Tissue-resident Lymphocytes Are Released During Hypothermic and Normothermic Machine Perfusion of Human Donor Kidneys.

BACKGROUND: Machine perfusion is the preferred preservation method for deceased donor kidneys. Perfusate fluid, which contains a complex mixture of components, offers potential insight into the organ's viability and function. This study explored immune cell release, particularly tissue-resident lymphocytes (TRLs), during donor kidney machine perfusion and its correlation with injury markers. METHODS: Perfusate samples from hypothermic machine perfusion (HMP; n = 26) and normothermic machine perfusion (NMP; n = 16) of human donor kidneys were analyzed for TRLs using flow cytometry. Residency was defined by expressions of CD69, CD103, and CD49as. TRL release was quantified exclusively in NMP. Additionally, levels of cell-free DNA, neutrophil gelatinase-associated lipocalin, and soluble E-cadherin (sE-cadherin) were measured in NMP supernatants with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Both HMP and NMP samples contained a heterogeneous population of TRLs, including CD4+ tissue-resident memory T cells, CD8+ tissue-resident memory T cells, tissue-resident natural killer cells, tissue-resident natural killer T cells, and helper-like innate lymphoid cells. Median TRL proportions among total CD45+ lymphocytes were 0.89% (NMP) and 0.84% (HMP). TRL quantities in NMP did not correlate with donor characteristics, perfusion parameters, posttransplant outcomes, or cell-free DNA and neutrophil gelatinase-associated lipocalin concentrations. However, CD103+ TRL release positively correlated with the release of sE-cadherin, the ligand for the CD103 integrin. CONCLUSIONS: Human donor kidneys release TRLs during both HMP and NMP. The release of CD103+ TRLs was associated with the loss of their ligand sE-cadherin but not with general transplant injury biomarkers.

Tissue-resident memory T cells in human kidney transplants have alloreactive potential.

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.

Virus-specific TRM cells of both donor and recipient origin reside in human kidney transplants.

Tissue-resident lymphocytes (TRLs) are critical for local protection against viral pathogens in peripheral tissue. However, it is unclear if TRLs perform a similar role in transplanted organs under chronic immunosuppressed conditions. In this study, we aimed to characterize the TRL compartment in human kidney transplant nephrectomies and examine its potential role in antiviral immunity. The TRL compartment of kidney transplants contained diverse innate, innate-like, and adaptive TRL populations expressing the canonical residency markers CD69, CD103, and CD49a. Chimerism of donor and recipient cells was present in 43% of kidney transplants and occurred in all TRL subpopulations. Paired single-cell transcriptome and T cell receptor (TCR) sequencing showed that donor and recipient tissue-resident memory T (TRM) cells exhibit striking similarities in their transcriptomic profiles and share numerous TCR clonotypes predicted to target viral pathogens. Virus dextramer staining further confirmed that CD8 TRM cells of both donor and recipient origin express TCRs with specificities against common viruses, including CMV, EBV, BK polyomavirus, and influenza A. Overall, the study results demonstrate that a diverse population of TRLs resides in kidney transplants and offer compelling evidence that TRM cells of both donor and recipient origin reside within this TRL population and may contribute to local protection against viral pathogens.

Th1-dominant cytokine responses in kidney patients after COVID-19 vaccination are associated with poor humoral responses.

Cytokines are regulators of the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the contribution of cytokine-secreting CD4+ and CD8+ memory T cells to the SARS-CoV-2-specific humoral immune response in immunocompromised kidney patients is unknown. Here, we profiled 12 cytokines after stimulation of whole blood obtained 28 days post second 100 µg mRNA-1273 vaccination with peptides covering the SARS-CoV-2 spike (S)-protein from patients with chronic kidney disease (CKD) stage 4/5, on dialysis, kidney transplant recipients (KTR), and healthy controls. Unsupervised hierarchical clustering analysis revealed two distinct vaccine-induced cytokine profiles. The first profile was characterized by high levels of T-helper (Th)1 (IL-2, TNF-α, and IFN-γ) and Th2 (IL-4, IL-5, IL-13) cytokines, and low levels of Th17 (IL-17A, IL-22) and Th9 (IL-9) cytokines. This cluster was dominated by patients with CKD, on dialysis, and healthy controls. In contrast, the second cytokine profile contained predominantly KTRs producing mainly Th1 cytokines upon re-stimulation, with lower levels or absence of Th2, Th17, and Th9 cytokines. Multivariate analyses indicated that a balanced memory T cell response with the production of Th1 and Th2 cytokines was associated with high levels of S1-specific binding and neutralizing antibodies mainly at 6 months after second vaccination. In conclusion, seroconversion is associated with the balanced production of cytokines by memory T cells. This emphasizes the importance of measuring multiple T cell cytokines to understand their influence on seroconversion and potentially gain more information about the protection induced by vaccine-induced memory T cells.

