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.

Alloreactive T cells to Assess Acute Rejection Risk in Kidney Transplant Recipients.

Memory T cells are important mediators of transplant rejection but are not routinely measured before or after kidney transplantation. The aims of this study were as follows: (1) validate whether pretransplant donor-reactive memory T cells are reliable predictors of acute rejection (AR) (2) determine whether donor-reactive memory T cells can distinguish AR from other causes of transplant dysfunction. Methods: Samples from 103 consecutive kidney transplant recipients (2018-2019) were obtained pretransplantation and at time of for-cause biopsy sampling within 6 mo of transplantation. The number of donor-reactive interferon gamma (IFN-γ) and interleukin (IL)-21-producing memory T cells was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay. Results: Of the 63 patients who underwent a biopsy, 25 had a biopsy-proven acute rejection (BPAR; 22 aTCMR and 3 aAMR), 19 had a presumed rejection, and 19 had no rejection. Receiver operating characteristic analysis showed that the pretransplant IFN-γ ELISPOT assay distinguished between patients who later developed BPAR and patients who remained rejection-free (area under the curve [AUC] 0.73; sensitivity 96% and specificity 41%). Both the IFN-γ and IL-21 assays were able to discriminate BPAR from other causes of transplant dysfunction (AUC 0.81; sensitivity 87% and specificity 76% and AUC 0.81; sensitivity 93% and specificity 68%, respectively). Conclusions: This study validates that a high number of donor-reactive memory T cells before transplantation is associated with the development of AR after transplantation. Furthermore, it demonstrates that the IFN-γ and IL-21 ELISPOT assays are able to discriminate between patients with AR and patients without AR at the time of biopsy sampling.

Natural Antibodies Are Associated With Rejection and Long-term Renal Allograft Loss in a Multicenter International Cohort.

BACKGROUND: Potentially harmful nonhuman leukocyte antigen antibodies have been identified in renal transplantation, including natural immunoglobulin G antibodies (Nabs) reactive to varied antigenic structures, including apoptotic cells. METHODS: In this retrospective, multicenter study, we assessed Nabs by reactivity to apoptotic cells in sera collected from 980 kidney transplant recipients across 4 centers to determine their association with graft outcomes. RESULTS: Elevated pretransplant Nabs were associated with graft loss (hazard ratio [HR] 2.71; 95% confidence interval [CI], 1.15-6.39; P = 0.0232), the composite endpoint of graft loss or severe graft dysfunction (HR 2.40; 95% CI, 1.13-5.10; P = 0.0232), and T cell-mediated rejection (odds ratio [OR] 1.77; 95% CI, 1.07-3.02; P = 0.0310). High pretransplant Nabs together with donor-specific antibodies (DSAs) were associated with increased risk of composite outcomes (HR 6.31; 95% CI, 1.81-22.0; P = 0.0039). In patients with high pretransplant Nabs, the subsequent development of posttransplant Nabs was associated with both T cell-mediated rejection (OR 3.64; 95% CI, 1.61-8.36; P = 0.0021) and mixed rejection (OR 3.10; 95% CI, 1.02-9.75; P = 0.0473). Finally, elevated pre- and posttransplant Nabs combined with DSAs were associated with increased risk of composite outcomes (HR 3.97; 95% CI, 1.51-10.43; P = 0.0052) and T cell-mediated rejection (OR 7.28; 95% CI, 2.16-25.96; P = 0.0016). CONCLUSIONS: The presence of pre- and posttransplant Nabs, together with DSAs, was associated with increased risk of poor graft outcomes and rejection after renal transplantation.

Systemic varicella zoster virus reactive effector memory T-cells impaired in the elderly and in kidney transplant recipients.

