This study investigates the role of antibody mediated rejection (AMR), and the complement system, within cardiac transplantation and it involvement with the development of CAV. Here, the presence of complement components was demonstrated within cardiac tissues taken through the transplant process and up regulated following brain death, through ischaemia and reperfusion of the graft. The deposition of complement within the first two years post transplantation was retrospectively associated with the future development of CA V. However, the deposition of complement alone did not reflect the occurrence of clinical AMR episodes. Antibodies within the tissues were also observed, with serum studies confirming the presence of auto reactive anti vimentin IgM, within the recipients. Developing the study further to investigate the CA V lesion, complement components and antibodies were co-localised to smooth muscle cells within the lesion. These data have led to the conclusion that antibody and complement deposition have a pivotal role in the cause and progression of CA V. Investigations into the source of the anti graft antibody, led to a six month post operative survey ofB cell population in cardiac recipients, leading to description of an altered B cell population within an immunosuppressed environment, with expansion in a little known CD21 10w B cell phenotype.

Background: Arterial allografts are used as vascular conduits in the treatment of prosthetic graft infection. Immunosuppression decreases their rupture risk rate. However, immunosuppression can be unprofitable in florid infection. Previously, we confirmed inhibition of cell-mediated destruction of rat aortic grafts by delayed use of tacrolimus. In this work, we studied the influence of this protocol on the antibody-mediated rejection.

This thesis synthesises the current literature on ABMR treatment and describes the development of dnDSA in kidney transplant recipients of low, moderate and high immune risk. Risk factors for dnDSA, including eplet mismatch load and early tacrolimus exposure, are explored with the aim of identifying strategies to prevent dnDSA and ABMR. Methods: A systematic review and meta-analysis of controlled trials for the treatment of ABMR is presented in Chapter 2. Chapter 3 estimates the incidence of dnDSA through a longitudinal cohort study with prospective DSA screening. The accuracy of high resolution HLA typing imputation compared to next generation sequencing (NGS) is assessed in Chapter 4. In Chapter 5, early tacrolimus exposure is explored as a risk factor for dnDSA. Results: No evidence for reduction in graft failure was found on pooled analysis of antibody removal, yet plasma exchange and IVIG (PLEX) have become the standard of care for acute ABMR. The incidence of dnDSA in recipients of low immunological risk was 16%, compared to 30% in moderate risk and 29% in high risk recipients. High resolution typing imputation performed poorly compared to NGS. Class II and HLA-DQ eplet mismatch load were associated with an increased risk of dnDSA. Compared to a mean tacrolimus concentration <11 ng/mL, mean concentrations 11-12.9 ng/mL over the first 90 days were associated with a decreased risk of dnDSA. Conclusions: The optimal treatment of ABMR remains unclear. Despite this, PLEX has become the standard of care for active ABMR. dnDSA developed in 16% of low-risk recipients and 30% of moderate and high-risk recipients. Risk factors for dnDSA included higher Class II eplet mismatch load, and lower early tacrolimus exposure. Collaborative studies are required to confirm the increased risk of dnDSA in higher immune-risk recipients, devise strategies for their prevention, and provide a platform for future trials in the prevention and treatment of ABMR.

Background Despite its clinical relevance, there is a lack of consensus regarding the definition of graft dysfunction (GD) in heart transplant (HT). Herein we aim to characterize clinical phenotypes of patients with GD, either acute or chronic, comparing their outcomes with stable patients. In addition, we explored the risk factors outcomes in GD patients. Methods The patients were divided in 3 groups: Group A - Patients who recently underwent HT (<5 years); Group B - Patients with clinical and instrumental signs of GD, regardless of the distance of HT; Group C - Stable patients (HT > 5 years). Primary Endpoints were: overall mortality, hospitalizations for cardiovascular events and hospitalization for all-causes. The Combined Endpoints was death or /and hospitalizations for cardiovascular events (CV hospitalization). Results We enrolled 134 consecutive HT patients. Patients with GD 32(24%) had significant higher prevalence of class NYHA >II, low EF, CAV, longer QRS and Qtc on the ECG (p<0.01) and donor specific antibodies (DSA) (all p<0.05), as compared with group A and C. Clinical presentation was highly heterogeneous: 6(19%) had acute presentation, 3 for acute rejection, and 3 for acute coronary syndromes; 21(66%) had chronic presentation: 17(53%) associated with CAV, and 4(13%) as chronic dysfunction after antibody-mediated rejection. During the 2y follow-up, GD patients showed higher mortality (P=0.01) and higher CVE hospitalization rate (54; P< 0.01) than patients in group A and C. Low EF, time from HT, and chronic clinical presentation (p<0.01) were risk factors for the combined endpoint Conclusions GD after HT is characterized by highly variable clinical presentation and is correlated with a particularly poor prognosis. CAV is the most frequent etiology, and DSA are more often found in patients with GD than in stable ones, but do not seem to influence outcome.

