Repeated COVID-19 Vaccination Drives Memory T- and B-cell Responses in Kidney Transplant Recipients: Results From a Multicenter Randomized Controlled Trial.

BACKGROUND: Insight into cellular immune responses to COVID-19 vaccinations is crucial for optimizing booster programs in kidney transplant recipients (KTRs). METHODS: In an immunologic substudy of a multicenter randomized controlled trial (NCT05030974) investigating different repeated vaccination strategies in KTR who showed poor serological responses after 2 or 3 doses of an messenger RNA (mRNA)-based vaccine, we compared SARS-CoV-2-specific interleukin-21 memory T-cell and B-cell responses by enzyme-linked immunosorbent spot (ELISpot) assays and serum IgG antibody levels. Patients were randomized to receive: a single dose of mRNA-1273 (100 µg, n = 25), a double dose of mRNA-1273 (2 × 100 µg, n = 25), or a single dose of adenovirus type 26 encoding the SARS-CoV-2 spike glycoprotein (Ad26.COV2.S) (n = 25). In parallel, we also examined responses in 50 KTR receiving 100 µg mRNA-1273, randomized to continue (n = 25) or discontinue (n = 25) mycophenolate mofetil/mycophenolic acid. As a reference, the data were compared with KTR who received 2 primary mRNA-1273 vaccinations. RESULTS: Repeated vaccination increased the seroconversion rate from 21% to 66% in all patients, which was strongly associated with enhanced levels of SARS-CoV-2-specific interleukin-21 memory T cells (odd ratio, 3.84 [1.89-7.78]; P < 0.001) and B cells (odd ratio, 35.93 [6.94-186.04]; P < 0.001). There were no significant differences observed in these responses among various vaccination strategies. In contrast to KTR vaccinated with 2 primary vaccinations, the number of antigen-specific memory B cells demonstrated potential for classifying seroconversion after repeated vaccination (area under the curve, 0.64; 95% confidence interval, 0.37-0.90; P = 0.26 and area under the curve, 0.95; confidence interval, 0.87-0.97; P < 0.0001, respectively). CONCLUSIONS: Our study emphasizes the importance of virus-specific memory T- and B-cell responses for comprehensive understanding of COVID-19 vaccine efficacy among KTR.

The role of interleukin-21 in COVID-19 vaccine-induced B cell-mediated immune responses in patients with kidney disease and kidney transplant recipients.

T-cell-mediated help to B cells is required for the development of humoral responses, in which the cytokine interleukin (IL)-21 is key. Here, we studied the mRNA-1273 vaccine-induced SARS-CoV-2-specific memory T-cell IL-21 response, memory B cell response, and immunoglobulin (Ig)G antibody levels in peripheral blood at 28 days after the second vaccination by ELISpot and the fluorescent bead-based multiplex immunoassay, respectively. We included 40 patients with chronic kidney disease (CKD), 34 patients on dialysis, 63 kidney transplant recipients (KTR), and 47 controls. We found that KTR, but not patients with CKD and those receiving dialysis, showed a significantly lower number of SARS-CoV-2-specific IL-21 producing T cells than controls (P < .001). KTR and patients with CKD showed lower numbers of SARS-CoV-2-specific IgG-producing memory B cells when compared with controls (P < .001 and P = .01, respectively). The T-cell IL-21 response was positively associated with the SARS-CoV-2-specific B cell response and the SARS-CoV-2 spike S1-specific IgG antibody levels (both Pearson r = 0.5; P < .001). In addition, SARS-CoV-2-specific B cell responses were shown to be IL-21 dependent. Taken together, we show that IL-21 signaling is important in eliciting robust B cell-mediated immune responses in patients with kidney disease and KTR.

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.

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.

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.

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.

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.

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.

Adipose Tissue-Derived Mesenchymal Stem Cells Have a Heterogenic Cytokine Secretion Profile.