Varicella zoster virus (VZV) infections cause varicella and subsequently herpes zoster upon reactivation. Immune-compromised individuals and the elderly are at high risk of developing herpes zoster due to waning of VZV-specific T-cell immunity. In the present study, a novel functional T-cell assay was developed to test the correlation between age and VZV-specific T-cell responses in peripheral blood from healthy individuals. Secondly, VZV-specific T-cell responses from renal transplant recipients were compared with healthy individuals. Monocytes were differentiated into mature monocyte-derived dendritic cells (moDCs) and were infected with VZV. T-cells were co-cultured with autologous moDCs infected with VZV and subjected to flowcytometric analysis to identify the phenotype (i.e., naïve [NA: CCR7(+) CD45RO(-) ], central [CM: CCR7(+) CD45RO(+) ] and effector memory [EM: CCR7(-) CD45RO(+) ] T-cells) and the frequency of VZV-reactive T-cell subsets by intra-cellular IFN-γ flowcytometry. In contrast to NA and CM T-cells, the frequency of VZV-reactive CD4 and CD8 EM T-cells was inversely correlated with age (P = 0.0007 and P = 0.01). No difference was found in the percentage of VZV-reactive CD4 NA, CM and EM T-cells between transplant recipients and controls. However, the percentage of VZV-reactive CD8 EM T-cells was significantly lower in transplant recipients compared to controls (P = 0.02). In conclusion, moDCs infected with VZV are efficient antigen presenting cells applicable to enumerate and characterize the phenotype and differentiation status of the systemic VZV-specific T-cell response ex-vivo. The data suggest that VZV-reactive EM T-cells are impaired in the elderly and renal transplant recipients.

Boosting the VZV-Specific Memory B and T Cell Response to Prevent Herpes Zoster After Kidney Transplantation.

Background: Solid organ transplant recipients are at high risk to develop (complicated) herpes zoster (HZ). Booster vaccination could prevent HZ. However, end-stage renal disease (ESRD) patients show poor immunological responses to vaccinations. We studied the effect of a live attenuated VZV booster vaccine on VZV-specific B and T cell memory responses in ESRD patients and healthy controls. NL28557.000.09, www.toetsingonline.nl. Methods: VZV-seropositive patients, aged ≥50 years, awaiting kidney transplantation, were vaccinated with Zostavax®. Gender and age-matched VZV-seropositive potential living kidney donors were included as controls. VZV-specific IgG titers were measured before, at 1, 3 and 12 months post-vaccination. VZV-specific B and T cell responses before, at 3 months and 1 year after vaccination were analysed by flow-cytometry and Elispot, respectively. Occurrence of HZ was assessed at 5 years post-vaccination. Results: 26 patients and 27 donors were included. Median VZV-specific IgG titers were significantly higher at all time-points post-vaccination in patients (mo 1: 3104 IU/ml [1967-3825], p<0.0001; mo 3: 2659 [1615-3156], p=0.0002; mo 12: 1988 [1104-2989], p=0.01 vs. pre: 1397 [613-2248]) and in donors (mo 1: 2981 [2126-3827], p<0.0001; mo 3: 2442 [2014-3311], p<0.0001; mo 12: 1788 [1368-2460], p=0.0005 vs. pre: 1034 [901-1744]. The patients' IgG titers were comparable to the donors' at all time-points. The ratio VZV-specific B cells of total IgG producing memory B cells had increased 3 months post-vaccination in patients (0.85 [0.65-1.34] vs. pre: 0.56 [0.35-0.81], p=0.003) and donors (0.85 [0.63-1.06] vs. pre: 0.53 [0.36-0.79], p<0.0001) and remained stable thereafter in donors. One year post-vaccination, the percentage of CD4+ central memory cells had increased in both patients (0.29 [0.08-0.38] vs. 0.12 [0.05-0.29], p=0.005) and donors (0.12 [0.03-0.37] vs. 0.09 [0.01-0.20], p=0.002) and CD4+ effector memory cells had increased in donors (0.07 [0.02-0.14] vs. 0.04 [0.01-0.12], p=0.007). Only 1 patient experienced HZ, which was non-complicated. Conclusion: VZV booster vaccination increases VZV-specific IgG titers and percentage VZV-specific memory T-cells for at least 1 year both in ESRD patients and healthy controls. VZV-specific memory B cells significantly increased in patients up to 3 months after vaccination. Prophylactic VZV booster vaccination prior to transplantation could reduce HZ incidence and severity after transplantation.

The human alloreactive CD4+ T-cell repertoire is biased to a Th17 response and the frequency is inversely related to the number of HLA class II mismatches.