Introdução: A associação entre a presença de anticorpo anti-HLA doador específico (DSA), em pacientes com prova cruzada negativa por citotoxicidade dependente de complemento (CDC), e a ocorrência de episódios de rejeição mediada por anticorpos (RMA) e menor sobrevida do enxerto já foi demonstrada por diversos autores. Entretanto,estimar a relevância clínica da presença desses anticorpos, em um determinado receptor, é um grande desafio e portanto novas estratégias de monitorização imunológicas são necessárias. Objetivo: O objetivo desse estudo foi monitorar a presença de DSA, bem como a variação dos seus títulos durante o primeiro ano após o transplante renal e correlacionar com episódios de rejeição aguda e função do enxerto ao final desse período. Metodologia: Foram analisados 389 soros de 71 pacientes incluídos no estudo. A pesquisa de DSA foi realizada utilizando os testes LABScreen single antigenbeads nas amostras correspondentes aos tempos: pré-transplante, 14, 30, 90, 180 e 365 dias após o transplante. Episódios de rejeição aguda comprovados por biópsia foram analisados de acordo com a classificação de Banff 2007. A taxa de filtração glomerular (TFG) ao final do primeiro ano foi estimada utilizando a fórmula Modificationof Diet in Renal Disease (MDRD). Os pacientes foram inicialmente separados em 3 grupos de diferentes riscos imunológicos (pré-transplante): A) DSA-, B) DSA+ com MFI >1000 e < 5000 e C) DSA+ com MFI > 5000. Num segundo momento, foram novamente agrupados de acordo com o perfil de mudança nos valores de MFI (intensidade de fluorescência média) ao longo do primeiro ano. Resultados: DSA estavam presentes pré-transplante em 15 pacientes. RMA foi mais frequente no grupo C (p = 0,02). De acordo com a variação dos títulos de DSA pós-transplante os pacientes foram novamente agrupados: grupo I) permaneceu DSA- durante todo acompanhamento = 50 pacientes, II) diminuiu ou manteve títulos de DSA em relação ao tempo zero = 13 pacientes e III) aumentou títulos em relação ao tempo zero = 8 pacientes (6 foram DSA de novo). Três pacientes dos grupos I e um paciente do grupo II apresentaram episódios de rejeição aguda celular. Não foi observada oscilação significativa nos títulos de anticorpos durante esses eventos. Nenhum paciente desse grupo apresentou episódio de RMA. Episódio de RMA ocorreu em dois pacientes do grupo III. Em ambos os pacientes foi detectado aumento significativo nos valores de MFI dos DSA em relação ao tempo zero. Não foi observada diferença significativa na TFG entre os grupos analisados nesse estudo. Entretanto, observou-se uma diferença estatisticamente significativa na TFG entre os pacientes que apresentaram episódio de rejeição aguda em relação aos que não tiveram, sendo menor nos primeiros (p = 0,04). Conclusão: A monitorização prospectiva dos anticorpos pode ajudar a identificar pacientes em maior risco para ocorrência de RMA e o aumento nos valores de MFI DSA deve ser interpretado como um sinal de alerta, sobretudo em pacientes previamente sensibilizados.
Introdução: A associação entre a presença de anticorpo anti-HLA doador específico (DSA), em pacientes com prova cruzada negativa por citotoxicidade dependente de complemento (CDC), e a ocorrência de episódios de rejeição mediada por anticorpos (RMA) e menor sobrevida do enxerto já foi demonstrada por diversos autores. Entretanto,estimar a relevância clínica da presença desses anticorpos, em um determinado receptor, é um grande desafio e portanto novas estratégias de monitorização imunológicas são necessárias. Objetivo: O objetivo desse estudo foi monitorar a presença de DSA, bem como a variação dos seus títulos durante o primeiro ano após o transplante renal e correlacionar com episódios de rejeição aguda e função do enxerto ao final desse período. Metodologia: Foram analisados 389 soros de 71 pacientes incluídos no estudo. A pesquisa de DSA foi realizada utilizando os testes LABScreen single antigenbeads nas amostras correspondentes aos tempos: pré-transplante, 14, 30, 90, 180 e 365 dias após o transplante. Episódios de rejeição aguda comprovados por biópsia foram analisados de acordo com a classificação de Banff 2007. A taxa de filtração glomerular (TFG) ao final do primeiro ano foi estimada utilizando a fórmula Modificationof Diet in Renal Disease (MDRD). Os pacientes foram inicialmente separados em 3 grupos de diferentes riscos imunológicos (pré-transplante): A) DSA-, B) DSA+ com MFI >1000 e < 5000 e C) DSA+ com MFI > 5000. Num segundo momento, foram novamente agrupados de acordo com o perfil de mudança nos valores de MFI (intensidade de fluorescência média) ao longo do primeiro ano. Resultados: DSA estavam presentes pré-transplante em 15 pacientes. RMA foi mais frequente no grupo C (p = 0,02). De acordo com a variação dos títulos de DSA pós-transplante os pacientes foram novamente agrupados: grupo I) permaneceu DSA- durante todo acompanhamento = 50 pacientes, II) diminuiu ou manteve títulos de DSA em relação ao tempo zero = 13 pacientes e III) aumentou títulos em relação ao tempo zero = 8 pacientes (6 foram DSA de novo). Três pacientes dos grupos I e um paciente do grupo II apresentaram episódios de rejeição aguda celular. Não foi observada oscilação significativa nos títulos de anticorpos durante esses eventos. Nenhum paciente desse grupo apresentou episódio de RMA. Episódio de RMA ocorreu em dois pacientes do grupo III. Em ambos os pacientes foi detectado aumento significativo nos valores de MFI dos DSA em relação ao tempo zero. Não foi observada diferença significativa na TFG entre os grupos analisados nesse estudo. Entretanto, observou-se uma diferença estatisticamente significativa na TFG entre os pacientes que apresentaram episódio de rejeição aguda em relação aos que não tiveram, sendo menor nos primeiros (p = 0,04). Conclusão: A monitorização prospectiva dos anticorpos pode ajudar a identificar pacientes em maior risco para ocorrência de RMA e o aumento nos valores de MFI DSA deve ser interpretado como um sinal de alerta, sobretudo em pacientes previamente sensibilizados.

La greffe cardiaque est le traitement ultime de l’insuffisance cardiaque. Le rejet aigu pose plusieurs problématiques, en particulier sa survenue imprévisible même sous traitement immunosuppresseur, et un diagnostic histologique qui nécessite des biopsies endomyocardiques (BEM) invasives répétées, et qui souffre de nombreuses limites. Le besoin de critères diagnostiques et prédictifs, idéalement non invasifs, nous a conduits à étudier le rejet aigu de greffe cardiaque sur le plan moléculaire. Nous avons caractérisé les profils d’expression génique (PEG) myocardiques et sanguins lors de différentes phases du rejet cellulaire (RC) et du rejet médié par les anticorps (RMA), par analyse sans a priori des transcriptomes sur puce à ADN. Par une première étude des PEG myocardiques menée sur une collection historique de BEM, nous avons montré la modification des PEG tissulaires lors du RC. Pour le même grade histologique, deux profils de RC aux degrés d’activation immunitaire différents ont été identifiés. De plus, les PEG myocardiques étaient modifiés dès un mois avant la survenue d’un RC, quand l’analyse histologique ne montrait encore aucune anomalie. Par une seconde étude conduite sur une collection prospective de BEM et échantillons sanguins, nous avons confirmé les résultats de la première étude, et de plus montré l’existence de modulations des PEG également dans le sang périphérique, aussi bien pendant un épisode de RC qu’un mois avant. Enfin pour la première fois la modulation tissulaire et périphérique des PEG a été montrée dans le RMA en transplantation cardiaque. L’existence de voies modulées dans les deux types de rejet devrait conduire à la recherche de biomarqueurs
Heart transplantation is the last treatment in case of terminal heart failure. Acute rejection after heart transplantation raises several issues due to its occurrence despite immunosuppressive therapies and the requirement of invasive and repeated endomyocardial biopsies (EMB) that have several histological grading limitations. The need of non-invasive diagnostic and predictive criteria led us to study the acute rejection of cardiac allograft using a molecular approach. We characterized myocardial and peripheral blood gene expression profiles (GEP) during acute cellular rejection (CR) and antibody-mediated rejection (AMR) by mean of microarray analyses. By a retrospective study conducted on a historical EMB collection, we first showed a strong immunologic modulation during CR. For the same CR histological grading, two transcriptional profiles were identified according to the inflammation level. Moreover, myocardial GEP modifications were observed one month before the occurrence of CR, while histological characteristics showed no abnormality. A second study conducted on a prospective collection of both EMB and peripheral blood samples confirmed the results obtained on EMB and showed peripheral blood GEP modulations during both CR and even one month earlier. Finally, we have also shown for the first time in heart transplantation, myocardial and peripheral GEP modulations in AMR. Identification of modulated pathways in both types of rejection should allow for the determination of rejection biomarkers

