Impaired antigen-specific B-cell responses after Influenza vaccination in kidney transplant recipients receiving co-stimulation blockade with Belatacept

Inhibidores de la calcineurina; Vacunación antigripal; Trasplante de riñónCalcineurin inhibitors; Influenza vaccination; Kidney transplantationInhibidors de la calcineurina; Vacunació antigripal; Trasplantament de ronyóEmerging data suggest that costimulation blockade with belatacept effectively controls humoral alloimmune responses. However, whether this effect may be deleterious for protective anti-infectious immunity remains poorly understood. We performed a mechanistic exploratory study in 23 kidney transplant recipients receiving either the calcineurin-inhibitor tacrolimus (Tac, n=14) or belatacept (n=9) evaluating different cellular immune responses after influenza vaccination such as activated T follicular Helper (Tfh), plasmablasts and H1N1 hemagglutinin (HA)-specific memory B cells (HA+mBC) by flow-cytometry, and anti-influenza antibodies by hemagglutination inhibition test (HI), at baseline and days 10, 30 and 90 post-vaccination. The proportion of CD4+CD54RA-CXCR5+ Tfh was lower in belatacept than Tac patients at baseline (1.86%[1.25-3.03] vs 4.88%[2.40-8.27], p=0.01) and remained stable post-vaccination. At M3, HA+mBc were significantly higher in Tac-treated patients (0.56%[0.32-1.49] vs 0.27%[0.13-0.44], p=0.04) and correlated with activated Tfh numbers. When stratifying patients according to baseline HA+mBc frequencies, belatacept patients with low HA+mBC displayed significantly lower HA+mBc increases after vaccination than Tac patients (1.28[0.94-2.4] vs 2.54[1.73-5.70], p=0.04). Also, belatacept patients displayed significantly lower seroprotection rates against H1N1 at baseline than Tac-treated patients (44.4% vs 84.6%) as well as lower seroconversion rates at days 10, 30 and 90 after vaccination (50% vs 0%, 63.6% vs 0%, and 63.6% vs 0%, respectively). We show the efficacy of belatacept inhibiting T-dependent antigen-specific humoral immune responses, active immunization should be highly encouraged before starting belatacept therapy.This work was supported by the Instituto de Salud Carlos III (ISCIII) (grant numbers ICI14/00242 and PI16/01321, PI19/01710) and by the European Union’s Horizon 2020 Research and innovation program (grant agreement 754995). Also, this work was partly supported by the SLT002/16/00183 grant, from the Department of Health of the Generalitat de Catalunya by the call “Accioí instrumental de programes derecerca orientats en l’àmbit de la recerca i la innovacioí en salut.” The authors thank the Research Centers of Catalonia (CERCA) Programme/Generalitat de Catalunya for institutional support. OB was awarded with an intensification grant from the “Instituto de Salud Carlos III” [INT19/00051]

On the clinical relevance of using complete high-resolution HLA typing for an accurate interpretation of posttransplant immune-mediated graft outcomes

HLA typing; Donor-specific antibodies; Kidney transplantationTipificación de HLA; Anticuerpos específicos del donante; Trasplante de riñónTipificació de HLA; Anticossos específics del donant; Trasplantament de ronyóComplete and high-resolution (HR) HLA typing improves the accurate assessment of donor–recipient compatibility and pre-transplant donor-specific antibodies (DSA). However, the value of this information to identify de novo immune-mediated graft events and its impact on outcomes has not been assessed. In 241 donor/recipient kidney transplant pairs, DNA samples were re-evaluated for six-locus (A/B/C/DRB1/DQB1+A1/DPB1) HR HLA typing. De novo anti-HLA antibodies were assessed using solid-phase assays, and dnDSA were classified either (1) as per current clinical practice according to three-locus (A/B/DRB1) low-resolution (LR) typing, estimating donor HLA-C/DQ typing with frequency tables, or (2) according to complete six-locus HR typing. The impact on graft outcomes was compared between groups. According to LR HLA typing, 36 (15%) patients developed dnDSA (LR_dnDSA+). Twenty-nine out of 36 (80%) were confirmed to have dnDSA by HR typing (LR_dnDSA+/HR_dnDSA+), whereas 7 (20%) did not (LR_dnDSA+/HR_dnDSA−). Out of 49 LR_dnDSA specificities, 34 (69%) were confirmed by HR typing whereas 15 (31%) LR specificities were not confirmed. LR_dnDSA+/HR_dnDSA+ patients were at higher risk of ABMR as compared to dnDSA− and LR_dnDSA+/HR_dnDSA− (logRank < 0.001), and higher risk of death-censored graft loss (logRank = 0.001). Both LR_dnDSA+ (HR: 3.51, 95% CI = 1.25–9.85) and LR_dnDSA+/HR_dnDSA+ (HR: 4.09, 95% CI = 1.45–11.54), but not LR_dnDSA+/HR_dnDSA− independently predicted graft loss. The implementation of HR HLA typing improves the characterization of biologically relevant de novo anti-HLA DSA and discriminates patients with poorer graft outcomes.This work was supported by the Biomarker-Driven Immunosuppression Minimization (BIO-DRIM) Consortium (EU FP7-health, grant agreement number 305147; FP7/2012-2017) and by the Instituto de Salud Carlos III (ISCIII) (grant numbers ICI14/00242, PI16/01321, and PI19/01710), co-funded by European Regional Development Fund (ERDF), a way to build Europe. Also, this work was partly supported by the SLT002/16/00183 grant, from the Department of Health of the Generalitat de Catalunya by the call “Acció instrumental de programes de recerca orientats en l’àmbit de la recerca i la innovació en salut”