Estimates of precursor frequency and assessment of functional characteristics of alloreactive CD4+ T cells are all biased by the need for long-term culture. In this study, direct visualization of human alloreactive CD4+ T cells on the single-cell level was achieved using cell surface expression of CD154 as a tool for identification. The average frequency of alloreactive CD154+CD4+ T cells among peripheral blood CD4+ T cells was 0.1%, with half of the cells displaying a naive phenotype. The proliferation capacity and expression of cytokines after allogeneic stimulation resided in these CD154+CD4+ T cells. The repertoire of alloreactive CD4+ T cells was biased to a Th17 response, and on average 24% of alloreactive CD154+CD4+ memory T cells produced interleukin-17 (IL-17) after polyclonal stimulation. Unexpectedly, mixed cell cultures from human leukocyte antigen (HLA)-identical donors also generated alloreactive CD154+CD4+ T cells and yielded the highest frequency compared with HLA-nonidentical combinations. Therefore, reactivity to minor histocompatibility antigens between HLA-identical subjects appears to be relatively common. Alloreactive HLA-identical T cells did not proliferate or express cytokines, but were driven to proliferation in the presence of exogenous IL-2.

A systematic review and meta-analysis of enzyme-linked immunosorbent spot (ELISPOT) assay for BK polyomavirus immune response monitoring after kidney transplantation.

BK virus (BKV) infection after kidney transplantation can cause BKV nephropathy (BKVAN) resulting in graft dysfunction and allograft loss. The treatment for BKVAN is reduction of the immunosuppressive load which increases the risk of kidney transplant rejection. There is no biomarker to monitor BKV activity besides BK viral load. The value of the Enzyme-Linked Immunosorbent Spot (ELISPOT) assay as a tool to monitor the recipient's anti-BKV immune response after transplantation was investigated systematically. Electronic databases, including MEDLINE, Scopus, and the Cochrane Central Register of Controlled Trials were searched for studies of ELISPOT evaluating the immune response against BKV. BKV status was categorized as "active BKV infection" and as "resolving BKV infection". Random-effects model meta-analysis was performed to determine the diagnostic performance of the ELISPOT assay, after stratifying patients into groups based on positive and negative ELISPOT results. One-hundred twenty-seven articles were identified of which nine were included. Patients with negative ELISPOT had an increased risk of having active BKV replication (odds ratio of 71.9 (95%-CI 31.0-167.1). Pooled sensitivity was 0.95 (95%-CI 0.89-0.98) and specificity was 0.88 (95%-CI 0.78-0.94). The standardized mean difference of the number of IFN-γ producing cells between patients with active BKV infection compared with patients who had resolving BKV infection was -2.09 (95%-CI -2.50, -1.68). The ELISPOT assay is a useful tool for BKV risk assessment and in combination with BKV load may support clinicians in guiding immunosuppressive therapy in patients with BKV replication.

Donor-specific ELISPOT assay for predicting acute rejection and allograft function after kidney transplantation: A systematic review and meta-analysis.

Acute rejection remains an important problem after kidney transplantation. Enzyme-linked immunosorbent spot (ELISPOT) assay has been investigated extensively and has shown promising results as a predictor of allograft rejection. The objective of this study was to systematically review and analyze the predictive value of the donor-specific ELISPOT assay to identify recipients at risk for acute rejection. Electronic databases were searched for studies reporting donor-specific ELISPOT and kidney transplantation outcomes. Odds ratio (OR) for acute rejection was calculated, along with standardized mean difference (SMD) of cytokine producing-cells between recipients with and without acute rejection. Pooled estimates were calculated using random-effect models. The positive ELISPOT cutoff frequencies were extracted from each study. From 665 articles found, 32 studies were included in the meta-analysis. IFN-γ was the most investigated cytokine (30 out of 32 studies). Patients with positive pre-transplantation donor-reactive IFN-γ ELISPOT had an OR of 3.3 for acute rejection (95%-CI 2.1 to 5.1), and OR of 6.8 (95%-CI 2.5 to 18.9) for post-transplantation ELISPOT. Recipients with rejection had significantly higher frequencies of pre- and post-transplantation cytokine producing-cells (SMD 0.47, 95%-CI 0.07 to 0.87 and SMD 3.68, 95%-CI 1.04 to 6.32, respectively). Pre-transplantation ELISPOT had a positive predictive value of 43% and a negative predictive value of 81% for acute rejection. A positive ELISPOT result was associated with a lower estimated glomerular filtration rate (SMD -0.59, 95%-CI -0.83 to -0.34). In conclusion, patients with a high frequency of donor-reactive IFN-γ ELISPOT are at higher risk for acute rejection. The donor-specific IFN-γ ELISPOT assay can serve as an immune-monitoring tool in kidney transplantation.

