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Background. Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C0). The intra-immune cellular concentration is potentially a better target than C0. 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.

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.

Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C0). The intra-immune cellular concentration is potentially a better target than C0. 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).. 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.. 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; P0.001). 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.. 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.

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.

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.

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.

Summary Blockade of the CD80/86-CD28 pathway by belatacept after kidney transplantation is associated with an increased risk of rejection compared with standard, calcineurin inhibitor (CNI)-based therapy. CD28− T cells, which express CD57, are not susceptible to belatacept treatment. High numbers of CD4+CD57+programmed death 1 (PD-1)− T cells pretransplantation have been associated with a higher chance of rejection, although conflicting data have been reported. To investigate the working mechanism behind this possible higher chance of rejection, we studied the expression of co-inhibitory molecules (CD223, CD244 and PD-1), proliferative capacity and cytotoxic potential of fluorescence activated cell sorted (FACS) CD4+CD57+PD-1− and CD8+CD57+PD-1− T cells, and their CD57− control populations, after alloantigen stimulation. The effect of belatacept on the cytotoxic capacity of pretransplantation peripheral blood mononuclear cells from 20 patients who received belatacept post-transplantation was also tested. Expression of co-inhibitory molecule CD223 increased by approximately 10-fold after allogeneic stimulation in all four T cell subsets. Proliferation and up-regulation of CD244 and PD-1 was observed for CD4+CD57−PD-1− T cells after allogeneic stimulation, but no up-regulation of these markers occurred on CD8+ T cells or CD4+CD57+PD-1− T cells. However, CD4+CD57+PD-1− T cells and, to a lesser extent, CD8+CD57+PD-1− T cells displayed higher cytotoxicity as indicated by granzyme B expression. Belatacept inhibited the cytotoxic potential of CD4+CD57+PD-1− T cells (median of inhibition 31%, P < 0·01) and CD8+CD57+PD-1− T cells (median of inhibition 10%, P < 0·05). In conclusion, alloantigen-activated CD4+CD57+PD-1− T cells exhibited a less proliferative but more cytotoxic profile than their CD57− counterparts. Their cytotoxic capacity can be inhibited partly by belatacept and was not associated with development of rejection 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.

Background. Belatacept, an inhibitor of the CD28-CD80/86 costimulatory pathway, allows for calcineurin-inhibitor free immunosuppressive therapy in kidney transplantation but is associated with a higher acute rejection risk than ciclosporin. Thus far, no biomarker for belatacept-resistant rejection has been validated. In this randomized-controlled trial, acute rejection rate was compared between belatacept-and tacrolimus-treated patients and immunological biomarkers for acute rejection were investigated. Methods. Forty kidney transplant recipients were 1:1 randomized to belatacept or tacrolimus combined with basiliximab, mycophenolate mofetil, and prednisolone. The 1-year incidence of biopsy-proven acute rejection was monitored. Potential biomarkers, namely, CD8(+) CD28(-), CD4(+) CD57(+) PD1(-), and CD8(+) CD28(++) end-stage terminally differentiated memory T cells were measured pretransplantation and posttranspla(n)tation and correlated to rejection. Pharmacodynamic monitoring of belatacept was performed by measuring free CD86 on monocytes. Results. The rejection incidence was higher in belatacept-treated than tacrolimus-treated patients: 55% versus 10% (P = 0.006). All 3 graft losses, due to rejection, occurred in the belatacept group. Although 4 of 5 belatacept-treated patients with greater than 35 cells CD8(+) CD28(++) end-stage terminally differentiated memory T cells/mu L rejected, median pretransplant values of the biomarkers did not differ between belatacept-treated rejectors and nonrejectors. In univariable Cox regressions, the studied cell subsets were not associated with rejection-risk. CD86 molecules on circulating monocytes in belatacept-treated patients were saturated at all timepoints. Conclusions. Belatacept-based immunosuppressive therapy resulted in higher and more severe acute rejection compared with tacrolimus-based therapy. This trial did not identify cellular biomarkers predictive of rejection. In addition, the CD28-CD80/86 costimulatory pathway appeared to be sufficiently blocked by belatacept and did not predict 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.