Introdução: A rejeição aguda mediada por anticorpos (RAMA) representa atualmente uma importante limitação para o sucesso do transplante renal. Seu diagnóstico é complexo e impreciso e avaliações moleculares com o desenvolvimento de biomarcadores não invasivos podem representar métodos promissores para seu diagnóstico. O objetivo do estudo foi avaliar, em pacientes transplantados renais, a expressão de genes relacionados à rejeição medida por anticorpos e celular, em tecido renal e no sangue periférico. Métodos: Estudo transversal com 56 pacientes transplantados renais divididos nas seguintes categorias diagnósticas de acordo com a classificação Banff 2007: RAMA, rejeição aguda celular (RAC), necrose tubular aguda (NTA), RAMA+RAC e normal. Foi utilizada a técnica de PCR Real-Time para a quantificação relativa dos genes: CD20, CD138, Fator de von Willebrand (FVW), TIM-3 e FOXP-3. Resultados: Pacientes com RAMA apresentaram, tanto no tecido renal quanto no sangue periférico, transcritos de mRNA para CD20 e TIM-3 significativamente aumentados (P<0,01), em relação aos grupos NTA e normal. Outros resultados com expressão significativamente maior na RAMA em relação ao grupo normal foram FOXP-3 no sangue (P<0,01), CD138 na biópsia (P<0,01) e FWV na biópsia e no sangue (P<0,05). As curvas ROC demonstraram áreas sobre a curva (ASC) de 0,950 (P<0,001) para CD20 no sangue periférico. Utilizando o ponto de corte 6,0 obtevese sensibilidade 94% e especificidade 88% para o diagnóstico de RAMA. CD138 no tecido renal apresentou ASC de 0, 905 (P<0,001), e com ponto de corte 6,0 encontrou-se sensibilidade 91% e especificidade 85%. Conclusão: A expressão de CD20, tanto em tecido renal como no sangue periférico, e de CD138 no tecido foram significativamente maiores em pacientes com RAMA. Mais estudos poderão confirmar estes achados e possibilitar a utilização da expressão destes e de outros genes como biomarcadores para o diagnóstico de RAMA.
Introduction: Acute antibody mediated rejection (ABMR) is currently a major limitation to the success of renal transplantation. Its diagnosis is complex and inaccurate and the development of non-invasive biomarkers can represent promising methods for that. The aim of this study was to evaluate, in kidney transplant patients, the expression of genes related to the antibody mediated rejection and cellular, in renal tissue and peripheral blood. Methods: Crosssectional study with 56 kidney transplant patients divided into the following diagnostic categories according by the Banff 2007 classification: ABMR, acute cellular rejection (ACR), acute tubular necrosis (ATN), ACR+ABMR and normal. We used Real Time PCR to quantify relative expression of genes: CD20, CD138, von Willebrand factor (vWF), FOXP-3 and TIM-3. Results: Patients with ABMR presented, both in renal tissue and in peripheral blood, CD20 and TIM-3 mRNA transcripts significantly increased (P <0.01), in relation to groups ATN and normal. Other results with significantly higher expression in ABMR in relation to the normal group were FOXP-3 in the peripheral blood (P <0.01), CD138 in tissue (P <0.01) and vWF renal tissue and blood (P <0.05). The ROC curves demonstrated area under the curve (AUC) of 0.950 (P <0.001) for CD20 in peripheral blood. Using the 6.0 cutoff point was obtained 94% sensitivity and 88% specificity for the diagnosis of RAMA. CD138 in renal tissue showed AUC 0, 905 (P <0.001), and 6.0 cutoff point was found 91% sensitivity and specificity 85%. Conclusion: The expression of CD20, both in renal tissue and in peripheral blood, and CD138 in tissue were significantly higher in patients with ABMR. More studies can confirm these findings and enable the use of the expression of these and other genes as biomarkers for the diagnosis of ABMR.

L'insuffisance rénale est un problème majeur de santé publique et la transplantation rénale est l’option thérapeutique principale, mais elle comporte le risque de rejet d'organe. Les cellules B jouent un rôle important dans le rejet médié par les anticorps (AMR). Au cours de l'AMR chronique, les structures lymphoïdes tertiaires, semblables aux centres germinatifs (GC), apparaissent dans l'organe rejeté, associées à la production des plasmocytes et des lymphocytes B mémoires spécifiques du donneur. Ces populations de lymphocyte B sont souvent mal contrôlées par les traitements actuels. La myeloid cell leukemia 1 (Mcl-1), un membre anti-apoptotique de la famille de B-cell lymphoma 2 (Bcl-2), est essentiel pour maintenir l’organisation de GC et de la différenciation des cellules B. Nous rapportons ici l'infiltration de cellules B exprimant Mcl-1 dans le rein de patients atteints d'AMR chronique, comme cela a été observé pour les cellules (pré) GC. Suite à l’abrogation de la signalisation du récepteur des cellules B (BCR), par l'inhibition de la spleen tyrosine kinase (SYK) nous avons observé une diminution de la viabilité des cellules GC, par l'intermédiaire d'une régulation de Mcl-1. La régulation négative de Mcl-1 est coordonnée au niveau de la transcription, potentiellement par l'intermédiaire du transducteur de signal et de l'activateur de la transcription 3 (STAT3), comme cela a été observé par (1) une translocation altérée de STAT3 dans le noyau suivant l'inhibition de SYK, et (2) les niveaux inférieurs de transcription de Mcl-1. Par ailleurs, la surexpression de Mcl-1 inhibe l'apoptose après l'inhibition du SYK. Des études avec des cellules B primaires, issues d'amygdales, ont confirmé que l'inhibition de SYK a diminué la survie cellulaire. Nous avons également constaté que l'inhibition du SYK a diminué les niveaux de protéines Mcl-1 dans les cellules B primaire, et que l’activation de ces cellules a été inhibée, tel que déterminé par l'expression de CD80 et des taux inférieurs de sécrétion d'IgG dans les cellules B primaires activées in vitro. Nos travaux suggèrent que la voie SYK-Mcl-1 peut offrir de nouvelles opportunités pour le traitement et la prévention de l'AMR
Renal failure is a major public health concern and renal transplantation is the main therapeutic option, however it comes with the risk of organ rejection. B-cells play an important role in antibody-mediated rejection (AMR). During chronic AMR, tertiary lymphoid germinal center (GC)-like structures appear in the rejected organ, associated with de novo production of donor-specific plasma and memory B-cells. Which are B-cell populations that are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma-2 (Bcl-2) family, is essential for maintaining the GC reaction and B-cell differentiation. We report here the infiltration of B-cells expressing Mcl-1 in the kidney of patients with chronic AMR, as observed for (pre-)GC cells. The impairment of B-cell receptor (BCR) signaling, by inhibition of spleen tyrosine kinase (SYK), reduced viability and Mcl-1 protein levels in GC like cells. This downregulation is coordinated at the transcriptional level, potentially via signal transducer and activator of transcription 3 (STAT3), as shown by (1) impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Moreover, overexpression of Mcl-1 prevented cells from entering apoptosis after SYK inhibition. In vitro studies with primary tonsillar B-cells confirmed that SYK inhibition decreased cell survival. We also found that SYK inhibition decreased Mcl-1 protein levels in primary B-cells, and that B-cell activation was inhibited, as determined by CD80 expression and lower levels of IgG secretion in tonsillar B-cells activated in vitro. Overall, our data suggest that the SYK-Mcl-1 pathway may provide new opportunities for the treatment and prevention of AMR