Pre-transplant donor-reactive IL-21 producing T cells as a tool to identify an increased risk for acute rejection.

Pre-transplant screening focuses on the detection of anti-HLA alloantibodies. Previous studies have shown that IFN-γ and IL-21 producing T cells are associated with the development of acute rejection (AR). The aim of this study, was to assess whether pre-transplant donor-reactive T cells and/or B cells are associated with increased rejection risk. Samples from 114 kidney transplant recipients (transplanted between 2010 and 2013) were obtained pre-transplantation. The number of donor-reactive IFN-γ and IL-21 producing cells was analyzed by ELISPOT assay. The presence of donor specific antibodies (DSA) was also determined before transplantation. Numbers of donor-reactive IFN-γ producing cells were similar in patients with or without AR whereas those of IL-21 producing cells were higher in patients with AR (p = 0.03). Significantly more patients with AR [6/30(20%)] had detectable DSA compared to patients without AR [5/84(5.9%), p = 0.03]. Multivariate logistic regression showed that donor age (OR 1.06), pre-transplant DSA (OR 5.61) and positive IL-21 ELISPOT assay (OR 2.77) were independent predictors of an increased risk for the development of AR. Aside from an advanced donor-age and pre-transplant DSA, also pre-transplant donor-reactive IL-21 producing cells are associated with the development of AR after transplantation.

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.

Herpes Zoster in Solid Organ Transplantation: Incidence and Risk Factors.

Background: Studies on herpes zoster (HZ) incidence in solid organ transplant (SOT) recipients report widely varying numbers. We investigated HZ incidence, severity, and risk factors in recipients of four different SOTs, with a follow-up time of 6-14 years. Methods: Records of 1,033 transplant recipients after first heart (HTx: n = 211), lung (LuTx: n = 121), liver (LiTx: n = 258) and kidney (KTx: n = 443) transplantation between 2000 and 2014 were analyzed for VZV-PCR, clinical signs of HZ, and complications. Results: HZ was diagnosed in 108 of 1,033 patients (10.5%): 36 HTx, 17 LuTx, 15 LiTx, and 40 KTx recipients. Overall HZ incidence rate after HTx (30.7 cases/1,000 person-years (PY)), LuTx (38.8 cases/1,000 PY), LiTx (22.7 cases/1,000 PY) and KTx (14.5 cases/1,000 PY) was significantly higher than in the general 50-70 year population. Multivariable analysis demonstrated age ≥50 years at transplantation (p = 0.038, RR 1.536), type of organ transplant (overall p = 0.002; LuTx p = 0.393; RR 1.314; LiTx p = 0.011, RR 0.444; KTx p = 0.034, RR 0.575), CMV prophylaxis (p = 0.043, RR 0.631) and type of anti-rejection therapy (overall p = 0.020; methylprednisolone p = 0.008, RR 0.475; r-ATG p = 0.64, RR1.194) as significant risk factors. Complications occurred in 33 of 108 (31%) patients (39% of HTx, 47% of LuTx, 20% of LiTx, 20% of KTx): post-herpetic neuralgia, disseminated disease, and cranial nerve involvement. Conclusion: HZ incidence and severity in SOT recipients are most pronounced after heart and lung transplantation, in older patients, and when CMV prophylaxis is lacking.

Migration of allosensitizing donor myeloid dendritic cells into recipients after liver transplantation.

