Martijn W.H.J. Demmers

Substantial proliferation of human renal tubular epithelial cell-reactive CD4+CD28null memory T cells, which is resistant to tacrolimus and everolimus

Background In spite of maintenance treatment with immunosuppressive drugs, tubulitis still occurs and can lead to structural kidney graft damage. We hypothesize that human renal tubular epithelial cells (TECs) trigger selective proliferation of recipient T-cell subsets with variable sensitivity to immunosuppressive drugs. Methods Recipient peripheral blood mononuclear cells were cocultured with donor-derived TECs for 7 days. The proliferation of the total CD4+ T-cell pool was assessed. Next, we analyzed which CD4+ T-cell subset proliferated and how this response was affected by tacrolimus, everolimus, prednisolone, and mycophenolic acid (MPA) in clinically relevant concentrations. Results CD4+ T-cell proliferation upon TEC encounter was mainly executed by memory T cells. Interestingly, 38%±7% of the proliferating CD4+ T-cell pool showed a CD28null phenotype. These proliferating CD4+CD28null memory T cells produced high levels of interferon-&ggr;, tumor necrosis factor-&agr;, and the cytolitic protease granzyme B. TEC-reactive CD4+ T-cell proliferation was significantly suppressed by tacrolimus, everolimus, prednisolone, and MPA (P<0.05). Surprisingly and in contrast to prednisolone and MPA, neither tacrolimus nor everolimus could inhibit the CD4+CD28null T-cell proliferative response. Conclusion Our data show substantial proliferation of TEC-reactive CD4+CD28null memory T cells, which are resistant to tacrolimus and everolimus. This phenomenon might play an important mechanistic role during cellular rejection under full immunosuppression.

Limited efficacy of immunosuppressive drugs on CD8+ T cell-mediated and natural killer cell-mediated Lysis of human renal tubular epithelial cells

Background Although CD8+ T cell-mediated and natural killer (NK) cell-mediated cytotoxicity against renal tubular epithelial cells (TECs) plays a crucial role during rejection, the degree of inhibition of these lytic immune responses by immunosuppressive drugs is unknown. We investigated the CD8 T-cell and NK cell responses induced by TECs in vitro and questioned how these processes are affected by immunosuppressive drugs. Methods Donor-derived TECs were co-cultured with recipient peripheral blood monocyte cells. Proliferation of CD8+ T cells and NK cell subsets was assessed using PKH dilution assay. CD107a degranulation and europium release assay were performed to explore CD8+-mediated and NK cell–mediated TEC lysis. Experiments were conducted in the absence or presence of tacrolimus (10 ng/mL), everolimus (10 ng/mL), and prednisolone (200 ng/mL). Results Tubular epithelial cells induce significant CD8+ T-cell and NK cell proliferation. All immunosuppressive drugs significantly inhibited TEC-induced CD8+ T-cell proliferation. Interestingly, prednisolone was the most powerful inhibitor of NK cell proliferation. CD8-mediated and NK cell–mediated early lytic responses were marked by strong degranulation after an encounter of unstimulated TECs, represented by a high cell surface expression of CD107a. However, with the use of interferon-&ggr;–activated and tumor necrosis factor-&agr;–activated TECs, the NK degranulation response was significantly reduced and CD8 degranulation response was even more enhanced (P<0.05). Tubular epithelial cell-induced CD8 degranulation and CD8-mediated TEC lysis were preferentially inhibited by tacrolimus and prednisolone, and not by everolimus. Although tacrolimus showed the most inhibitory effect on the degranulation of NK cells, NK cell-mediated TEC lysis was efficiently inhibited by prednisolone (P<0.05). Conclusion Overall, our data point to a limited efficacy of immunosuppressive drugs on CD8+ T cell-mediated and NK cell-mediated lysis of human renal TECs.

Differential effects of activated human renal epithelial cells on T-cell migration.