Background: Heart Transplantation (HTX) is the only curative treatment available for patients with end-stage heart failure (HF).During the first year post-transplantation more than 25% of patients will go through rejection episodes and will face the risk of developing rejection with consequent graft dysfunction with an increased morbidity and mortality. Preventing and treating acute rejection is the most central task for clinicians working with transplanted patients. The ISHLT 2005 and 2013 working formulations defined the histopathologic profile of three types of rejection: Cellular (ACR) Humoral (AMR) and Mixed (MIX). Nowadays serial endomyocardial biopsies (EMB) at decreasing intervals during the first year after transplantation and laboratory tests, such as Donor Specific Antibody (DSA) measurements, remain the gold-standard in diagnosing and monitoring acute rejection but they are morbid and prone to artefacts of sampling, interpretation and testing methodologies. Therefore this histopathological assessment needs integrative new biomarkers to characterize risk stratification for outcomes in heart transplantation. To date, the exact mechanisms involved in rejection after solid transplantation are not completely understood, so investigating process that contribute to acute allograft rejection and find effective biomarkers to diagnose, monitoring and predicting rejection will be of great value for the development of improved anti-rejection strategies. The advent of sequencing technology such as Next Generation Sequencing (NGS) is changing medical genomics by accelerating new disease biomarkers discovery. MicroRNAs (miRNAs) are small non-coding RNA molecules (19-24 nucleotides), highly conserved, which regulate genes expression at the post transcriptional level. Aim: The aim of this study is to identify MicroRNA (miRNAs) expression profile in the first year after heart transplantation (HTX) with Next Generation Sequencing (NGS) technology in formalin fixed paraffin-embedded (FFPE) endomyocardial Biopsies (EMBs), to characterize the three different types of allograft rejection classified as Cellular, Humoral and Mixed. Methods: Two groups of pts. were included: a study group of 19 pts. and a validation group of 14 pts. For each patient we selected the the first formalin fixed paraffin-embedded (FFPE) monitoring endomyocardial biopsies (EMBs) positive for each types of rejection. We excluded presensitized patients (pts) with previous implantation of Left Ventricular Assistance Device (LVAD) and with previous infections. EMBs were examined for the presence of rejection according to updated international classification criteria (ISHLT 2005 and 2013).The EMBs were classified in four groups: Acute Cellular Rejection (ACR) with 12 pts ACR: >=2R, pAMR:0, DSA: Neg ; Mixed with 6 pts ACR: >=2R, pAMR>1 (i+), DSA: Pos; Antibody Mediated Rejection (AMR) with 5 pts ACR: 0, pAMR>1 (i+), DSA: Pos; Control with 10 pts : ACR:0, pAMR:0, DSA: Neg. Small RNA fraction from the study group was sequenced with NGS Ion Proton in order to define the expression of mature miRNAs. We performed subsequent analysis with edgeR package comparing in pairs the groups to identify differentially expressed miRNAs in the different rejections. We selected 13 microRNAs according to bionformatic analysis as possible biomarckers and they have been confirmed by qRT-PCR in all the pts. With multivariate logistic regression analysis we created unique miRNA signatures as predictive model of each rejection. Moreover in situ PCR was carried out on the same EMBs to detect miRNAs expression and localization in cell types within the EMBs. Results: The identification of the best method of extraction for short non coding RNAs in FFPE EMBs was the first result I achieved. I tested different methods in house and commercial available kits and I modified the protocols to obtain good quality and adeguate quantity of RNA from FFPE tissue of small EMBs for the downstream application. With NGS we obtained and analysed more than 2257 mature microRNAs in all the biopsies of the study group. The three types of rejection and control groups were compared in pair with the un-supervised analysis showing a typical profile for each group of differentially expressed miRNAs; in particular: Mixed vs AMR: only 2 miRNAs overexpressed in the Mixed group suggesting a similarity between the two types. ACR vs AMR: 18 miRNAs overexpressed and 2 miRNAs under-expressed in the ACR. Mixed vs ACR : 7 miRNAs underexpressed and 39 miRNAs over-expressed in the ACR group. The analysis revealed that there are de-regulated microRNAs between the three rejections confirming our hypothesis that microRNAs can characterize the three pathological conditions. MiRNAs have been selected for further evaluation and validation, based on the number of reads resulting by NGS, on their highly significant FDR (< 0.05) or fold change, p-value and their involvement in relevant processes related to rejection as shown by a bioinformatic analysis based on validated target genes and reported in public databases such as TarBase (version 6.0) (111) , miRTarBase (112) , miRWalk (113), miRecords (114), DIANA-microT-CDS (115) , miRmap (116), miRDB (117) , TargetScan (118), and miRanda (119). At the end we selected 13 microRNAs. To validate the NGS data through qRT-PCR we enrolled other EMBs from 14 pts selected according to our criteria and we tested on all the 33 EMbs, both the study and validation cohort, the selected microRNAs. The validation analysis has shown a similar expression pattern for all microRNAs in particular: 6 hsa-miRNAs: 29c-3p/-29b-3p/199a-3p/190a-5p/27b-3p/302b-3p can differentiate all rejections compared to controls; 3 hsa-miRNAs: 31-5p/144-3p/218-5p are peculiar of AMR and MIX compared to control and ACR 2 hsa-miRNAs: 451a/208a-5p identify MIX compared to controls. Using miRNAs expression as co-variate and disease status as dependent variable we created logistic regression models: MIX:(miR-208a ,126-5p, 135a-5p); ACR:(miR-27b-3p, 29b-3p,199a-3p, 208a, 302b-3p); AMR: ( miR-208a, 29b-3p, 135a-5p, 144-3p) identifying with high specificity and sensitivity each types of rejection. Finally with in situ PCR we detected some of these microRNAs in different cell types: miR-29b-3p was mostly expressed in smooth muscle cells in ACR; miR-144-3p was expressed in macrophages and in endothelial cells; moreover the expression of this microRNA in macrophages was predominant and diffuse in the ACR compared to AMR. miR-126-5p was expressed in ACR and AMR samples not only in in endothelial cells but also in Cardiomyocytes and smooth muscle cells. For MicroRNA 451a we found a co-localization of signal in endothelial cells and in lymphocytes. Conclusions: This study demonstrate that MicroRNAs can be obtained easily from FFPE tissues, miRNAs differentially expressed are involved in pathophysiological mechanisms of rejection such as immune system cells cycle regulation and proliferation, , inflammatory pathways NFkB mediated and endothelial remodelling. According to our results the miRNAs up or down expressed modulate these pathways in a way peculiar for the different type of rejection. The regressive models might represent a powerful diagnostic tool and in situ detection of the miRNAs casts new light on the pathophysiological mechanisms of rejection. Moreover the expression of MiRNAs 144-3p, 126-5p, 29b-3p and 451a identified by in situ PCR in endothelial cells, smooth muscle and inflammatory cells are diagnostic and are potential pharmacological targets for rejections.
Contesto: Il trapianto di cuore è l'unico trattamento curativo disponibile per i pazienti con insufficienza cardiaca allo stadio terminale. Durante il primo anno dopo il trapianto più del 25% dei pazienti può subire episodi di rigetto e affrontare il rischio di sviluppare rigetto con conseguente disfunzione dell’ organo trapiantato con un aumento della morbilità e mortalità. Prevenire e trattare il rigetto acuto è l’ obiettivo principale per i medici che lavorano con pazienti trapiantati. Le linee guida ISHLT 2005 e 2013 hanno definito il profilo istopatologico di tre tipi di rigetto: Cellulare (ACR) Humoral (AMR) e Mixed (MIX). Al giorno d'oggi le biopsie endomiocardiche seriali (EMB) a intervalli decrescenti durante il primo anno dopo il trapianto e gli esami di laboratorio, come le misurazioni di anticorpi donatore specifici (DSA), rimangono parametri di riferimento nella diagnosi e nel monitoraggio del rigetto acuto, ma sono soggetti a artefatti dovuti alle metodologie di campionamento, interpretazione e test. Pertanto questa valutazione istopatologica necessita di nuovi biomarcatori integrativi per caratterizzare la stratificazione del rischio nel rigetto da trapianto di cuore. Ad oggi, i meccanismi esatti coinvolti nel rigetto dopo il trapianto non sono completamente compresi, quindi la ricerca sui processi che governano i meccanismi di rigetto e la scoperta di biomarcatori efficaci per diagnosticare, monitorare e prevedere il rigetto sarà di grande valore per lo sviluppo e miglioramento delle terapie contro il rigetto. L'avvento della tecnologia di sequenziamento come Next Generation Sequencing (NGS) sta cambiando la genomica medica accelerando la scoperta di nuovi biomarcatori di malattie. I microRNA (miRNA) sono piccole molecole di RNA non codificanti (19-24 nucleotidi), altamente conservate, che regolano l'espressione dei geni a livello post-trascrizionale. Obiettivo: Lo scopo di questo studio è identificare il profilo di espressione di MicroRNA (miRNA) nel primo anno dopo il trapianto di cuore (HTX) con la tecnologia Next Generation Sequencing (NGS) in biopsie endomiocardiche (EMB) fissate in formalina e incluse in paraffina (FFPE), per caratterizzare i tre diversi tipi di rigetto da trapianto di cuore classificati come Cellulare, Umorale e Misto. Metodi: due gruppi di pazienti (pz.) sono stati inclusi: un gruppo di studio di 19 pz. e un gruppo di validazione di 14. Per ogni paziente abbiamo selezionato la prima biopsia endomiocardica (EMB) fissata in formalina ed inclusa in paraffina (EMB) per ogni tipo di rigetto. Abbiamo escluso i pz. presensibilizzati con precedente impianto del dispositivo di assistenza ventricolare sinistro (LVAD) e con precedenti episodi di infezione. Le biopsie sono state esaminate per la presenza di rigetto secondo i criteri di classificazione internazionali aggiornati (ISHLT 2005 e 2013). Abbiamo quindi individuato quattro gruppi: Acute Cellular Rejection (ACR) con ACR a 12 punti:> = 2R, pAMR: 0, DSA: Neg; Misto con 6 pts ACR:> = 2R, pAMR> 1 (i +), DSA: Pos; Reiezione mediata da anticorpi (AMR) con 5 punti ACR: 0, pAMR> 1 (i +), DSA: Pos; Controllo con 10 punti: ACR: 0, pAMR: 0, DSA: Neg. La piccola frazione di RNA della coorte di studio è stata sequenziata con NGS Ion Proton per definire l'espressione dei miRNA maturi. Abbiamo eseguito un'analisi successiva con edgeR confrontando a coppie i gruppi per identificare i miRNA espressi differenzialmente nei diversi rigetti. Abbiamo selezionato 13 microRNA secondo l'analisi bionformatica come possibili biomarcatori i quali sono stati confermati da qRT-PCR in tutti i pz. Con l'analisi di regressione logistica multivariata abbiamo identificato gruppi univoci di miRNA come modelli predittivi specifici per ciascun rigetto. Inoltre, la PCR in situ è stata eseguita sulle stesse EMBs per rilevare l'espressione e la localizzazione dei miRNA nei tipi di cellule all'interno delle EMBs. Risultati: l'identificazione del miglior metodo di estrazione di microRNA da EMBs FFPE è stato il primo risultato che ho raggiunto. Ho testato diversi metodi sia manuali che kit commerciali e ho modificato i protocolli per ottenere una buona qualità e una quantità adeguata di microRNA per l'applicazioni successive. Con NGS abbiamo ottenuto e analizzato oltre 2257 microRNA maturi in tutte le biopsie del gruppo di studio. I tre tipi di gruppi di controllo e di rigetto sono stati confrontati in coppia con l'analisi non supervisionata che mostra per ciascun gruppo un profilo tipico di miRNA differenzialmente espressi; in particolare: Misto vs AMR: solo 2 miRNA sovraespressi nel gruppo Misto suggeriscono una somiglianza tra i due tipi di rigetto. ACR vs AMR: 18 miRNA sovraespressi e 2 miRNA sottoespressi nell'ACR. Mixed vs ACR: 7 miRNAs non sovraespressi e 39 miRNA sovraespressi nel gruppo ACR. L'analisi ha rivelato che ci sono microRNA de-regolati tra i tre tipi di rigetto confermando la nostra ipotesi che i microRNA possano caratterizzare le tre condizioni patologiche. I MiRNA sono stati selezionati per un'ulteriore valutazione e convalida, in base al numero di reads risultanti da NGS, sulla loro FDR significativa (<0,05), fold change, p-value e il loro coinvolgimento in processi rilevanti correlati al rigetto come mostrato dalle analisi bioinformatiche basate su geni target validati e riportati in database pubblici come TarBase (versione 6.0) (111), miRTarBase (112), miRWalk (113), miRecords (114), DIANA-microT-CDS (115), miRmap (116) , miRDB (117), TargetScan (118) e miRanda (119). Alla fine abbiamo selezionato 13 microRNA. Per validare i dati NGS tramite qRT-PCR abbiamo arruolato altri EMBs da 14 pz. selezionati in base ai nostri criteri e abbiamo testato su tutte le 33 EMbs, sia quelle della coorte di studio che quelle della coorte di validazione, i microRNA selezionati. L'analisi di validazione ha mostrato un pattern di espressione simile per tutti i microRNA in particolare: 6 hsa-miRNA: 29c-3p / -29b-3p / 199a-3p / 190a-5p / 27b-3p / 302b-3p possono differenziare tutti i rigetti rispetto a controlli; 3 hsa-miRNA: 31-5p / 144-3p / 218-5p sono peculiari di AMR e MIX rispetto al controllo e ACR 2 hsa-miRNA: 451a / 208a-5p identificano MIX rispetto ai controlli. Usando l'espressione di miRNA e la condizione patologica come variabili dipendenti abbiamo creato modelli di regressione logistica: MIX: (miR-208a, 126-5p, 135a-5p); ACR: (miR-27b-3p, 29b-3p, 199a-3p, 208a, 302b-3p); AMR: (miR-208a, 29b-3p, 135a-5p, 144-3p) che identificano con alta specificità e sensibilità ciascun tipo di rigetto. Infine con PCR in situ abbiamo rilevato alcuni di questi microRNA in diversi tipi di cellule: miR-29b-3p era principalmente espresso nelle cellule muscolari lisce in ACR; miR-144-3p era espresso nei macrofagi e nelle cellule endoteliali; inoltre l'espressione di questo microRNA nei macrofagi era predominante e diffusa nell'ACR rispetto all'AMR. Il miR-126-5p è risultato espresso in campioni ACR e AMR non solo nelle cellule endoteliali ma anche nei cardiomiociti e nelle cellule muscolari lisce. Per il MicroRNA 451a abbiamo trovato una co-localizzazione del segnale nelle cellule endoteliali e nei linfociti. Conclusioni: Questo studio dimostra che i microRNA possono essere ottenuti facilmente dai tessuti fissati in formalina e inclusi in paraffina, i miRNA differenzialmente espressi sono coinvolti in meccanismi patofisiologici del rigetto quali regolazione e proliferazione del ciclo cellulare del sistema immunitario, vie infiammatorie mediate da NFkB e rimodellamento endoteliale. Secondo i nostri risultati, i miRNA sovra o sotto espressi hanno mostrato una modulazione di questi processi in un modo peculiare per ciascun tipo di rigetto. I modelli di regressione logistica identificati potrebbero rappresentare un potente strumento diagnostico e il rilevamento in situ dei miRNA getta nuova luce sui meccanismi patofisiologici del rigetto. Inoltre l'espressione di MiRNA 144-3p, 126-5p, 29b-3p e 451a identificati mediante PCR in situ in cellule endoteliali, cellule muscolari lisce e infiammatorie è diagnostica e costituisce un potenziale bersaglio farmacologico contro il rigetto da trapianto di cuore.