It is thought, but there is no evidence, that myeloid dendritic cells (MDCs) of donor origin migrate into the recipient after clinical organ transplantation and sensitize the recipient's immune system by the direct presentation of donor allo-antigens. Here we show prominent MDC chimerism in the recipient's circulation early after clinical liver transplantation (LTx) but not after renal transplantation (RTx). MDCs that detach from human liver grafts produce large amounts of pro-inflammatory [tumor necrosis factor alpha and interleukin 6 (IL-6)] and anti-inflammatory (IL-10) cytokines upon activation with various stimuli, express higher levels of toll-like receptor 4 than blood or splenic MDCs, and are sensitive to stimulation with a physiological concentration of lipopolysaccharide (LPS). Upon stimulation with LPS, MDCs detaching from liver grafts prime allogeneic T cell proliferation and production of interferon gamma but not of IL-10. Soluble factors secreted by liver graft MDCs amplify allogeneic T helper 1 responses. In conclusion, after clinical LTx, but not after RTx, prominent numbers of donor-derived MDCs migrate into the recipient's circulation. MDCs detaching from liver grafts produce pro-inflammatory and anti-inflammatory cytokines and are capable of stimulating allogeneic T helper 1 responses, and this suggests that MDC chimerism after clinical LTx may contribute to liver graft rejection rather than acceptance.

Preeclampsia is associated with increased cytotoxic T-cell capacity to paternal antigens.

OBJECTIVE: During an uncomplicated pregnancy the conceptus is a semiallogeneic entity in which rejection is prevented by suppression of the maternal immune system. We hypothesized that this suppression is disturbed in patients with preeclampsia and that a maternal immune response to fetal (foreign/paternal) antigens in the fetal-maternal interface may be responsible for local inflammation, with subsequent endothelial dysfunction and systemic disease. STUDY DESIGN: Blood samples were obtained from 14 women with preeclampsia (cases), 14 gestational-age and parity-matched women with uncomplicated pregnancies (controls), and their partners. We determined the partner-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) and the CTLpf directed to unrelated partners with uncomplicated pregnancies. We measured the CTLpf in peripheral blood mononuclear cells (PBMCs) from cases and controls using limited-dilution assays. In addition, proliferation was tested in a mixed-lymphocyte culture (MLR). RESULTS: The partner-specific CTLpf was significantly higher in cases compared with controls (median, 183 [15-338] vs 67 [9-232] per million PBMCs, P = .02). In contrast, in women with uncomplicated pregnancies, the partner-specific CTLpf was down-regulated compared with the CTLpf directed to an unrelated partner who fathered uncomplicated pregnancies (P = .02). No difference was found in partner-specific MLR response between cases and controls. CONCLUSION: These results suggest that women with preeclampsia have a higher cytotoxic T-cell response to paternal antigens compared with pregnant controls. This insufficiently suppressed immune response may eventually lead to the development of preeclampsia.

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.

Non-HLA T-cell reactivity during the first year after HLA-identical living-related kidney transplantation.

BACKGROUND: It has been reported that donor-reactive T-cell responses may decrease during the first year after HLA-mismatched organ transplantation. We wondered whether donor-reactive T-cell responses directed to minor histocompatibility antigens (mHAgs) or other non-HLA antigens also decrease after HLA-identical living-related (LR) kidney transplantation. METHODS: We studied donor-reactive T-cell responses by IFN-gamma and granzyme B (GrB) Elispot assays in 15 HLA-identical LR kidney transplant recipients before, six months and one yr after transplantation. Third-party reactivity was used as control. Patient and donor peripheral blood mononuclear cells were typed for 11 known mHAgs. RESULTS: During the study period, 60% and 36% of the patients demonstrated donor-reactive IFN-gamma and GrB producing cells (pc), respectively. The number of donor-reactive IFN-gamma and GrB pc was significantly lower than the number of third-party reactive IFN-gamma and GrB pc. After transplantation, donor-reactivity and third-party reactivity were comparable to pre-transplant values. No relation was found in mHAg mismatches between donor and recipient and donor-reactive T-cell response. CONCLUSIONS: Donor-reactivity could be detected before and after HLA-identical LR kidney transplantation, but was not related with the number of mHAg mismatches, and did not decrease after transplantation.

The role of follicular T helper cells in the humoral alloimmune response after clinical organ transplantation.

Over the past decade, antibody-mediated or humoral rejection in combination with development of de novo donor-specific antibodies (DSA) has been recognized as a distinct and common cause of transplant dysfunction and is responsible for one-third of the failed allografts. Detailed knowledge of the mechanisms that initiate and maintain B-cell driven antidonor reactivity is required to prevent and better treat this antidonor response in organ transplant patients. Over the past few years, it became evident that this response largely depends on the actions of both T follicular helper (Tfh) cells and the controlling counterparts, the T follicular regulatory (Tfr) cells. In this overview paper, we review the latest insights on the functions of circulating (c)Tfh cells, their subsets Tfh1, Tfh2 and Tfh17 cells, IL-21 and Tfr cells in antibody mediated rejection (ABMR). This may offer new insights in the process to reduce de novo DSA secretion resulting in a decline in the incidence of ABMR. In addition, monitoring these cell populations could be helpful for the development of biomarkers identifying patients at risk for ABMR and provide novel therapeutic drug targets to treat ABMR.