P-glycoprotein, FK-binding Protein-12, and the Intracellular Tacrolimus Concentration in T-lymphocytes and Monocytes of Kidney Transplant Recipients.

BACKGROUND: . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C 0 ). The intra-immune cellular concentration is potentially a better target than C 0 . However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp). METHODS: . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus. RESULTS: . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5-33.4] versus 131.0 [73.5-225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2-1.7] versus 1.2 [1.1-1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2-1.7] versus 1.5 [1.4-2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells. CONCLUSIONS: . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement.

Tacrolimus Monotherapy is Safe in Immunologically Low-Risk Kidney Transplant Recipients: A Randomized-Controlled Pilot Study.

In this randomized-controlled pilot study, the feasibility and safety of tacrolimus monotherapy in immunologically low-risk kidney transplant recipients was evaluated [NTR4824, www.trialregister.nl]. Low immunological risk was defined as maximal 3 HLA mismatches and the absence of panel reactive antibodies. Six months after transplantation, recipients were randomized if eGFR >30 ml/min, proteinuria <50 mg protein/mmol creatinine, no biopsy-proven rejection after 3 months, and no lymphocyte depleting therapy given. Recipients were randomized to tacrolimus/mycophenolate mofetil (TAC/MMF) or to taper and discontinue MMF at month 9 (TACmono). 79 of the 121 recipients were randomized to either TACmono (n = 38) or TAC/MMF (n = 41). Mean recipient age was 59 years and 59% received a living donor transplant. The median follow-up was 62 months. After randomization, 3 TACmono and 4 TAC/MMF recipients experienced a biopsy-proven rejection. At 5 years follow-up, patient survival was 84% in TACmono versus 76% in TAC/MMF with death-censored graft survival of 97% for both groups and no differences in eGFR and proteinuria. Eleven TACmono recipients had an infectious episode versus 22 TAC/MMF recipients (p < 0.03). Donor-specific anti-HLA antibodies were not detected during follow-up in both groups. Tacrolimus monotherapy in selected immunologically low-risk kidney transplant recipients appears safe and reduces the number of infections.

High Tacrolimus Intrapatient Variability and Subtherapeutic Immunosuppression are Associated With Adverse Kidney Transplant Outcomes.

BACKGROUND: Kidney transplant recipients with high intrapatient variability (IPV) in tacrolimus (Tac) exposure experience more rejection and reduced graft survival. To understand the underlying pathophysiology of this association, the authors investigated whether patients with high tacrolimus IPV have a more activated immune system than patients with low IPV. In addition, exposure to tacrolimus and mycophenolic acid (MPA) was studied in relation to rejection and graft survival. METHODS: At the time of patient inclusion (5-7 years post-transplantation), the frequency of donor-reactive cells was determined by enzyme-linked immunosorbent assay, and the development of donor-specific anti-Human Leukocyte Antigen antibodies (DSA) was measured by Luminex Single Antigen assay. Tacrolimus IPV was retrospectively calculated between 6 and 12 months and the exposure to tacrolimus and MPA was determined between 1 and 5 years post-transplantation. RESULTS: A total of 371 kidney transplant recipients were included in this study, of whom 56 developed a rejection episode after 12 months and 60 experienced graft failure after 5-7 years. No correlations were found between tacrolimus IPV or immunosuppression exposure and the number of donor-reactive cells after 5 years of transplantation. DSA were detected more often in patients with low exposure to both tacrolimus and MMF [4/21 (19%) versus 17/350 (4.9%), P = 0.04]. In this cohort, neither tacrolimus IPV nor low overall immunosuppression exposure was associated with a higher incidence of rejection. However, regression analysis showed that a higher tacrolimus IPV was associated with an increased incidence of graft failure (odds ratio = 1.03, P = 0.02). CONCLUSIONS: This study verifies the relationship between high tacrolimus IPV and impaired kidney allograft survival in long-term follow-up. DSA was also found to be more prevalent in patients with subtherapeutic concentrations of tacrolimus and MPA. An increased prevalence of donor-specific alloreactivity is yet to be demonstrated in patients with high IPV.

