Functional Consequences of Memory Inflation after Solid Organ Transplantation.

CMV is a major infectious complication following solid organ transplantation. Reactivation of CMV leads to memory inflation, a process in which CD8 T cells expand over time. Memory inflation is associated with specific changes in T cell function, including increased oligoclonality, decreased cytokine production, and terminal differentiation. To address whether memory inflation during the first year after transplantation in human subjects alters T cell differentiation and function, we employed single-cell-matched TCRαß and targeted gene expression sequencing. Expanded T cell clones exhibited a terminally differentiated, immunosenescent, and polyfunctional phenotype whereas rare clones were less differentiated. Clonal expansion occurring between pre- and 3 mo posttransplant was accompanied by enhancement of polyfunctionality. In contrast, polyfunctionality and differentiation state were largely maintained between 3 and 12 mo posttransplant. Highly expanded clones had a higher degree of polyfunctionality than rare clones. Thus, CMV-responsive CD8 T cells differentiated during the pre- to posttransplant period then maintained their differentiation state and functional capacity despite posttransplant clonal expansion.

Evolution of Cytomegalovirus-Responsive T Cell Clonality following Solid Organ Transplantation.

CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αß sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

Reduced T-cell Numbers and Elevated Levels of Immunomodulatory Cytokines in Metastatic Prostate Cancer Patients De Novo Resistant to Abiraterone and/or Enzalutamide Therapy.

Currently, there are two Food and Drug Administration (FDA)-approved drugs for androgen deprivation therapy (ADT) of metastatic castration-resistant prostate cancer (mCRPC) patients: abiraterone and enzalutamide. However, our understanding of the effect of these therapies on the immune system in mCRPC patients remains limited. Here, we examined how abiraterone and enzalutamide treatment affects levels of soluble immune mediators in plasma and in circulating immune cells of 44 mCRPC patients. We found that the baseline levels of cytokines fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10), and IL-6 were significantly lower in ADT-sensitive compared to de novo resistant patients. In addition, resistant patients showed significantly lower T cell frequencies. When comparing the levels of cytokines over the course of treatment, we observed that the levels of proinflammatory mediators, such as interferon-γ (IFN-γ), IL-5, macrophage inflammatory protein 1 alpha (MIP-1α), and tumor necrosis factor alpha (TNFα), were significantly increased in the ADT-sensitive patients. At the same time, the abiraterone/enzalutamide therapy did not reduce the percentage of tolerogenic myeloid cell populations, such as polymorphonuclear myeloid-derived suppressor cells, which retained unaltered expression of programmed death-ligand 1 (PD-L1) and B7-H3. Overall, our results suggest that certain immune markers, such as IL-6 and the frequency of effector T cells, could be predictive of therapeutic response to ADT therapies in mCRPC patients.

Value of monitoring circulating donor-reactive memory B cells to characterize antibody-mediated rejection after kidney transplantation.

Antibody-mediated rejection (ABMR) is defined by specific histopathological lesions and evidence of circulating donor-specific antibodies (DSA). Although DSA are not always detectable, monitoring donor-reactive memory B cells (mBC) could identify patients at risk of developing ABMR. Peripheral donor-reactive mBC using a novel HLA B cell ELISpot assay, serum DSA, and numbers of different B cell subsets were assessed in 175 consecutive kidney transplants undergoing either for-cause or 6- and 24-month surveillance biopsies for their association with main histological lesions of ABMR and impact on allograft outcome. In 85 incident for-cause biopsies, high frequencies of donor-reactive mBC were detected in all 16 (100%) acute ABMR/DSA+ and most chronic ABMR, with or without DSA (24/30[80%] and 21/29[72.4%], respectively). In a longitudinal cohort of 90 nonsensitized patients, a progressively higher expansion of donor-reactive mBC than de novo DSA was observed at 6 and 24 months (8.8% vs 7.7% and 15.5% vs 11.1%, respectively) and accurately identified patients with ongoing subclinical ABMR (area under the curve = 0.917 and area under the curve = 0.809, respectively). An unsupervised hierarchical cluster analysis revealed a strong association between donor-reactive mBC with main fundamental allograft lesions associated with ABMR and conferred a significant deleterious impact on graft outcome. Monitoring donor-reactive mBC may be useful to further characterize humoral rejection after kidney transplantation.