Background Renal tubular epithelial cells (TECs) are one of the main targets of inflammatory insults during interstitial nephritis and kidney transplant rejection. While Th1 cells are know to be essential in the pathogenesis of rejection, the role of Th17 is still under debate. We hypothesize that TECs modulate the outcome of rejection process by production of distinct chemokines and cytokines that determine the attraction of different T-cell subsets. Therefore, we studied differential effects of activated human renal epithelial cells on T-cell migration. Methods Human primary TECs were stimulated by IFN-γ and TNF-α in vitro. Chemokines and cytokines produced by activated TECs were measured using Luminex or ELISA. Chemotaxis assay was performed using activated peripheral blood mononuclear cells composed of CD4+CXCR3+ and CD4+CCR6+ T cells migrating towards stimulated and unstimulated TECs. Results While activated TECs secreted abundant amounts of the pro-inflammatory cytokines IL-6 and IL-8, the T helper cell differentiation cytokines IL-1β, IL-12p70, IL-23 or TGF-β1 were not produced. The production of Th1 chemokines CXCL9, CXCL10 and CCL5 were significantly upregulated after TEC stimulation. In contrast, Th17 chemokine CCL20 could not be detected. Finally, activated TECs attracted significantly higher numbers of CD4+CXCR3+ T cells as compared to unstimulated TECs. No migration of CD4+CCR6+ T cells could be observed. Conclusion Activated primary renal tubular epithelial cells do not attract Th17 cells nor produce cytokines promoting Th17 cell differentiation in our experimental system mimicking the proinflammatory microenvironment of rejection.

FoxP3 T Cells and the Pathophysiologic Effects of Brain Death and Warm Ischemia in Donor Kidneys

BACKGROUND AND OBJECTIVES Forkhead box P3 regulatory T cells control inflammatory responses, but it remains unclear whether they inhibit brain death-initiated inflammation and tissue injury in deceased kidney donors. DESIGN, SETTING, PARTICIPANTS, MEASUREMENT: To study the actions of regulatory T cells at various stages of the donation and transplantation procedure, forkhead box P3, regulatory and inflammatory cytokine expression, and tissue injury markers were determined in time 0 kidney biopsies from deceased and living donors. Additionally, the interaction between forkhead box P3+ T cells and kidney injury molecule-1 by activated primary tubular epithelial cells was studied. RESULTS After cold storage, the deceased donor kidneys expressed the higher mRNA levels of kidney injury molecule-1 and CD3ε. In these samples, the inflammatory cytokines IL-8 and IFN-γ and markers associated with regulation (forkhead box P3, TGF-β, and IL-10) were highly expressed compared with living donor kidneys. Correlations were found between mRNA expression levels of forkhead box P3 and kidney injury molecule-1 and forkhead box P3 and IFN-γ. Immunohistochemical analysis confirmed the presence of forkhead box P3+ T cells in donor kidneys. Renal function (analyzed by serum creatinine levels) at the first week posttransplantation correlated with kidney injury molecule-1 and forkhead box P3 mRNA levels. In vitro studies showed that kidney injury molecule-1 expression by primary tubular epithelial cells was 63% (mean) lower when cocultured with regulatory T cells compared with control T cells. CONCLUSIONS These results show that donor forkhead box P3+ T cells infiltrate the deceased donor kidney, where they may control inflammatory and injury responses.

Human renal tubular epithelial cells suppress alloreactive T cell proliferation

Renal tubular epithelial cells (TECs) are one of the main targets of alloreactive T cells during acute rejection. We hypothesize that TECs modulate the outcome of alloimmunity by executing immunosuppressive effects in order to dampen the local inflammation. We studied whether TECs possess immunosuppressive capacities and if indoleamine 2,3‐dioxygenase (IDO) might play a role in suppressing T cell alloreactivity. Next, we studied the role of programmed death ligand 1 (PD‐L1) and intercellular adhesion molecule‐1 (ICAM‐1 with regard to TEC‐related immunomodulatory effects. CD3/CD28 and alloactivated peripheral blood mononuclear cells were co‐cultured with activated TECs. We analysed CD4+ and CD8+ T cell proliferation and apoptosis in the absence or presence of IDO inhibitor 1‐methyl‐L‐tryptophan (1‐L‐MT), anti‐PD‐L1 and anti‐ICAM‐1. Further, we examined whether inhibition of T cell proliferation was cell–cell contact‐dependent. We found that TECs dose‐dependently inhibited CD4+ and CD8+ T cell proliferation (P < 0·05). Activated TECs showed significantly increased IDO activity and up‐regulated PD‐L1 and ICAM‐1 expression. Suppressed CD4+ and CD8+ T cell proliferation was only partially restored or failed to restore using 1‐L‐MT. Activated TECs increased early and late apoptosis of proliferating CD4+ and CD8+ T cells; only CD4+ T cell apoptosis was statistically affected by 1‐L‐MT. Transwell experiments revealed that TEC‐mediated immunosuppression is cell–cell contact‐dependent. We found that anti‐ICAM‐1 affected only CD4+ T cell apoptosis and not T cell proliferation. Our data show that TECs suppress both CD4+ and CD8+ T cell proliferation contact dependently. Interestingly, inhibition of proliferation and enhancement of apoptosis of T cell subsets is differentially regulated by indoleamine 2,3‐dioxygenase and ICAM‐1, with no evidence for the involvement of PD‐L1 in our system.