Le rejet à médiation humorale (ABMR) est aujourd’hui reconnu comme la première cause de perte du greffon rénal au-delà de la première année. Les anticorps anti-HLA spécifiques du donneur de novo (DSAdn) sont le facteur de risque principal d’ABMR après transplantation rénale. Ils sont au centre des mécanismes physiopathologiques impliqués au cours de cette pathologie. Cependant, l’évolution clinique après la détection d’un DSAdn est extrêmement hétérogène suggérant que tous n’ont pas la même pathogénicité. Plusieurs caractéristiques des DSAdn ont été identifiées comme étant associées à un risque plus élevé d’ABMR ou de perte du greffon comme la « force » des anticorps (évaluée par la MFI au test Luminex Single Antigen), leur capacité à fixer et activer la voie classique du complément et la détection d’une sous-classe IgG3. Dans ce travail de thèse, j’ai étudié le rôle de la répartition des différentes sous-classe IgG et du profil de glycosylation des DSA au cours de l’ABMR, grâce à la mise au point d’une technique innovante basée sur la spectrométrie de masse.Dans la première partie de ce travail, nous avons mis en évidence que les DSA étaient toujours composés des quatre sous-classes d’IgG mais avec une répartition variable selon les patients. La distribution des sous-classes des DSA était différente de celle des IgG totales avec plus d’IgG1, plus d’IgG3, plus d’IgG4 mais moins d’IgG2. Une proportion élevée d’IgG3 (>6.4%) était significativement associée à la présence d’un ABMR, à la sévérité histologique de l’ABMR avec plus de dépôts de complément et plus d’inflammation de la microcirculation (glomérulite et capillarite péri-tubulaire) et au déclin du débit de filtration glomérulaire, indépendamment des autres caractéristiques du DSA, en particulier de la valeur de MFI.Dans la deuxième partie de ce travail, nous avons montré pour la première fois une association entre le profil de glycosylation des DSA et le risque d’ABMR. Le groupe de patients présentant un ABMR avaient des DSA dont les sous-classes IgG1 et IgG3 exhibaient un profil de glycosylation pro-inflammatoire associant une plus faible galactosylation des IgG1, une plus faible sialylation des IgG3 et une proportion plus élevée de GlcNAc en position bissectrice. L’hyposialylation des IgG3 semble un facteur prometteur pour prédire du risque d’ABMR. Ces résultats ouvrent aussi potentiellement la voie à de nouvelles stratégies thérapeutiques qui sont particulièrement attendues, l’efficacité des thérapeutiques utilisées actuellement étant souvent décevante
Antibody-mediated rejection (ABMR) is now recognized as the leading cause of graft loss beyond the first year. De novo donor-specific anti-HLA antibodies (DSAdn) are the main risk factor for ABMR after kidney transplantation. However, the clinical course after the detection of a DSAdn is extremely heterogeneous, suggesting that not all DSA have the same pathogenicity. Several characteristics of DSAdn have been identified as being associated with a higher risk of ABMR or graft loss such as the “strength” of the antibodies (evaluated by the MFI in the Luminex Single Beads Antigen test), their ability to activate the classical complement pathway and the detection of IgG3 subclass. In this work, we studied the role of the DSA subclasses distribution and the DSA glycosylation profile in ABMR occurrence and graft outcomes. For this, we developed an innovative method for DSA characterization based on mass-spectrometry.In the first part of this work, we highlighted that the DSAs were always composed of the four IgG subclasses but with a variable distribution. The distribution of subclasses was specific to DSA with more IgG1, more IgG3, more IgG4 but less IgG2 compared to total IgG. A high proportion of IgG3 (> 6.4%) was significantly associated with ABMR occurrence and with ABMR severity (more complement deposition and more microvascular inflammation) and with the decline of the glomerular filtration rate, independently to other DSA characteristics, in particular the MFI value.In the second part of this work, we showed for the first time an association between the glycosylation profile of DSA and the risk of ABMR. DSA from ABMR+ patients exhibited a pro-inflammatory glycosylation profile, associating a lower galactosylation of IgG1, a lower sialylation of IgG3 and a higher proportion of bisecting GlcNAc. Hyposialylation of IgG3 appears to be a promising factor in the ABMR risk prediction. These results also potentially pave the way to new therapeutic strategies which are particularly expected, the effectiveness of current therapies often being disappointing