A multicolour HLA-specific B-cell FluoroSpot assay to functionally track circulating HLA-specific memory B cells.

Emerging evidence suggests that donor-reactive memory B cells (mBC) play a key role inducing antibody-mediated rejection (ABMR) after solid organ transplantation and show a broader antigen repertoire than plasma cells thus, being potentially present even in absence of donor-specific antibodies. Therefore, the development of novel immune assays capable of quantifying circulating donor-reactive mBC in organ transplantation is highly warranted. We developed a novel HLA-specific B-cell FluoroSpot assay capable of enumerating multiple HLA-specific Antibody Secreting Cells (ASC) originated from circulating mBC after in-vitro polyclonal activation. We performed a thorough characterization of distinct selective in-vitro mBC activation methods based on either the TLR7,8 agonist R848 plus Interleukin-2 or an anti-CD40 agonist monoclonal antibody, assessed optimal activation culture conditions, cell sources, activation time-frame as well as the advantage of measuring HLA-specific IgG-ASC as compared to HLA-IgG Ab detected in supernatants of in-vitro stimulated B-cell to characterize anti-HLA alloreactivity. Notably, using fluorescently-labeled multimerized HLA molecules as detection matrix, we show the ability of this assay to precisely quantify multiple anti-HLA mBC specificities. In conclusion, evaluating circulating donor-reactive mBC using new technology may provide novel insight of the pathogenesis of humoral rejection and may help identifying transplant recipients at high risk of allograft rejection.

Refinement of humoral immune monitoring in kidney transplantation: the role of "hidden" alloreactive memory B cells.

The advent of novel sensitive assays assessing circulating anti-human leukocyte antigen (HLA) antibodies has allowed recognizing humoral alloimmunity as the main immune-mediated mechanism responsible for allograft rejection and graft loss in kidney transplantation. However, current immune-monitoring techniques, exclusively focusing on circulating anti-HLA antibodies, may underestimate the magnitude of humoral immune response as they exclude the memory B-cell (mBC) pool. Different biological compartments are involved in the intricate mechanisms triggering humoral alloimmune responses even in absence of detectable circulating alloantibodies. Recent studies in animal models as well as in clinical kidney transplantation have shown the key role of this B-cell subset triggering allograft rejection, thus emphasizing the value of recognizing antidonor mBC both as a biomarker of allosensitization and as therapeutic targets. Therefore, considerable efforts are being made among the transplant research community to better understand the role, hierarchy, and impact of mBC in the context of organ transplantation. In this review article, we provide a deep insight into the biology of mBC as well as main evidence of their role orchestrating allograft rejection. Also, we provide a thorough description of main immune-monitoring tools aiming at tracking mBC and a rational for their potential use to refine current humoral immune-risk assessment in kidney transplantation.

Effector Antitumor and Regulatory T Cell Responses Influence the Development of Nonmelanoma Skin Cancer in Kidney Transplant Patients.