Association Between the Intracellular Tacrolimus Concentration in CD3 + T Lymphocytes and CD14 + Monocytes and Acute Kidney Transplant Rejection.

BACKGROUND: Intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) (TAC [PBMC] ) has been proposed to better represent its active concentration than its whole blood concentration. As tacrolimus acts on T lymphocytes and other white blood cells, including monocytes, we investigated the association of tacrolimus concentration in CD3 + T lymphocytes (TAC [CD3] ) and CD14 + monocytes (TAC [CD14] ) with acute rejection after kidney transplantation. METHODS: From a total of 61 samples in this case-control study, 28 samples were obtained during biopsy-proven acute rejection (rejection group), and 33 samples were obtained in the absence of rejection (control group). PBMCs were collected from both cryopreserved (retrospectively) and freshly obtained (prospectively) samples. CD3 + T lymphocytes and CD14 + monocytes were isolated from PBMCs, and their intracellular tacrolimus concentrations were measured. RESULTS: The correlation between tacrolimus whole-blood and intracellular concentrations was poor. TAC [CD3] was significantly lower than TAC [CD14] (median 12.8 versus 81.6 pg/million cells; P < 0.001). No difference in TAC [PBMC] (48.5 versus 44.4 pg/million cells; P = 0.82), TAC [CD3] (13.4 versus 12.5 pg/million cells; P = 0.28), and TAC [CD14] (90.0 versus 72.8 pg/million cells; P = 0.27) was found between the rejection and control groups. However, freshly isolated PBMCs showed significantly higher TAC [PBMC] than PBMCs from cryopreserved samples. Subgroup analysis of intracellular tacrolimus concentrations from freshly isolated cells did not show a difference between rejectors and nonrejectors. CONCLUSIONS: Differences in TAC [CD3] and TAC [CD14] between patients with and without rejection could not be demonstrated. However, further optimization of the cell isolation process is required because a difference in TAC [PBMC] between fresh and cryopreserved cells was observed. These results need to be confirmed in a study with a larger number of patients.

Monitoring intracellular tacrolimus concentrations and its relationship with rejection in the early phase after renal transplantation.

INTRODUCTION: After kidney transplantation, rejection and drug-related toxicity occur despite tacrolimus whole-blood pre-dose concentrations ([Tac]blood) being within the target range. The tacrolimus concentration within peripheral blood mononuclear cells ([Tac]cells) might correlate better with clinical outcomes. The aim of this study was to investigate the correlation between [Tac]blood and [Tac]cells, the evolution of [Tac]cells and the [Tac]cells/[Tac]blood ratio, and to assess the relationship between tacrolimus concentrations and the occurrence of rejection. METHODS: In this prospective study, samples for the measurement of [Tac]blood and [Tac]cells were collected on days 3 and 10 after kidney transplantation, and on the morning of a for-cause kidney transplant biopsy. Biopsies were reviewed according to the Banff 2019 update. RESULTS: Eighty-three [Tac]cells samples were measured of 44 kidney transplant recipients. The correlation between [Tac]cells and [Tac]blood was poor (Pearson's r = 0.56 (day 3); r = 0.20 (day 10)). Both the dose-corrected [Tac]cells and the [Tac]cells/[Tac]blood ratio were not significantly different between days 3 and 10, and the median inter-occasion variability of the dose-corrected [Tac]cells and the [Tac]cells/[Tac]blood ratio were 19.4% and 23.4%, respectively (n = 24). Neither [Tac]cells, [Tac]blood, nor the [Tac]cells/[Tac]blood ratio were significantly different between patients with biopsy-proven acute rejection (n = 4) and patients with acute tubular necrosis (n = 4) or a cancelled biopsy (n = 9; p > 0.05). CONCLUSION: Tacrolimus exposure and distribution appeared stable in the early phase after transplantation. [Tac]cells was not significantly associated with the occurrence of rejection. A possible explanation for these results might be related to the low number of patients included in this study and also due to the fact that PBMCs are not a specific enough matrix to monitor tacrolimus concentrations.

Natural Antibodies and Alloreactive T Cells Long after Kidney Transplantation.