Mesenchymal stem cells derived from adipose tissue (ASC) have immune regulatory function, which makes them interesting candidates for cellular therapy. ASC cultures are however heterogeneous in phenotype. It is unclear whether all ASC contribute equally to immunomodulatory processes. ASC are also responsive to cytokine stimulation, which may affect the ratio between more and less potent ASC populations. In the present study, we determined IL-6 receptor (CD126 and CD130 subunits) and IFN-γ receptor (CD119) expression on ASC by flow cytometry. The production of IL-6 and IFN-γ was measured by ELISA and the frequency of IL-6 and IFN-γ secreting cells by ELISPOT. The results showed that ASC did not express CD126, and only 10-20% of ASC expressed CD130 on their surface, whereas 18-31% of ASC expressed CD119. ASC produced high levels of IL-6 and 100% of ASC were capable of secreting IL-6. Stimulation by IFN-γ or TGF-ß had no effect on IL-6 secretion by ASC. IFN-γ was produced by only 1.4% of ASC, and TGF-ß significantly increased the frequency to 2.7%. These results demonstrate that ASC cultures are heterogeneous in their cytokine secretion and receptor expression profiles. This knowledge can be employed for selection of potent, cytokine-producing, or responsive ASC subsets for cellular immunotherapy.

Humoral and cellular response after varicella vaccination in VZV IgG seronegative kidney transplant candidates.

BACKGROUND: In immunocompromised patients, primary infection with VZV may have a disastrous clinical course. Vaccination of VZV-seronegative patients on the waiting list for renal transplantation may prevent severe disease. However, the immunologic response of end-stage renal disease patients to peptide vaccines is far from optimal. Our question was whether end-stage renal disease patients with undetectable VZV-IgG levels were able to mount an adequate humoral and cellular response to a live attenuated varicella vaccine. METHODS: Kidney transplant candidates with undetectable VZV levels were vaccinated twice with a live attenuated varicella vaccine at an interval of 6weeks. VZV IgG levels were analysed till 2years after vaccination. The VZV-specific T-cell reactivity was determined prior to vaccination and after transplantation. RESULTS: Seventy-seven percent (40/52) of the vaccinees reached positive VZV-IgG levels after vaccination (responders). Eighty-two percent (9/11) showed an increase in VZV-specific CD4+ memory T-cells (both central and effector memory cells). The percentage VZV-specific CD8+ memory T-cells did not increase. None of the non-responders suffered from primary VZV after transplantation. No severe vaccine-related adverse events were reported, only spontaneously resolving local skin irritation. CONCLUSION: The live attenuated varicella vaccine evokes positive VZV IgG-levels and VZV-specific memory T-cells in VZV-seronegative potential kidney transplant candidates.

Herpes zoster after lung transplantation boosts varicella zoster virus-specific adaptive immune responses.

BACKGROUND: Varicella zoster virus (VZV)-specific memory T cells are significantly lower in transplant recipients than in controls. In addition, VZV-specific immunoglobulin G titers are significantly lower after than before transplantation. Data on the incidence and timing of herpes zoster (HZ) after lung transplantation are limited. This study had two aims: first, we investigated the incidence and severity of HZ after lung transplantation; second, we determined the systemic VZV-specific T-cell and B-cell memory responses before and after HZ. METHODS: The records of 119 patients who underwent transplantation were analyzed for post-transplant HZ. The VZV-specific B-cell and T-cell memory responses of 5 patients before and after HZ were compared with 5 patients without HZ by enzyme-linked immunospot assay and flow cytometry, respectively. RESULTS: HZ was clinically diagnosed and confirmed by polymerase chain reaction on blister fluids and/or plasma in 17 transplant recipients. Uncomplicated cutaneous HZ was present in 12 patients, and 5 patients had disseminated HZ, of whom 1 died. The incidence of HZ after transplantation (38.2 cases/1,000 patient-years) was significantly higher than the age-matched healthy population (7-8 cases/1,000 patient-years). The frequency of VZV-specific immunoglobulin G-producing B cells (p = 0.06) and the percentage of VZV-specific CD4 and CD8 memory T cells increased after HZ to higher frequencies than in patients without HZ (p = 0.03). This was mainly attributed to VZV-reactive effector memory CD4 T cells (p = 0.02) and central memory (p = 0.02) and effector memory (p = 0.03) CD8 T cells. CONCLUSIONS: Lung transplant recipients are highly prone to develop HZ with severe complications. Despite deep immunosuppression, HZ boosted their systemic VZV-specific B-cell and T-cell memory responses.