El trasplante renal es el tratamiento de elección en los pacientes con enfermedad renal crónica avanzada porque mejora la supervivencia del paciente, su calidad de vida e implica menores costes si lo comparamos con el tratamiento sustitutivo mediante diálisis. En los últimos años, los avances en la inmunosupresión, la mejora en las técnicas quirúrgicas y el mejor conocimiento de la inmunología del trasplante han permitido incrementar la supervivencia del paciente y del injerto a corto plazo, con menor impacto en las tasas de pérdida a medio y largo plazo. Se han producido dos importantes avances en la última década: el desarrollo de nuevas técnicas de detección de anticuerpos frente a antígenos HLA y la caracterización histológica del rechazo mediado por anticuerpos. El objetivo de la presente tesis ha sido profundizar en el conocimiento del rechazo mediado por anticuerpos abordando nuevos aspectos desde estos dos puntos de vista: estudio serológico de los anticuerpos HLA y caracterización histológica del rechazo humoral. Para ello, hemos analizado una amplia cohorte de pacientes trasplantados con estudio de anticuerpos antiHLA pre y postrasplante determinados mediante tecnología Luminex. Los pacientes con anticuerpos dirigidos frente al donante detectados pretrasplante presentan una peor supervivencia del injerto y mayor riesgo de rechazo mediado por anticuerpos; y este riesgo sigue incrementado tanto si persiste como si no persiste el anticuerpo después del trasplante o aparece de novo. Por otro lado, hemos realizado el estudio de otros anticuerpos considerados clásicamente menos inmunogénicos y con poca relevancia en el campo del trasplante hasta la fecha, pero que las nuevas técnicas en fase sólida permiten identificar, como son los anticuerpos frente a los antígenos HLA DP. En nuestra experiencia, el 10% de los pacientes trasplantados presentan anticuerpos antiHLA-DP detectados por Luminex, tanto pre como postrasplante. La presencia de estos anticuerpos no parece modificar el impacto en la supervivencia del injerto. Desde el punto de vista histológico, hemos demostrado que el rechazo mediado por anticuerpos es un diagnóstico frecuente en las biopsias tardías de injerto renal realizadas por indicación según los criterios de la clasificación de Banff 2013, y confiere un peor pronóstico que otras categorías histológicas. Finalmente, hemos profundizado en el análisis de la categoría histológica de rechazo mediado por anticuerpos comparando la clasificación de Banff 2009 con la nueva definición de cambios mediados por anticuerpos de la clasificación de Banff 2013. Según nuestros resultados, la clasificación de Banff 2013 proporciona un diagnóstico más preciso del rechazo mediado por anticuerpos.
Kidney transplantation is considered the treatment of choice for patients with end-stage renal disease. It is associated with improved survival, better quality of life and reduced costs when compared with dialysis. Throughout the years, the progress in immunosuppression, the improvement in surgical techniques and a better understanding of transplant immunology have produced an increase in both patient and graft survival short-term, notwithstanding in medium and long-term outcomes. Two major developments have taken place over the last decade: the development of new techniques to detect HLA antibodies, and the histological characterization of antibody-mediated rejection. The aim of this thesis has been to expand the knowledge of antibody-mediated rejection by addressing the issue from these two points of view: HLA antibody serological testing with new techniques and histological characterization of humoral rejection. To that end, we have analyzed a large cohort of transplant patients with pre and post-transplant anti-HLA antibodies determined by Luminex technologies. Patients with preformed donor specific antibodies show worse graft survival and greater risk of antibody-mediated rejection, regardless potential DSA clearing after transplantation. Furthermore, we evaluated the impact of typically less immunogenic antibodies believed to be less relevant in the transplant field thus far, such as HLA DP antibodies, albeit detectable by newer solid phase techniques. In our experience, 10% of transplant patients show HLA DP antibodies as detected by Luminex assay both pre and post-transplant. The presence of these antibodies does not seem to modify graft survival Histologically, we have shown that antibody-mediated rejection is a common diagnosis most often seen in late kidney graft biopsies according to the Banff 2013 classification criteria; antibody-mediated rejection also shows worse prognosis compared to other histological categories. Eventually, we have delved into the analysis of the antibody-mediated rejection category by comparing the Banff 2009 classification to the new antibody-mediated changes from Banff 2013. According to our results, Banff 2013 classification provides a more accurate diagnosis of antibody-mediated rejection.