BACKGROUND: The relationship between circulating effector memory T and B cells long after transplantation and their susceptibility to immunosuppression are unknown. To investigate the impact of antirejection therapy on T cell-B cell coordinated immune responses, we assessed IFN-γ-producing memory cells and natural antibodies (nAbs) that potentially bind to autoantigens on the graft. METHODS: Plasma levels of IgG nAbs to malondialdehyde (MDA) were measured in 145 kidney transplant recipients at 5-7 years after transplantation. In 54 of these patients, the number of donor-reactive IFN-γ-producing cells was determined. 35/145 patients experienced rejection, 18 of which occurred within 1 year after transplantation. RESULTS: The number of donor-reactive IFN-γ-producing cells and the levels of nAbs were comparable between rejectors and nonrejectors. The nAbs levels were positively correlated with the number of donor-reactive IFN-γ-producing cells (r s = 0.39, p=0.004). The positive correlation was only observed in rejectors (r s = 0.53, p=0.003; nonrejectors: r s = 0.24, p=0.23). Moreover, we observed that intravenous immune globulin treatment affected the level of nAbs and this effect was found in patients who experienced a late ca-ABMR compared to nonrejectors (p=0.008). CONCLUSION: The positive correlation found between alloreactive T cells and nAbs in rejectors suggests an intricate role for both components of the immune response in the rejection process. Treatment with intravenous immune globulin impacted nAbs.

Immunosuppression Has Long-Lasting Effects on Circulating Follicular Regulatory T Cells in Kidney Transplant Recipients.

Background: FoxP3+ follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector immune responses against transplanted tissue. Methods: To understand the biology of Tfr cells in kidney transplant patients treated with tacrolimus and mycophenolate mofetil (MMF) combination immunosuppression, we measured circulating (c)Tfh and cTfr cells in peripheral blood by flow cytometry in n = 211 kidney transplant recipients. At the time of measurement patients were 5-7 years after transplantation. Of this cohort of patients, 23.2% (49/211) had been previously treated for rejection. Median time after anti-rejection therapy was 4.9 years (range 0.4-7 years). Age and gender matched healthy individuals served as controls. Results: While the absolute numbers of cTfh cells were comparable between kidney transplant recipients and healthy controls, the numbers of cTfr cells were 46% lower in immunosuppressed recipients (p < 0.001). More importantly, in transplanted patients, the ratio of cTfr to cTfh was decreased (median; 0.10 vs. 0.06), indicating a disruption of the balance between cTfr and cTfh cells. This shifted balance was observed for both non-rejectors and rejectors. Previous pulse methylprednisolone or combined pulse methylprednisolone + intravenous immunoglobulin anti-rejection therapy led to a non-significant 30.6% (median) and 51.2% (median) drop in cTfr cells, respectively when compared to cTfr cell numbers in transplant patients who did not receive anti-rejection therapy. A history of alemtuzumab therapy did lead to a significant decrease in cTfr cells of 85.8% (median) compared with patients not treated with anti-rejection therapy (p < 0.0001). No association with tacrolimus or MMF pre-dose concentrations was found. Conclusion: This cross-sectional study reveals that anti-rejection therapy with alemtuzumab significantly lowers the number of cTfr cells in kidney transplant recipients. The observed profound effects by these agents might dysregulate cTfr functions.

Inhibition of T Helper Cell Differentiation by Tacrolimus or Sirolimus Results in Reduced B-Cell Activation: Effects on T Follicular Helper Cells.

The effect of immunosuppressive drugs on the generation of T follicular helper (Tfh) cells, specialized in supporting B-cell differentiation, is largely unknown. We examined whether the calcineurin inhibitor tacrolimus (TAC) and the mammalian target of rapamycin (mtor) inhibitor sirolimus (SRL) inhibit Tfh cell differentiation, and affect subsequent B-cell functions. Isolated naive T cells were polarized into Tfh-like cells in the presence of TAC or SRL. To demonstrate their functionality, we co-cultured these cells with isolated B cells in the presence of alloantigen and studied the activation and differentiation of these B cells. Tfh-like cells were defined as CD4+CXCR5+ T cells, expressing immunoinhibitory programmed death protein 1 (pd1) and inducible T-cell costimulator (icos). We found that TAC and SRL significantly inhibited Tfh-like cell differentiation. Therapeutic concentrations of TAC and SRL reduced the percentage of pd1+ and icos+ Tfh cells compared to controls. In addition, T cells grown in the presence of TAC or SRL expressed less IL-21 and provided less B-cell help. TAC and SRL both inhibited Tfh-dependent alloantigen-activated B-cell proliferation and differentiation into plasma cells and transitional B cells. In conclusion, TAC and SRL inhibited the differentiation of naive T cells into functional Tfh-like cells, a finding that can be extrapolated to immunosuppressive regimens in transplant patients.

The Effects of an IL-21 Receptor Antagonist on the Alloimmune Response in a Humanized Mouse Skin Transplant Model.