Donor-Derived Renal Tubular Epithelial Cells Induce Recipient Memory T-Cell Proliferation with a CD28null Phenotype Which Is not Susceptible to Tacrolimus: 1475

Introduction: It is known that memory T-cell activation can result in a donor-specific effector immune response. Memory T cells are considered to be sensitive to calcineurin inhibition. Clinically tubulitis still occurs despite treatment with potent immunosuppressive drugs. Here we investigated the proliferative responses and tissue damaging capacities of recipient T cells induced by donor-derived renal tubular epithelial cells (TECs). In addition, we studied their susceptibility to currently used immunosuppressive drugs. Materials and methods: Recipient PBMCs were co-cultured with donorderived TECs for 7 days (N=5). As control, autologous co-cultures of donor PBMCs and donor-derived TECs were used. By flow cytometry the proliferative response of CD3, CD4, CD8 naïve (CD45RO-CCR7+), effector memory (CD45RO+CCR7-), central memory (CD45RO+CCR7+) and effector memory RA (EMRA, CD45RO-CCR7-) was measured. In addition, we determined the tissue damaging capacities of proliferating T cells by intracellular staining for Granzyme B, IFN-γ and TNF-α. Finally we assessed the inhibitory effect of tacrolimus (10ng/ml), everolimus (10ng/ml) and prednisolone (200ng/ml) on the induced T-cell proliferation. Results: Up to 8.3% ± 1.9% of the recipient CD4+ T-cells and 5.7% ± 1.2% of the CD8+ T-cells mounted a proliferative response upon donorderived TECs encounter. This response was mainly executed by effector memory CD4+ and CD8+ T cells with a CD28null phenotype comprising of 40-50% ± 8.2% of the proliferating T-cell pool. The CD28null memory T cells expressed the serine protease Granzyme B (34.7-40.9%) and produced after polyclonal stimulation the proinflammatory cytokine IFN-γ (5.3-13.1%) and TNF-α (43.0-53.6%). Tacrolimus (10 ng/ml) suppressed donor-induced PBMC proliferation by 20% as compared to 80% inhibition of mixed leukocyte reaction (MLR)-induced alloreactive immune response. Surprisingly, tacrolimus could not inhibit the donor-induced proliferation of CD28null effector memory T cell, whereas everolimus and prednisolone inhibited CD4+CD28null T-cell proliferation by 37% ± 12.3% and 75% ± 1.1%, respectively. CD8+CD28null T-cell proliferation was not affected by any of the drugs tested. Conclusion: Donor-derived TECs induce recipient memory T-cell proliferation. Tacrolimus only slightly inhibits this proliferative response, while CD28null proliferation was not inhibited at all. 1002

Donor-Derived Tubular Epithelial Cells Induce Class I Restricted Alloreactivity in Kidney Transplant Recipients: 1488

Introduction: Although it is known from in vitro studies that human renal tubular epithelial cells (TEC) have stimulatory capacities their effect on alloreactivity in organ transplant patients is unknown. In the present study, the immunostimulatory effect of donor TEC on recipient anti-donor T-cell reactivity was examined by analysing the function and characteristics T cell subsets before and after clinical kidney transplantation. Material and methods: Recipient T-cell reactivity against donor TECs was investigated in pre-transplant and post-transplant co-culture system and transwell experiments of 6 living-kidney donor-recipient pairs. For TEC/PBMC co-culture, recipient PBMCs vs donor TEC (allogeneic coculture) and donor PBMCs vs donor TEC (autologous co-culture) were used. By flow cytometry the proliferative response of CD3, CD4, CD8 naïve (CD45ROCCR7+), effector memory (CD45RO+CCR7+), central memory (CD45RO+CCR7-) and effector memory RA (EMRA, CD45ROCCR7-) was measured. Results: After stimulation by TEC a allogeneic response was measured in the CD8+ T-cell subset, but was not found in CD4+ T-cells. No autologous induced CD8+ T-cell proliferation was found. The proliferative response in the pre-transplantation co-culture was 5.7% ± 1.2 and in the post-transplantation co-culture a response of 6.6% ± 3.1 was found. In addition, transwell experiments revealed that the TEC induced CD8+ T-cell proliferation was cell-cell contact dependent. Co-cultured CD8+ T-cells also expressed the activation marker CD69. Additionally, the vast majority of the CD8+ responding T-cells where of the memory phenotype; effector memory T cells (47%), EMRA (25%) and of central memory T cells (13%). No proliferation of the naïve CD8+ T-cell was found. Conclusion: Donor-derived TECs induce a class I restricted effector memory T-cell response in kidney transplant recipients. 1553