Contexte : La réponse allo-immune induite par la transplantation à partir d'un donneur génétiquement différent est un obstacle majeur au succès de la greffe. Notre objectif est de caractériser les différents phénotypes de rejet d'allogreffe rénale et d'identifier la façon dont chacun est associé aux anticorps anti-HLA. Nous avons également évalué l’impact de certaines propriétés de ces anticorps, comme leur intensité ou leur capacité à fixer le complément, sur l'échec des allogreffes rénales. Pour finir, nous avons étudié l’impact pronostic des formes indolentes de rejets ainsi que l’apport des nouvelles technologies d’analyses transcriptomique des biopsies de patients transplantés. Méthodes : Nous avons utilisé une approche en population, basée sur l’étude de larges cohortes de receveurs de greffes rénales. L’étude concomitante des données immunologiques et histologiques, nous a permis de corréler les caractéristiques des anticorps anti-HLA circulants aux phénotypes lésionnels. Résultats : Nous avons identifié et caractérisé 4 types distincts de rejet : les rejets vasculaires médiés par les lymphocytes T (9%) et par les anticorps (21%), non reconnus par les classifications internationales, et les rejets cellulaires (46%) et humoraux sans vascularite (24%). Le risque de perte de greffons est le plus important dans les cas de rejet vasculaire médié par anticorps. Les anticorps dirigés contre le donneur (DSA) fixant le complément induisent un phénotype histologique plus sévère, dominé par des lésions inflammatoires et plus de dépôts de la fraction C4d du complément dans les greffons. En leur présence, le risque de perte de greffons est augmenté de 3,7 fois (IC95 1,9-7,2). Les formes indolentes de rejet médié par les anticorps sont également associées à un risque accru de perte du greffon. L’utilisation d’approches moléculaires permet d’améliorer la stratification du risque au sein du groupe des patients présentant des rejets humoraux. Conclusion : Ce travail répond à un besoin clinique pressant dans le domaine de la transplantation, celui de déterminer l’impact clinique des anticorps anti-HLA et d’améliorer la stratification du risque immunologique en se basant sur leurs propriétés et l’utilisation de nouvelles technologies pour mieux caractériser l’activité et le stade des rejets humoraux
Background : The alloimmune response induced by transplantation from a donor who differs genetically from the kidney recipient has always been the major obstacle to graft success. The present work aimed to improve characterization of kidney-allograft rejection phenotypes and identify how each one is associated with anti-HLA antibodies. We also sought to determine whether characteristics of these antibodies i.e., their levels or complementbinding ability, might play a role in kidney allograft failure. Finally, we evaluated the clinical relevance of indolent forms of ABMR and the clinical relevance of new genes expression technologies to stratify the kidney recipients at risk for failure. Methods : We used a population-based approach in precisely phenotyped cohorts of kidney recipients. The design of our study, which is based on the concomitant evaluation of immunologic and histologic data, permits a precise connection of circulating anti-HLA antibodies with a phenotype of graft injury. Findings : We identified four distinct patterns of kidney allograft rejection: T cell-mediated vascular rejection (9%), antibody-mediated vascular rejection (21%), not included in international classifications, T cell- (46%) and antibody-mediated rejection without vasculitis (24%). Risk of graft loss was 9.07 times (95CI 3.6-19.7) higher in antibody-mediated vascular rejection than in T-cell mediated rejections (p<0.0001). Patients with post-transplant complement-binding DSA had more severe graft injury phenotype with higher inflammation and increased deposition of complement fraction C4d. They have the poorest graft survival with 3.7 fold increased risk of graft loss (95CI 1.9-7.2). Subclinical ABMR is a truncated for of rejection associated with risk of kidney allograft failure. Gene expression assessment in kidney allografts with early ABMR improves classification of individuals at risk for kidney allograft loss. Conclusion : This work addresses the unmet need of the deleterious impact of anti-HLA antibodies and the improvement of risk stratification in kidney transplantation. Recognition of distinct phenotypes could lead to the development of new treatment strategies. Gene expression assessment appears useful to evaluate disease activity, disease state and prediction of failure

La transplantation rénale est le meilleur traitement de l’insuffisance rénale chronique terminale, que ce soit en termes d’espérance ou de qualité de vie. Malgré les progrès réalisés en immunologie de la transplantation et dans la gestion globale des patients transplantés, la principale étiologie de perte de greffon reste le rejet, et en particulier le rejet humoral.L’évaluation du risque de rejet humoral repose principalement sur le dosage des anticorps anti-HLA dirigés contre le greffon. Pourtant, il apparait que ces anticorps ont un faible pouvoir prédictif de l’incidence et du pronostic du rejet, ce qui pourrait être expliqué par une hétérogénéité de leurs caractéristiques intrinsèques. Ces caractéristiques dépendent des cellules responsables de leur sécrétion, plasmocytes à courte et longue durée de vie, et donc indirectement des cellules responsables du maintien du pool de ces cellules sécrétrices d’anticorps : les lymphocytes B mémoires. Il a été montré en pathologies infectieuses que ces lymphocytes B mémoires sont hétérogènes en termes de phénotype, de fonction, de degré de clonalité et de diversification de leur BCR (B-cell receptor). Néanmoins, ceci n’a pas encore été analysé en transplantation rénale. Un des objectifs de cette thèse était d’étudier le degré d’hétérogénéité des lymphocytes B mémoires HLA-spécifiques chez des patients immunisés en attente de transplantation rénale. Pour ce faire, une analyse en cellule unique de lymphocytes B mémoires HLA-spécifiques de patients présentant différents contextes et degré d’immunisation a été réalisée, dans le but d’identifier leurs caractéristiques phénotypiques et transcriptomiques et la diversification de leur répertoire BCR.La deuxième partie des travaux s’est concentrée sur les modalités diagnostiques du rejet de greffe rénale. Depuis quelques années, des outils de biologie moléculaire sont disponibles, permettant d’évaluer des centaines de transcrits exprimés dans le tissu de biopsie. Ces outils donnent la possibilité de décrire de nouvelles voies physiopathologiques, et potentiellement d’améliorer le diagnostic du rejet, en particulier le rejet humoral. Néanmoins, leur utilisation en pratique courante est restreinte du fait de leur faible disponibilité, des difficultés à interpréter les données produites, et de leur coût. De plus, du fait de cette sous-utilisation en pratique clinique, leur impact exact dans la prise en charge des patients n’est pas déterminé. Au cours de cette thèse, un outil de diagnostic moléculaire ayant des caractéristiques compatibles avec une utilisation en pratique clinique a été développé. Celui-ci permet de diagnostiquer le rejet et de le classer en rejet humoral ou cellulaire. Dans un second temps, cet outil a été confronté à des situations cliniques litigieuses, afin d’évaluer son intérêt en pratique courante.À travers ces travaux, cette thèse vise d’une part à améliorer la compréhension de la réponse humorale en transplantation rénale, afin de contribuer à terme à mieux stratifier le risque immunologique en transplantation, et d’autre part à améliorer les modalités diagnostiques du rejet en aidant à la généralisation des outils de biologie moléculaire appliqués aux biopsies de greffons rénaux
Kidney transplantation is the best treatment of end-stage renal disease, improving life quality and quantity. Despite advances in pathophysiological knowledge of kidney transplant immunology, kidney transplant rejection remains the major cause of allograft dysfunction, especially antibody-mediated rejection.Antibody-mediated rejection risk assessment is based on the evaluation of donor-specific anti-HLA antibodies. However, these antibodies have a poor predictive value for incidence and prognosis of rejection. This could be explained by the heterogeneity of their intrinsic characteristics. These characteristics depend on cells responsible for their secretion, which include short- and long- lived plasma cells. Consequently, they indirectly depend on the cells responsible for maintaining the pool of these antibody-secreting cells, such as memory B cells. In infectious diseases, it is known that memory B cells are heterogeneous in terms of phenotype, function, degree of clonality, and diversification of their B-cell receptor (BCR). However, this heterogeneity has not been examined in the context of kidney transplantation.The aim of the first part of this thesis was to study the heterogeneity of HLA-specific memory B cells in sensitised patients on kidney transplant waiting list. To this end, single-cell analysis of HLA-specific memory B cells from patients with various aetiologies and degrees of immunisation was performed. This led to their phenotypic and transcriptomic characterisation and to the assessment of their BCR repertoire.The second part of this thesis was dedicated to the diagnosis of kidney transplant rejection.In recent years, biopsy-based transcriptomics has emerged, enabling the assessment of hundreds of transcripts in kidney biopsy tissue. These tools provide the opportunity to elucidate new physiopathological pathways and potentially enhance the diagnosis of rejection, especially humoral rejection. However, their application in clinical practice is still limited due to their restricted availability, required expertise for data processing and interpretation, and cost. Furthermore, their exact impact on patient management remains undetermined. Here, a molecular diagnostic tool with characteristics suitable for clinical use was developed. This tool enables the diagnosis of rejection and its classification between antibody-mediated and T-cell mediated rejection. Subsequently, this tool was assessed in ambiguous clinical situations to evaluate its impact in clinical practice.Through these studies, this thesis focused on enhancing our understanding of the humoral response in renal transplantation, which could help improving immunological risk stratification in transplantation. Additionally, it aimed to improve biopsy-based transcriptomics in the diagnosis of kidney transplant rejection