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.

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 IL2rγ-/-Rag2-/- mice were transplanted with human skin followed by adoptive transfer of human allogeneic splenocytes. Control animals were treated with a PBS vehicle while the other group was treated with a humanized anti-IL-21R antibody (αIL-21R).Results: In the PBS 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.Conclusion: These findings demonstrate that blockade of IL-21 signaling can delay allograft rejection in a humanized skin transplantation model.

Summary Memory B cells play a pivotal role in alloreactivity in kidney transplantation. Follicular T helper (Tfh) cells play an important role in the differentiation of B cells into immunoglobulin-producing plasmablasts [through interleukin (IL)-21]. It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney transplant patients, therefore we investigated the absolute numbers and function of peripheral Tfh cells (CD4POSCXCR5POS T cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA), and the presence of Tfh cells in rejection biopsies. After transplantation peripheral Tfh cell numbers remained stable, while their IL-21-producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral Tfh cells still had the capacity to induce B cell differentiation and immunoglobulin production, which could be inhibited by an IL-21-receptor-antagonist. After transplantation the quantity of Tfh cells was the highest in patients with pre-existent DSA. In kidney biopsies taken during rejection, Tfh cells co-localized with B cells and immunoglobulins in follicular-like structures. Our data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu.

BACKGROUND: Interleukin-17 (IL-17) is regarded as a major effector cytokine with pro-inflammatory actions. It has pleiotropic and environment-specific functions by promoting adaptive cytotoxic T-lymphocyte responses during inflammation. Therefore, it is tempting to speculate that IL-17 plays a major role in inflammatory responses in transplant recipients. We questioned whether IL-17 is expressed in the transplanted heart during acute rejection (AR), or during immunologic quiescence, and which graft-infiltrating lymphocytes produce IL-17. In addition, we analyzed donor-specific IL-17-producing cells in peripheral blood cells in comparable periods after transplantation. METHODS: Endomyocardial biopsies from heart transplant recipients with early or late AR or in an immunologic quiescence period were analyzed for the presence of IL-17 mRNA. In addition, the capacity of graft-infiltrating lymphocytes (GILs) to produce IL-17 was analyzed. Moreover, we determined the frequency of donor-reactive IL-17-producing peripheral blood mononuclear cells (PBMCs) using an Elispot assay. RESULTS: Twenty-one percent (14 of 67) of the biopsies assessed were positive for IL-17 mRNA. Thirteen of 41 biopsies were observed in the early period (

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.

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

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 of αIL-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.

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.

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.

Background. Belatacept has been associated with an increased acute rejection rate after kidney transplantation. This case report sheds light on the possible immunological mechanisms underlying this phenomenon by analyzing the immunological mechanisms in patient serum, peripheral blood mononuclear cells, rejected kidney tissue, and graft infiltrating cells. Methods. A 61-year-old woman treated with belatacept, who received her first kidney transplant from her husband was admitted with an acute, vascular rejection 56 days after transplantation which necessitated a transplantectomy. Histology and immunohistochemistry were performed on biopsy and explant tissue. CD86 expression on peripheral monocytes was assessed. Using Ficoll density methods, peripheral blood, and graft infiltrating lymphocytes were isolated and phenotyped. Results. The explant showed a vascular rejection (Banff ACR grade III) and a perivascular infiltrate mostly consisting of T cells. No evidence for antibodymediated rejection was found. In contrast to the peripheral blood monocytes, CD86 was still expressed by part of the mononuclear cells in the explant. Isolated graft cells were mostly CCR7?CD45RO+ effector memory CD4+ and CD8+ T cells (60-70%). CD28-positive as CD28-negative T cells were present in the explant, showing a great IFN-? production capacity and expressing granzyme B. Conclusions. We postulate that this glucocorticoid-resistant cellular rejection occurring under belatacept was predominantly mediated by cytotoxic memory T cells, which are less susceptible to costimulatory blockade by belatacept, or resulted from incomplete CD80/86 blockade at the tissue level.