BACKGROUND: Chronic immunosuppression promotes nonmelanocytic squamous cell carcinoma (SCC) after kidney transplantation. Adaptive and innate immunity play a key role controlling tumor growth and are influenced by different immunosuppressive agents. We hypothesized that functional impairment of tumor-specific T cell responses due to calcineurin inhibitors (CNI) could contribute to SCC development, whereas conversion to mammalian target of rapamycin inhibitors (mTOR-i) could recover this protective immune response. METHODS: Peripheral tumor-specific T cell responses against main SCC-derived antigens using the IFN-γ enzyme-linked immunospot assay and intratumor (IT) and circulating immune phenotypes (CD4 + T, CD8 + T, CD20 + B, CD56 + NK, FOXP3 + regulatory T [Treg] cells) were explored in a cross-sectional analysis in 59 kidney transplant patients with SCC on CNI (KT-CNI-SCC) or mTOR-i (KT-mTORi-SCC), 25 nontransplants developing SCC (NoKT-SCC) and 6 healthy controls. Moreover, 25 KT-CNI-SCC were switched to mTOR-i and evaluated after 12 months. RESULTS: Kidney transplant patients showed lower IT infiltrates and tumor-specific T cell responses than NoKT-SCC, and intratumoral and circulating FOXP3 + Treg cells were higher in KT-mTORi-SCC (P < 0.05). Tumor-specific T cell responses were significantly lower in KT-CNI-SCC than KT-mTORi-SCC and NoKT-SCC and predicted SCC relapses (area under the curve = 0.837; P < 0.05). One-year after mTOR-i conversion, a significant increase in FOXP3 + Treg cell numbers and tumor-specific T cell responses were observed, reaching similar levels than KT-mTORi-SCC and NoKT-SCC patients. CONCLUSIONS: Tumor-specific T cell responses are strongly impaired in CNI-treated patients but recover after mTOR-i conversion, reducing SCC relapses.

Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation.

The accurate evaluation of donor-specific antibodies (DSAs) has allowed a precise identification of sensitized patients at risk of antibody-mediated rejection (ABMR). However, the scale of the humoral response is not always fully addressed, as it excludes the complete memory B-cell (mBC) pool such as that caused by antigen-specific mBC. Using a novel B-cell ELISpot assay approach, we assessed circulating mBC frequencies against class I and II HLA antigens in highly sensitized and nonsensitized patients in the waiting list for kidney transplantation. Also, kidney transplant patients undergoing ABMR were evaluated for the presence of donor-specific mBCs both at the time of rejection and before transplantation. For this purpose, 278 target HLA-sp antigens from 70 patients were studied and compared to circulating HLA-sp antibodies. Both class I and II HLA-sp mBC frequencies were identified in highly sensitized individuals but not in nonsensitized and healthy individuals, many years after first sensitization. Also, high donor-specific mBC responses were clearly found both during ABMR and before transplantation, regardless of circulating DSA. The higher the donor-specific mBC response, the more aggressive the allograft rejection. Thus, assessing donor-specific mBC frequencies may be relevant to better refine patient alloimmune-risk stratification, and provides new insight into the mechanisms of the adaptive humoral alloimmune response taking place in kidney transplantation.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury.

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte-endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Pre-transplant donor-specific T-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy.

Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.

Preformed frequencies of cytomegalovirus (CMV)-specific memory T and B cells identify protected CMV-sensitized individuals among seronegative kidney transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection remains a major complication after kidney transplantation. Baseline CMV risk is typically determined by the serological presence of preformed CMV-specific immunoglobulin (Ig) G antibodies, even though T-cell responses to major viral antigens are crucial when controlling viral replication. Some IgG-seronegative patients who receive an IgG-seropositive allograft do not develop CMV infection despite not receiving prophylaxis. We hypothesized that a more precise evaluation of pretransplant CMV-specific immune-sensitization using the B and T-cell enzyme-linked immunospot assays may identify CMV-sensitized individuals more accurately, regardless of serological evidence of CMV-specific IgG titers. METHODS: We compared the presence of preformed CMV-specific memory B and T cells in kidney transplant recipients between 43 CMV IgG-seronegative (sR(-)) and 86 CMV IgG-seropositive (sR(+)) patients. Clinical outcome was evaluated in both groups. RESULTS: All sR(+) patients showed a wide range of CMV-specific memory T- and B-cell responses. High memory T- and B-cell frequencies were also clearly detected in 30% of sR(-) patients, and those with high CMV-specific T-cell frequencies had a significantly lower incidence of late CMV infection after prophylactic therapy. Receiver operating characteristic curve analysis for predicting CMV viremia and disease showed a high area under the receiver operating characteristic curve (>0.8), which translated into a high sensitivity and negative predictive value of the test. CONCLUSIONS: Assessment of CMV-specific memory T- and B-cell responses before kidney transplantation among sR(-) recipients may help identify immunized individuals more precisely, being ultimately at lower risk for CMV infection.