An overview of T follicular cells in transplantation: spotlight on their clinical significance.

Introduction: For late stage organ failure patients, transplantation is the best option to increase life expectancy with a superior quality of life. Unfortunately, after transplantation many patients are at risk of cellular and antibody-mediated rejection (ABMR). The latter is initiated by donor specific antibodies (DSA) which depend on the actions of B cells, T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells that are present in the germinal center of lymphoid organs.Areas covered: In this overview paper, we discuss the biology and function of Tfh and Tfr cells in lymphoid tissues, transplanted organs and their circulating counterparts. We report on their relevance to alloimmunity and on the effects of immunosuppressive drugs on these immunocompetent cell populations.Expert opinion: Growing knowledge about the actions of Tfh and Tfr allows for a better understanding of the immunological mechanisms of ABMR after organ transplantation. This understanding feeds the hypothesis that immunosuppressive drugs targeting the actions of Tfh cells have huge therapeutic potential. This new concept in the treatment of the humoral rejection response will improve graft and patient survival after organ transplantation.

Impact of low tacrolimus exposure and high tacrolimus intra-patient variability on the development of de novo anti-HLA donor-specific antibodies in kidney transplant recipients.

Introduction: De novo donor-specific antibodies (dnDSA) directed against HLA are a major contributing factor to the chronic deterioration of renal allograft function. Several factors, including the degree of HLA matching, younger recipient age, and past sensitization events have been shown to increase the risk for the development of dnDSA. The development of dnDSA is also strongly associated with modifications in the immunosuppressive regimen, non-adherence, and under-immunosuppression.Areas covered: Tacrolimus is widely used after solid organ transplantation (SOT) and in recent years, both a high intra-patient variability in tacrolimus exposure and low tacrolimus exposure have been found to be associated with a higher risk of dnDSA development in kidney transplant recipients. This article provides an overview of current findings published in the recent 5 years regarding the relationship between tacrolimus exposure and variation therein and the development of dnDSA.Expert opinion: In this review, we describe how combining data on tacrolimus intra-patient variability and mean pre-dose concentration may be an effective tool to identify kidney transplant recipients who are at higher risk of developing dnDSA.

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.

Tapering immunosuppressive therapy significantly improves in vivo cutaneous delayed type hypersensitivity responses.

Immunosuppressive therapy affects cell-mediated immunity and thereby increases the frequency of infections and malignancies in transplanted patients. We questioned whether reducing the immunosuppressive dose in stable kidney transplant patients has an in vivo effect on cutaneous delayed type hypersensitivity responses (DTH) reflecting cell-mediated immunity. We measured DTH responses to recall antigens (Tetanus, Diphteria, Streptococcus, Tuberculin, Candida, Trychophyton, Proteus, glycerin control) on the volar surface of the forearm in patients before and after successful reduction (50%) of the dose of mycophenolate mofetil (MMF) or azathioprine (AZA). In addition, we tested healthy individuals who were age- and sex-matched to the patient group. Results of the skin reaction test were calculated as the sum in millimeters (mm) of all positive reactions (score), and as the number of positive antigens. Patients treated with a high dose of MMF or AZA had a significantly lower test score compared to healthy controls (p=0.01). Also the number of positive antigens was reduced in patients compared to healthy controls (p=0.02). After reduction of the MMF or AZA dose, the test score and the number of positive antigens increased significantly (p=0.02, p=0.01, respectively) to comparable scores of healthy controls. Additionally, the mycophenolic acid (MPA) trough level was negatively correlated with the test score (p=0.006) and number of positive antigens (p=0.004). In conclusion, successful tapering of the MMF or AZA dose in kidney transplant patients more than 2 years after transplantation favorably affects the in vivo DTH response, reflecting an improvement of the general immunity, facilitating the defense against infection and malignancies.