BACKGROUND: Interleukin 21 (IL-21) is involved in regulating the expansion and effector function of a broad range of leukocytes, including T cells and B cells. In transplantation, the exact role of IL-21 in the process of allograft rejection is unknown. To further explore this, the aim of this study is to test the effect of an IL-21 receptor (IL-21R) blocking antibody on the early phase of allograft rejection in a humanized skin transplantation model in mice reconstituted with human T and B cells. METHODS: Immunodeficient Balb/c IL2rγRag2 mice were transplanted with human skin followed by adoptive transfer of human allogeneic splenocytes. Control animals were treated with a phosphate buffered saline vehicle while the other group was treated with a humanized anti-IL-21R antibody (αIL-21R). RESULTS: In the phosphate buffered saline-treated animals, human skin allografts were infiltrated with lymphocytes and developed a thickened epidermis with increased expression of the inflammatory markers Keratin 17 (Ker17) and Ki67. In mice treated with αIL-21R, these signs of allograft reactivity were significantly reduced. Concordantly, STAT3 phosphorylation was inhibited in this group. Of note, treatment with αIL-21R attenuated the process of T and B cell reconstitution after adoptive cellular transfer. CONCLUSIONS: These findings demonstrate that blockade of IL-21 signaling can delay allograft rejection in a humanized skin transplantation model.

Characterization of donor and recipient CD8+ tissue-resident memory T cells in transplant nephrectomies.

Tissue-resident memory T (TRM) cells are characterized by their surface expression of CD69 and can be subdivided in CD103+ and CD103- TRM cells. The origin and functional characteristics of TRM cells in the renal allograft are largely unknown. To determine these features we studied TRM cells in transplant nephrectomies. TRM cells with a CD103+ and CD103- phenotype were present in all samples (n = 13) and were mainly CD8+ T cells. Of note, donor-derived TRM cells were only detectable in renal allografts that failed in the first month after transplantation. Grafts, which failed later, mainly contained recipient derived TRM cells. The gene expression profiles of the recipient derived CD8+ TRM cells were studied in more detail and showed a previously described signature of tissue residence within both CD103+ and CD103- TRM cells. All CD8+ TRM cells had strong effector abilities through the production of IFNγ and TNFα, and harboured high levels of intracellular granzyme B and low levels of perforin. In conclusion, our results demonstrate that donor and recipient TRM cells reside in the rejected renal allograft. Over time, the donor-derived TRM cells are replaced by recipient TRM cells which have features that enables these cells to aggressively respond to the allograft.

The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection.

Interleukin (IL)-21 supports induction and expansion of CD8+ T cells, and can also regulate the differentiation of B cells into antibody-producing plasma cells. We questioned whether the number of circulating donor-specific IL-21 producing cells (pc) can predict kidney transplant rejection, and evaluated this in two different patient cohorts. The first analysis was done on pre-transplantation samples of 35 kidney transplant recipients of whom 15 patients developed an early acute rejection. The second study concerned peripheral blood mononuclear cell (PBMC) samples from 46 patients obtained at 6 months after kidney transplantation of whom 13 developed late rejection. Significantly higher frequencies of donor-specific IL-21 pc were found by Elispot assay in both patients who developed early and late rejection compared to those without rejection. In addition, low frequencies of donor-specific IL-21 pc were associated with higher rejection-free survival. Moreover, low pre-transplant donor-specific IL-21 pc numbers were associated with the absence of anti-HLA antibodies. Donor-reactive IL-21 was mainly produced by CD4+ T cells, including CD4+ follicular T helper cells. In conclusion, the number of donor-specific IL-21 pc is associated with an increased risk of both early and late rejection, giving it the potential to be a new biomarker in kidney transplantation.

Analysis of NFATc1 amplification in T cells for pharmacodynamic monitoring of tacrolimus in kidney transplant recipients.