The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts. Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0,2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall destruction and infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies. The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20,7±6,7 cells, P <0,001) compared to both immunosuppressed groups (TAC1 5,9±5,5 cells, TAC7 6,1±5,1 cells). Anti-MHC antibodies class I and II level in peripheral blood...

Kidney transplantation is the best treatment for patients with end-stage renal failure. The main problem of kidney transplantation is however the development of a cellular and antibody-mediated (humoral) rejection. During the last decade, thanks to the advanced immunosuppression, prognosis of survival and function of transplanted organs has significantly improved. Nevertheless, humoral rejection remains very serious obstacle in high-risk patients, because it can permanently damage the graft. Therefore, before transplantation it is necessary to stratify patients into high and low risk groups for development of antibody-mediated rejection. Current immunogenetic tests performed before transplantation include, in addition to HLA typing, detection of panel-reactive antibodies. However, this test does not provide information about B cells which participate in the humoral response of the kidney recipient. Therefore, in the presented thesis we studied B cell reactivity and its regulation in transplanted patients. In this retrospective analysis we measured levels of the B cell activating factor, a cytokine regulating the function of B lymphocytes (BAFF). Current reports suggest that BAFF could serve as a marker of humoral rejection. Furthermore, we focused on B lymphocytes and their capacity to produce...

Kidney transplantation is the treatment of choice for patients with end stage renal failure and is associated with prolonged survival of patients and better quality of life than long-term dialysis. Simultaneously, however, transplantation carries the risk of immunological complications leading to graft rejection. A serious problem in patients after organ transplantation is the development of humoral rejection, which is most often associated with the presence of antibodies specific to HLA antigens, particularly against mismatched HLA antigens of the organ donor. In certain cases antibodies may be specific to antigens expressed on endothelial cells, not on lymphocytes, like MICA, MICB, ICAM, and up till now unidentified tissue-specific antigens. Humoral rejection has significantly worse prognosis for the transplanted kidney than cellular rejection, and therefore its timely diagnosis is of great importance for the subsequent choice of appropriate therapy. The diagnosis of humoral rejection is based on the simultaneous detection of C4d deposits in the peritubular capillaries of the transplanted kidney and the finding of antibodies specific to the mismatched antigens of the donor (donor specific antibodies, DSA). The aim of our retrospective study was to contribute to improvement of the diagnosis of acute and...

Since its beginning, graft rejection remains the key problem of solid organ transplantation. This reaction of the recipient's immune system against mismatched antigens of the transplanted organ causes graft damage and consequently loss of its function. Rejection involves cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Among the genetic factors which may have a prognostic value in rejection risk evaluation are the Human Leukocyte Antigens (HLA) genotype, the Killer Immunoglobuline-like Receptor (KIR) gene repertoir, cytokine and other gene polymorphisms. These factors could be screened for before transplantation to find the best possible combination of genetic characteristics of the donor and recipient and to reveal patients with "risky" genotypes, who may need more intensive immunosuppression and more careful post-transplant follow-up. Molecular factors, such as HLA and non-HLA antibodies, soluble CD30 molecule (sCD30), Hepatocyte Growth Factor (HGF) and other cytokines, measured before and/or after transplantation in the recipient's blood may be helpful for rejection risk estimation and may also be used as post-transplant rejection onset markers. In our study, we focused on some of the above mentioned factors. We found that ethnicity plays a significant role in the...

Kidney transplantation is the most appropriate treatment for end-stage kidney failure. The risk of graft failure in retransplanted patients is generally higher than in first-transplant patients due to immunological and non-immunological reasons. An important risk factor to consider for retransplant patients is their sensitization, i.e. the presence of antibodies directed to HLA antigens of previous donor(s). For that reason, a project called Forbidden (Non-acceptable) Antigens was launched by IKEM with the aim of reducing the incidence of acute cellular and antibody-mediated rejection in retransplant patients. Work on the project was carried out between the years 2011-2013. Forbidden antigens were defined as mismatched HLA antigens of previous kidney donor(s) against which patients waiting for retransplantation produced antibodies. The aim of this diploma thesis is to evaluate whether the incidence of rejection is lower in patients with forbidden HLA antigens in comparison with a control cohort, where no forbidden antigens are defined. 234 patients (162 males and 72 females) were included in the study. Almost all tested patients were producing HLA antibodies (90.2%) and forbidden antigens were determined in 71.4% of patients. In a control group of 267 patients waiting for their first transplantation, the...

Indiana University-Purdue University Indianapolis (IUPUI)
Solid organ transplantation is limited by available donor allografts. Pig to human transplantation, xenotransplantation, could potentially solve this problem if physiologic and immunologic incompatibilities are overcome. Genetic modifications of pigs have proven valuable in the study of xenotransplantation by improving pig to human compatibility. More genetic targets must be identified for clinical success. First, this study examines platelet homeostasis incompatibilities leading to acute thrombocytopenia in liver xenotransplantation. Mechanisms for xenogeneic thrombocytopenia were evaluated using liver macrophages, Kupffer cells, leading to identification of CD18, beta-2 integrin, as a potential target for modification. When disruption of CD18 was accomplished, human platelet binding and clearance by pig Kupffer cells was inhibited. Further, human and pig platelet surface carbohydrates were examined demonstrating significant differences in carbohydrates known to be involved with platelet homeostasis. Carbohydrate recognition domains of receptors responsible for platelet clearance Macrophage antigen complex-1 (CD11b/CD18) and Asialoglycoprotein receptor 1 in pigs were found to be different from those in humans, further supporting the involvement of platelet surface carbohydrate differences in xenogeneic thrombocytopenia. Second, immunologic incompatibilities due to antibody recognition of antigens resulting in antibody-mediated rejection were studied. Identification of relevant targets was systematically approached through evaluation of a known xenoantigenic protein fibronectin from genetically modified pigs. N-Glycolylneuraminic acid, a sialic acid not found in humans, was expressed on pig fibronectin and was identified as an antigenic epitope recognized by human IgG. These studies have provided further insight into xenogeneic thrombocytopenia and antibody-mediated rejection, and have identified potential targets to improve pig to human transplant compatibility.