Human CMV-specific T-cell responses in kidney transplantation; toward changing current risk-stratification paradigm.

Despite the great efficacy of current antiviral preventive strategies, hCMV infection is still a major complication after renal transplantation, significantly challenging patient and graft survival. This issue seems to be explained because of the rather poor immunologic monitoring of the antiviral immune response. An important body of evidence has shown that monitoring the hCMV-specific T-cell response, at different time points of the transplant setting, seems to add crucial information for predicting the risk of viral infection, thus potentially helping individualization of therapeutic decision-making in clinical transplantation. While several immune-cellular assays have shown its capability for accurately monitoring hCMV-specific T-cell responses, only few such as the IFN-γ ELISPOT and the ELISA based technology assays might be reliable for its application in the clinic. Nonetheless, an important effort has to be made among the transplant community to standardize and validate such immune assays. Noteworthy, large-scale prospective randomized trials are highly warranted to ultimately introduce them in current clinical practice as a part of the highly desired personalized medicine.

Prospective assessment of antidonor cellular alloreactivity is a tool for guidance of immunosuppression in kidney transplantation.

Current characterization of the immune risk in renal transplant patients is only focused on the assessment of preformed circulating alloantibodies; however, alloreactive memory T cells are key players in mediating allograft rejection. Immune monitoring of antidonor alloreactive memory/effector T cells using an IFN-γ Elispot has been shown to distinguish patients at risk for immune-mediated graft dysfunction, suggesting a potential tool for immunosuppression individualization. In this nonrandomized study, we prospectively assessed donor and nondonor T-cell alloreactivity in 60 highly alloreactive patients receiving calcineurin inhibitor-based immunosuppression and in non-T-cell alloreactive transplant recipients treated with a calcineurin inhibitor-free regimen. The impact was evaluated using 1-year allograft outcome. We found a strong association between ongoing antidonor T-cell alloreactivity and histological lesions of acute T cell-mediated rejection in 6-month protocol biopsies, distinguishing those patients with better 1-year graft function, regardless of immunosuppression regimen. Interestingly, evidence for enhanced immune regulation, driven by circulating Foxp3-demethylated regulatory T cells, was only observed among patients achieving antidonor T-cell hyporesponsiveness. Thus, prospective evaluation of donor-specific T-cell sensitization may add crucial information on the alloimmune state of transplanted patients to be used in daily clinical practice.

Costimulatory blockade with mTor inhibition abrogates effector T-cell responses allowing regulatory T-cell survival in renal transplantation.

The advent of novel immunosuppressive strategies in renal transplantation, with immunomodulatory properties, might facilitate long-term allograft survival. T-cell depletion, costimulation-blockade and mTor inhibition have been shown to favour anti-donor hyporesponsiveness. Recently, the combination of rATG, belatacept (Bela) and sirolimus (SRL) has been used in kidney transplantation, showing very low incidence of acute rejection and excellent 12-month graft and patient survival. Herein, we have analysed the 1-year evolution of memory/effector and regulatory T cells and assessed the donor-specific T-cell alloimmune response in a group of these patients and compared with others treated with a calcineurin-inhibitor(CNI)-based (rATG/tacrolimus/MMF), and two other Bela-based regimens (rATG/Bela/MMF and basiliximab/Bela/MMF/steroids). During the first year after transplantation, patients receiving rATG/Bela/SRL had significantly higher percentage of Tregs upon the memory T-cell compartment and showed a potent anti-donor suppressive activity. In an in vitro naive and memory/effector T-cell co-culture, the combination of costimulation-blockade and SRL could abrogate both antigen-specific T-cell responses as efficiently as using a CNI drug. The combination of T-cell depletion, costimulation-blockade and mTor inhibition seems to be able to allow Treg survival and inhibit donor-specific alloreactive effector immune responses after kidney transplantation in humans.