BACKGROUND: Therapeutic drug monitoring (TDM) of tacrolimus, based on blood concentrations, shows an imperfect correlation with the occurrence of rejection. Here, we tested whether measuring NFATc1 amplification, a member of the calcineurin pathway, is suitable for TDM of tacrolimus. MATERIALS AND METHODS: NFATc1 amplification was monitored in T cells of kidney transplant recipients who received either tacrolimus- (n = 11) or belatacept-based (n = 10) therapy. Individual drug effects on NFATc1 amplification were studied in vitro, after spiking blood samples of healthy volunteers with either tacrolimus, belatacept or mycophenolate mofetil. RESULTS: At day 30 after transplantation, in tacrolimus-treated patients, NFATc1 amplification was inhibited in CD4+ T cells expressing the co-stimulation receptor CD28 (mean inhibition 37%; p = 0.01) and in CD8+CD28+ T cells (29% inhibition; p = 0.02), while this was not observed in CD8+CD28- T cells or belatacept-treated patients. Tacrolimus pre-dose concentrations of these patients correlated inversely with NFATc1 amplification in CD28+ T cells (rs = -0.46; p < 0.01). In vitro experiments revealed that 50 ng/ml tacrolimus affected NFATc1 amplification by 58% (mean; p = 0.02). CONCLUSION: In conclusion, measuring NFATc1 amplification is a direct tool for monitoring biological effects of tacrolimus on T cells in whole blood samples of kidney transplant recipients. This technique has potential that requires further development before it can be applied in daily practice.

Differential T Cell Signaling Pathway Activation by Tacrolimus and Belatacept after Kidney Transplantation: Post Hoc Analysis of a Randomised-Controlled Trial.

Pharmacokinetic immunosuppressive drug monitoring poorly correlates with clinical outcomes after solid organ transplantation. A promising method for pharmacodynamic monitoring of tacrolimus (TAC) in T cell subsets of transplant recipients might be the measurement of (phosphorylated) p38MAPK, ERK1/2 and Akt (activated downstream of the T cell receptor) by phospho-specific flow cytometry. Here, blood samples from n = 40 kidney transplant recipients (treated with either TAC-based or belatacept (BELA)-based immunosuppressive drug therapy) were monitored before and throughout the first year after transplantation. After transplantation and in unstimulated samples, p-p38MAPK and p-Akt were inhibited in CD8+ T cells and p-ERK in CD4+ T cells but only in patients who received TAC-based therapy. After activation with PMA/ionomycin, p-p38MAPK and p-AKT were significantly inhibited in CD4+ and CD8+ T cells when TAC was given, compared to pre-transplantation. Eleven BELA-treated patients had a biopsy-proven acute rejection, which was associated with higher p-ERK levels in both CD4+ and CD8+ T cells compared to patients without rejection. In conclusion, phospho-specific flow cytometry is a promising tool to pharmacodynamically monitor TAC-based therapy. In contrast to TAC-based therapy, BELA-based immunosuppression does not inhibit key T cell activation pathways which may contribute to the high rejection incidence among BELA-treated transplant recipients.

Pharmacodynamic Monitoring of Tacrolimus-Based Immunosuppression in CD14+ Monocytes After Kidney Transplantation.

BACKGROUND: Monocytes significantly contribute to ischemia-reperfusion injury and allograft rejection after kidney transplantation. However, the knowledge about the effects of immunosuppressive drugs on monocyte activation is limited. Conventional pharmacokinetic methods for immunosuppressive drug monitoring are not cell type-specific. In this study, phosphorylation of 3 signaling proteins was measured to determine the pharmacodynamic effects of immunosuppression on monocyte activation in kidney transplant patients. METHODS: Blood samples from 20 kidney transplant recipients were monitored before and during the first year after transplantation. All patients received induction therapy with basiliximab, followed by tacrolimus (TAC), mycophenolate mofetil, and prednisolone maintenance therapy. TAC whole-blood predose concentrations were determined using an antibody-conjugated magnetic immunoassay. Samples were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and phosphorylation of p38MAPK, ERK, and Akt in CD14 monocytes was quantified by phospho-specific flow cytometry. RESULTS: Phosphorylation of p38MAPK and Akt in monocytes of immunosuppressed recipients was lower after 360 days compared with before transplantation in the unstimulated samples [mean reduction in median fluorescence intensity 36%; range -28% to 77% for p-p38MAPK and 20%; range -22% to 53% for p-Akt; P < 0.05]. P-ERK was only decreased at day 4 after transplantation (mean inhibition 23%; range -52% to 73%; P < 0.05). At day 4, when the highest whole-blood predose TAC concentrations were measured, p-p38MAPK and p-Akt, but not p-ERK, correlated inversely with TAC (rs = -0.65; P = 0.01 and rs = -0.58; P = 0.03, respectively). CONCLUSIONS: Immunosuppressive drug combination therapy partially inhibits monocyte activation pathways after kidney transplantation. This inhibition can be determined by phospho-specific flow cytometry, which enables the assessment of the pharmacodynamic effects of immunosuppressive drugs in a cell type-specific manner.