Tacrolimus CYP3A Single-Nucleotide Polymorphisms and Preformed T- and B-Cell Alloimmune Memory Improve Current Pretransplant Rejection-Risk Stratification in Kidney Transplantation

Rechazo agudo; Genética; InmunobiologíaRebuig agut; Genètica; ImmunobiologiaAcute rejection; Genetics; ImmunobiologyAchieving fast immunosuppression blood exposure after kidney transplantation is key to abrogating both preformed and de novo anti-donor humoral and cellular alloresponses. However, while tacrolimus (TAC) is the cornerstone immunosuppressant inhibiting adaptive alloimmunity, its blood exposure is directly impacted by different single-nucleotide polymorphisms (SNPs) in CYP3A TAC-metabolizing enzymes. Here, we investigated how functional TAC-CYP3A genetic variants (CYP3A4*22/CYP3A5*3) influence the main baseline clinical and immunological risk factors of biopsy-proven acute rejection (BPAR) by means of preformed donor-specific antibodies (DSAs) and donor-specific alloreactive T cells (DSTs) in a large European cohort of 447 kidney transplants receiving TAC-based immunosuppression. A total of 70 (15.7%) patients developed BPAR. Preformed DSAs and DSTs were observed in 12 (2.7%) and 227 (50.8%) patients, respectively. According to the different CYP3A4*22 and CYP3A5*3 functional allele variants, we found 4 differential new clusters impacting fasting TAC exposure after transplantation; 7 (1.6%) were classified as high metabolizers 1 (HM1), 71 (15.9%) as HM2, 324 (72.5%) as intermediate (IM), and 45 (10.1%) as poor metabolizers (PM1). HM1/2 showed significantly lower TAC trough levels and higher dose requirements than IM and PM (p < 0.001) and more frequently showed TAC underexposure (<5 ng/ml). Multivariate Cox regression analyses revealed that CYP3A HM1 and IM pharmacogenetic phenotypes (hazard ratio (HR) 12.566, 95% CI 1.99-79.36, p = 0.007, and HR 4.532, 95% CI 1.10-18.60, p = 0.036, respectively), preformed DSTs (HR 3.482, 95% CI 1.99-6.08, p < 0.001), DSAs (HR 4.421, 95% CI 1.63-11.98, p = 0.003), and delayed graft function (DGF) (HR 2.023, 95% CI 1.22-3.36, p = 0.006) independently predicted BPAR. Notably, a significant interaction between T-cell depletion and TAC underexposure was observed, showing a reduction of the BPAR risk (HR 0.264, 95% CI 0.08-0.92, p = 0.037). Such variables except for DSAs displayed a higher predictive risk for the development of T cell-mediated rejection (TCMR). Refinement of pretransplant monitoring by incorporating TAC CYP3A SNPs with preformed DSAs as well as DSTs may improve current rejection-risk stratification and help induction treatment decision-making.This work was supported by the Instituto de Salud Carlos III (ISCIII) (grant numbers PI16/01321, PI19/01710, and PI18/P1740) (co-funded by European Regional Development Fund, ERDF, a way to build Europe). Also, this work was partly supported by the SLT002/16/00183 grant, from the Department of Health of the Generalitat de Catalunya by the call “Acció instrumental de programes de recerca orientats en l’àmbit de la recerca i la innovació en salut.” The authors thank the Research Centers of Catalonia (CERCA) Programme/Generalitat de Catalunya for institutional support. OB was awarded an intensification grant from the “Instituto de Salud Carlos III” [NT19/00051]

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

[eng] 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.[spa] 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

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

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