An immunological basis for chronic histiocytic intervillositis in recurrent fetal loss.

PROBLEM: Chronic histiocytic intervillositis (CHIV) is a rare type of placental pathology that is associated with reproductive loss at all gestational ages. The aim of the study was to investigate the relationship between the severity of CHIV and the outcome of pregnancy and to compare the immune response between CHIV patients and controls to explore an immunological origin of CHIV. METHOD OF STUDY: Microscopic slides were reviewed and scored according to a previously published grading system in 30 pregnancies of 22 CHIV patients. Partner-specific mixed lymphocyte reactions, cytotoxic T-lymphocyte precursor frequencies (CTLpf), and anti-HLA antibodies were determined in four patients and seven controls. RESULTS: Higher CHIV scores are associated with worse pregnancy outcome. CHIV patients demonstrated a higher CTLpf against their partner compared to non-complicated pregnancies (P = 0.03). The CTLpf was extremely high in 75% of the patients. Antipaternal HLA antibodies were only present in 75% of the CHIV patients compared to none of the controls (P = 0.02). CONCLUSION: CHIV scores seem to be associated with the severity of adverse pregnancy outcome. High antipaternal cellular (T-cell) and humoral (B-cell) response to partner-specific CTLpf and the presence of anti-HLA antibodies directed to the partner suggest an immunologic origin of CHIV.

Human mesenchymal stem cells are susceptible to lysis by CD8(+) T cells and NK cells.

There is growing interest in the use of mesenchymal stem cells (MSCs) to improve the outcome of organ transplantation. The immunogenicity of MSCs is, however, unclear and is important for the efficacy of MSC therapy and for potential sensitization against donor antigens. We investigated the susceptibility of autologous and allogeneic MSCs for lysis by CD8(+) T-lymphocytes and NK cells in a kidney transplant setting. MSCs were derived from adipose tissue of human kidney donors and were CD90(+), CD105(+), CD166(+), and HLA class I(+). They showed differentiation ability and immunosuppressive capacity. Lysis of MSCs by peripheral blood mononuclear cells (PBMCs), FACS-sorted CD8(+) T cells, and NK cells was measured by europium release assay. Allogeneic MSCs were susceptible for lysis by cytotoxic CD8(+) T cells and NK cells, while autologous MSCs were lysed by NK cells only. NK cell-mediated lysis was inversely correlated with the expression of HLA class I on MSCs. Lysis of autologous MSCs was not dependent on culturing of MSCs in FBS, and MSCs in suspension as well as adherent to plastic were lysed by NK cells. Pretransplant recipient PBMCs did not lyse donor MSCs, but PBMCs isolated 3, 6, and 12 months after transplantation showed increasing lysing ability. After 12 months, CD8(+) T-cell-mediated lysis of donor MSCs persisted, indicating there was no evidence for desensitization against donor MSCs. Lysis of MSCs is important to take into account when MSCs are considered for clinical application. Our results suggest that the HLA background of MSCs and timing of MSC administration are important for the efficacy of MSC therapy.

Deficient TNF-alpha and IFN-gamma production correlates with nondetectable donor-specific cytotoxicity after clinical kidney transplantation.

BACKGROUND: We previously reported that no cytotoxic T-lymphocyte precursor frequencies (CTLpf) were found in 60% of patients on azathioprine or mycophenolate mofetil+Pred long after kidney transplantation. We questioned whether the absence of donor-specific CTLpf was associated with low levels of stimulatory Th1 (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma) or high levels of regulatory Th2 (interleukin [IL]-4, IL-6, IL-10) cytokines. METHODS: In this study, peripheral blood monocyte cells (PBMC) were stimulated with irradiated donor cells. After 7 days, cytokine production was determined by cytokine bead array, and CTLpf by limiting dilution assay. RESULTS: Patients with detectable CTLpf (> or =10/10(6) PBMC) had significantly higher levels of TNF-alpha (P=0.04) and IFN-gamma (P=0.02) than patients with nondetectable CTLpf (<10/10(6) PBMC). Donor-reactive IL-4, IL-6, and IL-10 production was comparable in both patient groups. Additionally, CTLpf was positively correlated with TNF-alpha (rs=0.54, P=0.0003) and IFN-gamma (rs=0.64, P<0.0001) production. CONCLUSION: The absence of donor-specific CTLp after transplantation correlates with low levels of stimulatory cytokines, not with elevated levels of regulatory cytokines.

