Tracking preformed serological and T-cell alloimmune memory together with donor/recipient Molecular Human Leukocyte Antigen (HLA) disparity to improve immune-risk stratification in Kidney Transplantation

Abstract

INTRODUCTION: The presence of a donor-specific alloimmune response negatively impacts allograft outcomes, being associated to risk of rejection and premature graft loss. Alloimmunity can be both preformed (memory) or can develop de novo after transplantation. The immune assays currently used in clinical practice to evaluate alloimmunity have several limitations and do not allow a complete and precise assessment of those two responses at time of transplantation. The hypothesis of this doctoral thesis is that at the time of kidney transplantation, an accurate characterization of pretransplant anti-donor alloimmune sensitization using highly sensitive assays tracking both serological memory and circulating donor-reactive memory T cells together with the assessment of the susceptibility to de novo alloimmune activation assessing the degree of donor/recipient HLA matching at the molecular level, would improve current immune-risk stratification and ultimately guide transplant physicians individualizing immunosuppressive therapies. OBJECTIVES: - To compare the accuracy of different immune-assays evaluating the preformed serological immunity (circulating donor(HLA)-specific antibodies), either individually or in combination and their value predicting distinct kidney graft outcomes. - To investigate the development and kinetics of primary T-cell alloreactivity after transplantation by detection of alloreactive IFN-γ producing T cells using an Enzyme-link ImmunoSpot (ELISPOT) assay and evaluate their predominant antigen presenting pathways. - To analyze the impact of donor/recipient HLA molecular mismatching on the generation of de novo donor-specific alloimmunity both at humoral and T-cell level after transplantation using distinct bioinformatic algorithms. - To evaluate the value of assessing preformed donor-reactive IFN-γ-producing T cells and donor/recipient Molecular HLA mismatching to identify kidney transplant recipients at low risk of rejection when receiving reduced immunosuppression based on tacrolimus monotherapy. METHODS: we performed two retrospective clinical studies and one prospective multicenter biomarker-guided study (CELLIMIN). The predictive capacity of different assays to detect pretransplant donor-specific antibodies (DSA) has been evaluated: flow cytometry crossmatch, solid phase assays and complement activating (C3d) capacity of DSA in vitro. Furthermore, the presence of alloreactive T cells in vitro has been assessed by Interferon-γ ELISPOT before and after transplantation. Donor/recipient HLA incompatibility has been evaluated with different informatic algorithms: Amino acid mismatch score, HLA-Matchmaker eplet mismatches and PIRCHE-II scores. It has been assessed the impact of the results of those algorithms on the prediction of primary alloimmunity both at the serological and T-cell level. Last, in a prospective study guided by biomarkers assessing both pretransplant serological and T-cell alloimmunity we randomized low-risk patients to receive either immunosuppression based on tacrolimus monotherapy or standard of care (steroids, Mycophenolate mofetil and tacrolimus). MAIN RESULTS: DSA with high mean fluorescence intensity (MFI) and those fixing complement in vitro predict higher rejection risk. The most accurate serological assays to predict transplant outcomes were a combination of DSA detected by solid phase assay and flow cytometry crossmatch. All the informatic HLA molecular mismatch algorithms precisely predicted risk of humoral primary alloimmunity. Similarly, a higher molecular incompatibility (especially by PIRCHE-II score) predicted risk of de novo T-cell activation. Finally, in the CELLIMIN trial, we observed that patients without preformed alloreactivity (neither serological or T cell-mediated) displayed significantly lower risk of acute rejection as compared to patients with preformed cellular alloreactivity and receiving the same standard of care immunosuppression. However, patients without serological/T cell preformed alloreactivity receiving minimized immunosuppression with tacrolimus monotherapy showed significantly higher incidence of acute rejection especially those with high molecular HLA mismatch at the DQ level. CONCLUSIONS: A complete and accurate study of the donor-specific preformed immune responses both at the serological and T-cell level, together with the assessment of the molecular HLA incompatibility, could improve stratification of the alloimmune risk in a more precise way, finally allowing adapted individualization of immunosuppression.

Las respuestas inmunológicas donante-especificas impactan negativamente en la evolución del aloinjerto renal. Estas pueden ser preformadas o activarse de novo tras el trasplante. Las técnicas inmunológicas disponibles en la clínica presentan limitaciones que no permiten una evaluación completa y precisa de esas respuestas. La hipótesis de esta tesis doctoral es que una evaluación de la memoria inmunológica mediante nuevas herramientas diagnosticas junto con estudios de compatibilidad HLA donante/receptor a nivel molecular para predecir el riesgo de aloinmunidad de novo, mejorarían la estratificación del riesgo inmunológico y permitirían personalizar la terapia inmunosupresora. Se han usado diferentes metódicas de detección de anticuerpos donante-específicos (DSA) pre-trasplante: cross-match por citometría de flujo, técnicas de fase solida y capacidad de los DSA de fijar complemento (C3d) in vitro y se ha medido la presencia de células T aloreactivas in vitro mediante ELISPOT Interferon(IFN)-y antes y después del trasplante. La incompatibilidad molecular HLA se ha valorado mediante algoritmos informáticos: incompatibilidad de aminoácidos, HLAMatchmaker y PIRCHE-II. Por ultimo, en un ensayo clínico prospectivo, guiado por biomarcadores de alorespuesta pre-trasplante (serológica y celular T) se han aleatorizado pacientes de bajo riesgo a recibir monoterapia con tacrolimus o tratamiento inmunosupresor convencional y comparado el riesgo de rechazo. La combinación de DSA (por fase solida) y cross-match por citometría son las técnicas que mejor se asocian el riesgo de pérdida del injerto, mientras que los DSA con elevado índice de fluorescencia y los que fijan complemento se asocian al riesgo de rechazo. Todos los algoritmos de incompatibilidad molecular HLA se asocian al riesgo de aloreactividad humoral primaria post-trasplante. De forma parecida, la incompatibilidad molecular (sobretodo por PIRCHE-II) se relaciona al riesgo de generar respuesta T donante-especifica de novo. En el ensayo CELLIMIN, los pacientes sin aloreactividad pre-trasplante (DSA/aloractividad T) presentaron inferior riesgo de rechazo. Sin embrago, aquellos pacientes que recibieron tacrolimus monoterapia presentaron una mayor incidencia de rechazo, especialmente en presencia de elevada incompatibilidad de epletos HLA-DQ. Un estudio completo de las respuestas de memoria tanto serológica como celular T donante-específica, junto con la evaluación de la incompatibilidad HLA a nivel molecular, podrían estratificar más precisamente el riesgo inmunológico de cada receptor frente a su donante y permitir adaptar el tratamiento inmunosupresor de una forma personalizada.