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The intracellular tacrolimus concentration in CD3+ T lymphocytes is proposed to be a better representative of the active component of tacrolimus than the whole blood concentration. However, intracellular measurements are complicated. Therefore, the aim of this study was to describe the relationship between intracellular and whole blood tacrolimus concentrations in a population pharmacokinetic model. Twenty‐eight de novo kidney transplant recipients, treated with a once‐daily oral extended‐release tacrolimus formulation, were followed during the first‐month post‐transplantation. Additional whole blood and intracellular tacrolimus concentrations were measured at day 6 ± 1 (pre‐dose, 4 and 8 hours post‐dose) and day 14 ± 3 (pre‐dose) post‐transplantation. Pharmacokinetic analysis was performed using nonlinear mixed effects modeling software (NONMEM). The ratio between intracellular (n = 109) and whole blood (n = 248) concentrations was best described by a two‐compartment whole blood model with an additional intracellular compartment without mass transfer from the central compartment. The ratio remained stable over time. Prednisolone dose influenced the absorption rate of tacrolimus, while hemoglobin, CYP3A4*22 allele carrier, and CYP3A5 expresser status were associated with the oral clearance of tacrolimus (P‐value < 0.001). Furthermore, the intracellular tacrolimus concentrations were correlated with the intracellular production of interleukin‐2 (P‐value 0.015). The intracellular tacrolimus concentration can be predicted from a measured whole blood concentration using this model, without the need for repeated intracellular measurements. This knowledge is particularly important when the intracellular concentration is ready to be implemented into clinical practice, to overcome the complexities of cell isolation and analytical methods. [ABSTRACT FROM AUTHOR]

This article discusses the prevalence and clinical implications of endothelial cell (EC) chimerism in kidney transplants. EC chimerism refers to the presence of both donor and recipient ECs in the transplanted kidney. The study presents an improved method using flow cytometry to accurately detect and quantify EC chimerism. The researchers found that EC chimerism was present in 58% of the kidney transplant samples, with the proportion of recipient-derived cells varying. They also observed an association between the time elapsed since transplantation and the presence of EC chimerism, as well as a higher prevalence of EC chimerism in younger recipients. The study suggests that recipients with high levels of EC chimerism may require less immunosuppressive medication. Further research is needed to explore the dynamics of EC chimerism and its impact on graft outcomes. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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 >30ml/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. [ABSTRACT FROM AUTHOR]

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 (rs = 0.39, p = 0.004). The positive correlation was only observed in rejectors (rs = 0.53, p = 0.003 ; nonrejectors: rs = 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Humoral alloreactivity has been recognized as a common cause of kidney transplant dysfunction. B-cell activation, differentiation, and antibody production are dependent on IL-21+CXCR5+follicular T-helper (Tfh) cells. Here, we studied whether belatacept, an inhibitor of the costimulatory CD28-CD80/86-pathway, interrupts the crosstalk between Tfh- and B-cells more efficiently than the calcineurin inhibitor tacrolimus. The suppressive effects of belatacept and tacrolimus on donor antigen-driven Tfh-B-cell interaction were functionally studied in peripheral blood mononuclear cells from 40 kidney transplant patients randomized to a belatacept- or tacrolimus-based immunosuppressive regimen. No significant differences in uncultured cells or donor antigen-stimulated cells were found between belatacept- and tacrolimus-treated patients in the CXCR5+Tfh cell generation and activation (upregulation of PD-1). Belatacept and tacrolimus in vitro minimally inhibited Tfh-cell generation (by ~6-7%) and partially prevented Tfh-cell activation (by ~30-50%). The proportion of IL-21+-activated Tfh-cells was partially decreased by in vitro addition of belatacept or tacrolimus (by ~60%). Baseline expressions and proportions of activated CD86+ B-cells, plasmablasts, and transitional B-cells after donor antigen stimulation did not differ between belatacept- and tacrolimus-treated patients. Donor antigen-driven CD86 upregulation on memory B-cells was not fully prevented by adding belatacept in vitro (~35%), even in supratherapeutic doses. In contrast to tacrolimus, belatacept failed to inhibit donor antigen-driven plasmablast formation (~50% inhibition vs. no inhibition, respectively, p < 0.0001). In summary, donor antigen-driven Tfh-B-cell crosstalk is similar in cells obtained from belatacept- and tacrolimus-treated patients. Belatacept is, however, less potent in vitro than tacrolimus in inhibiting Tfh-cell-dependent plasmablast formation. [ABSTRACT FROM AUTHOR]