After discontinuation of calcineurin inhibitors, tapering of mycophenolate mofetil further impairs donor-directed cytotoxicity.

BACKGROUND: Recently, we described a significant decrease in donor-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) after discontinuation of calcineurin inhibitors (CNI), while the proliferative capacity in mixed lymphocyte culture (MLC), and the number of interferon-gamma (IFN-gamma) producing cells (pc) in Elispot remained unchanged. METHODS: We tested T-cell reactivity in CNI free patients with stable renal graft function, on mycophenolate mofetil (MMF) or azathioprine (AZA) plus prednisone, who were tapered to 50% of their MMF or AZA dose. RESULTS: Furthermore, tapering of the MMF or AZA dose resulted in a decrease of donor-reactive CTLpf in all patients with detectable CTLpf. Detectable numbers decreased from a median of 32 to 8 CTLp/10(6) peripheral blood mononuclear cell (PBMC). No effect on third-party reactive CTLpf was found, while the T-cell reactivity to donor and third-party cells as tested in MLC and in IFN-gamma Elispot was not affected either by tapering of immunosuppression. Third-party reactivity was significantly higher than donor-specific reactivity in all tests. A control group showed no changes in any of the in vitro assays. CONCLUSION: Both withdrawal of CNI and tapering of MMF or AZA dose decreases the donor-specific CTLpf. Our data suggest that reduction of immunosuppression results in a specific decrease of donor-directed cytotoxic capacity of immunocompetent cells, while their proliferation and cytokine production capacity remained unchanged. Immunosuppression hinders development of cytotoxic non-responsiveness.

Interleukin-21: an interleukin-2 dependent player in rejection processes.

BACKGROUND: Interleukin (IL)-21 is the most recently described cytokine that signals via the common cytokine receptor (gammac), is produced by activated CD4+ T-cells, and regulates expansion and effector function of CD8+ T-cells. MATERIALS: To explore the actions of IL-21 with other gammac-dependent cytokines in alloreactivity, mRNA expression of IL-21, IL-21R alpha-chain, and IL-2 proliferation and cytotoxicity was measured after stimulation in mixed lymphocyte reactions. Additionally, IL-21 and IL-21R alpha-chain expression was studied in biopsies of heart transplant patients. RESULTS: Analysis of mRNA expression levels of allostimulated T-cells showed a 10-fold induction of IL-21 and IL-21R alpha-chain. Interestingly, induction of IL-21 was highly dependent on IL-2 (as in the presence of anti-IL-2, anti-IL-2R alpha-chain, and the immunosuppressive drugs cyclosporine A, tacrolimus, and rapamycin) the transcription of IL-21 was almost completely inhibited, whereas in the presence of exogenous IL-2 the mRNA expression of IL-21 was even more upregulated. IL-21 functioned as a costimulator for IL-2 to augment proliferation and cytotoxic responses, while blockade of the IL-2 route abrogated these functions of IL-21. Blockade of the IL-21 route by anti-IL-21R alpha-chain monoclonal antibodies inhibited the proliferation of alloactivated T-cells. Also, in vivo alloreactivity was associated with IL-21/IL-21R alpha-chain expression. After heart transplantation, the highest intragraft IL-21, IL-21R alpha-chain, and IL-2 mRNA expression levels were measured during acute rejection (P<0.001, P=0.01, P=0.03). CONCLUSION: IL-21 is a critical cytokine for IL-2 dependent immune processes. Blockade of the IL-21 pathway may provide a new perspective for the treatment of allogeneic responses in patients after transplantation.