Interaction between T follicular helper (Tfh) cells and B cells is complex and involves various pathways, including the production of IL-21 by the Tfh cells. Secretion of IL-21 results in B cell differentiation toward immunoglobulin-producing plasmablasts. In patients after kidney transplantation, the formation of alloantibodies produced by donor antigen-activated B cells are a major cause of organ failure. In this allogeneic response, the role of IL-21-producing Tfh cells that regulate B cell differentiation is unknown. Here, we tested, in an alloantigen-driven setting, whether Tfh cell help signals control B cell differentiation with its dependency on IL-21. Pre-transplantation patient PBMCs were sorted into pure CD4posCXCR5pos Tfh cells and CD19posCD27pos memory B cells and stimulated with donor antigen in the presence or absence of an IL-21 receptor (IL-21R) antagonist (αIL-21R). Donor antigen stimulation initiated expression of the activation markers inducible co-stimulator (ICOS) and programmed death 1 (PD-1) on Tfh cells and a shift toward a mixed Tfh2 and Tfh17 phenotype. The memory B cells underwent class switch recombination and differentiated toward IgM- and IgG-producing plasmablasts. In the presence of αIL-21R, a dose-dependent inhibition of STAT3 phosphorylation was measured in both T and B cells. Blockade of the IL-21R did not have an effect on PD-1 and ICOS expression on Tfh cells but significantly inhibited B cell differentiation. The proportion of plasmablasts decreased by 78% in the presence of αIL-21R. Moreover, secreted IgM and IgG2 levels were significantly lower in the presence ofα L-21R. In conclusion, our results demonstrate that IL-21 produced by alloantigen-activated Tfh cells controls B cell differentiation toward antibody producing plasmablasts. The IL-21R might, therefore, be a useful target in organ transplantation to prevent antigen-driven immune responses leading to graft failure. [ABSTRACT FROM AUTHOR]

Monocytes and macrophages play key roles in many disease states, including cellular and humoral rejection after solid organ transplantation (SOT). To suppress alloimmunity after SOT, immunosuppressive drug therapy is necessary. However, little is known about the effects of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA) on monocyte activation and function. Here, the effect of these immunosuppressants on monocytes was investigated by measuring phosphorylation of three intracellular signaling proteins which all have a major role in monocyte function: p38MAPK, ERK and Akt. In addition, biological functions downstream of these signaling pathways were studied, including cytokine production, phagocytosis and differentiation into macrophages. To this end, blood samples from healthy volunteers were spiked with diverse concentrations of tacrolimus and MPA. Tacrolimus (200 ng/ml) inhibited phosphorylation of p38MAPK by 30% (mean) in CD14+ monocytes which was significantly less than in activated CD3+ T cells (max 60%; p < 0.05). This immunosuppressive agent also partly inhibited p-AKT (14%). MPA, at a therapeutic concentration showed the strongest effect on p-AKT (27% inhibition). p-ERK was inhibited with a maximum of 15% after spiking with either tacrolimus or MPA. The production of IL-1β and phagocytosis by monocytes were not affected by tacrolimus concentrations, whereas MPA did inhibit IL-1β production by 50%. Monocyte/macrophage polarization was shifted to an M2-like phenotype in the presence of tacrolimus, while MPA increased the expression of M2 surface markers, including CD163 and CD200R, on M1 macrophages. These results show that tacrolimus and MPA do not strongly affect monocyte function, apart from a change in macrophage polarization, to a clinically relevant degree. [ABSTRACT FROM AUTHOR]

Background: The co-stimulatory inhibitor of the CD28-CD80/86-pathway, belatacept, allows calcineurin-inhibitor-free immunosuppression in kidney transplantation. However, aggressive T-cell mediated allogeneic responses have been observed in belatacept-treated patients, which could be explained by effector-memory T-cells that lack membrane expression of CD28, i.e. CD28-negative (CD28NULL) T-cells. CD28-positive (CD28POS) T-cells that down regulate their surface CD28 after allogeneic stimulation could also pose a threat against the renal graft. The aim of this study was to investigate this potential escape mechanism for CD28POS T-cells under belatacept treatment. Materials & Methods: PBMCs, isolated T-cell memory subsets and isolated CD28POS T-cells were obtained from end-stage renal disease (ESRD) patients and co-cultured with allo-antigen in the presence of belatacept to mimic allogeneic reactions in kidney-transplant patients under belatacept treatment. As a control, IgG was used in the absence of belatacept. Results: Despite high in vitro belatacept concentrations, a residual T-cell growth of ±30% was observed compared to the IgG control after allogeneic stimulation. Of the alloreactive T-cells, the majority expressed an effector-memory phenotype. This predominance for effector-memory T-cells within the proliferated cells was even larger when a higher dose of belatacept was added. Contrary to isolated naïve and central-memory T cells, isolated effector-memory T cells could not be inhibited by belatacept in differentiation or allogeneic IFNγ production. The proportion of CD28-positive T cells was lower within the proliferated T cell population, but was still substantial. A fair number of the isolated initially CD28POS T-cells differentiated into CD28NULL T-cells, which made them not targetable by belatacept. These induced CD28NULL T-cells were not anergic as they produced high amounts of IFNγ upon allogeneic stimulation. The majority of the proliferated isolated originally CD28POS T-cells, however, still expressed CD28 and also expressed IFNγ. Conclusion: This study provides evidence that, apart from CD28NULL T-cells, also CD28POS, mostly effector-memory T-cells can mediate allogeneic responses despite belatacept treatment. [ABSTRACT FROM AUTHOR]