On Patients Who Purchase Organ Transplants Abroad.

The international transplant community portrays organ trade as a growing and serious crime involving large numbers of traveling patients who purchase organs. We present a systematic review about the published number of patients who purchased organs. With this information, we discuss whether the scientific literature reflects a substantial practice of organ purchase. Between 2000 and 2015, 86 studies were published. Seventy-six of these presented patients who traveled and 42 stated that the transplants were commercial. Only 11 studies reported that patients paid, and eight described to what or whom patients paid. In total, during a period of 42 years, 6002 patients have been reported to travel for transplantation. Of these, only 1238 were reported to have paid for their transplants. An additional unknown number of patients paid for their transplants in their native countries. We conclude that the scientific literature does not reflect a large number of patients buying organs. Organ purchases were more often assumed than determined. A reporting code for transplant professionals to report organ trafficking networks is a potential strategy to collect and quantify cases.

A Randomized Controlled Trial Comparing the Efficacy of Cyp3a5 Genotype-Based With Body-Weight-Based Tacrolimus Dosing After Living Donor Kidney Transplantation.

Patients expressing the cytochrome P450 (CYP) 3A5 gene require a higher tacrolimus dose to achieve therapeutic exposure compared with nonexpressers. This randomized-controlled study investigated whether adaptation of the tacrolimus starting dose according to CYP3A5 genotype increases the proportion of kidney transplant recipients being within the target tacrolimus predose concentration range (10-15 ng/mL) at first steady-state. Two hundred forty living-donor, renal transplant recipients were assigned to either receive a standard, body-weight-based or a CYP3A5 genotype-based tacrolimus starting dose. At day 3, no difference in the proportion of patients having a tacrolimus exposure within the target range was observed between the standard-dose and genotype-based groups: 37.4% versus 35.6%, respectively; p = 0.79. The proportion of patients with a subtherapeutic (i.e. <10 ng/mL) or a supratherapeutic (i.e. >15 ng/mL) Tac predose concentration in the two groups was also not significantly different. The incidence of acute rejection was comparable between both groups (p = 0.82). Pharmacogenetic adaptation of the tacrolimus starting dose does not increase the number of patients having therapeutic tacrolimus exposure early after transplantation and does not lead to improved clinical outcome in a low immunological risk population.

Human adipose tissue-derived mesenchymal stem cells abrogate plasmablast formation and induce regulatory B cells independently of T helper cells.

Mesenchymal or stromal stem cells (MSC) interact with cells of the immune system in multiple ways. Modulation of the immune system by MSC is believed to be a therapeutic option for autoimmune disease and transplant rejection. In recent years, B cells have moved into the focus of the attention as targets for the treatment of immune disorders. Current B-cell targeting treatment is based on the indiscriminate depletion of B cells. The aim of this study was to examine whether human adipose tissue-derived MSC (ASC) interact with B cells to affect their proliferation, differentiation, and immune function. ASC supported the survival of quiescent B cells predominantly via contact-dependent mechanisms. Coculture of B cells with activated T helper cells led to proliferation and differentiation of B cells into CD19(+) CD27(high) CD38(high) antibody-producing plasmablasts. ASC inhibited the proliferation of B cells and this effect was dependent on the presence of T cells. In contrast, ASC directly targeted B-cell differentiation, independently of T cells. In the presence of ASC, plasmablast formation was reduced and IL-10-producing CD19(+) CD24(high) CD38(high) B cells, known as regulatory B cells, were induced. These results demonstrate that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B-cell response in immune disease.

Interviews With Patients Who Traveled From Macedonia/Kosovo, The Netherlands, and Sweden for Paid Kidney Transplantations.

BACKGROUND: Patients travel worldwide for paid kidney transplants. Although transplantations abroad are not always illegal, they are commonly perceived to be illegal and unethical involving risks. AIM: We aimed to describe the motivations and experiences of patients who traveled abroad for paid kidney transplantations and to examine how these transplantations were facilitated. METHODS: We interviewed 22 patients who traveled from Macedonia/Kosovo, the Netherlands, and Sweden for paid kidney transplantations between years 2000 and 2009. RESULTS: Patients traveled because of inadequate transplant activity in their domestic countries and dialysis-related complaints. However, 6 patients underwent preemptive transplantations. Cultural factors such as patients' affinity with destination countries, feelings of being discriminated against by the health-care system, and family ties also help explain why patients travel abroad. Seven of the 22 patients went to their country of origin. They were able to organize their transplantations by arranging help from family and friends abroad who provided contacts of caregivers there and who helped cover the costs of their transplants. The costs varied from €5000 to €45 000 (US$6800-US$61 200). Seven patients paid the hospital, 5 paid their doctor, 4 paid a broker, and 6 paid their donors. CONCLUSION: Research should include interviews with brokers, transplant professionals, and other facilitators to achieve a full picture of illegally performed transplantations.

Reporting Organ Trafficking Networks: A Survey-Based Plea to Breach the Secrecy Oath.

Patients travel worldwide to purchase kidneys. Transplant professionals can play a role in identifying kidney purchase. However, due to the tension between their rights and obligations, a lack of understanding and knowledge exists on how to prevent and report purchase. We present the results of a national survey that describes transplant professionals' experiences, attitudes, behaviors, conflicts of duties, legal knowledge and needs for guidelines toward patients who purchase kidneys abroad. Second, we clarify professionals' rights and obligations regarding organ purchase and propose actions that they can take to report purchase. Of the 100/241 (42%) professionals who treated patients who traveled to a country outside the European Union for a kidney transplant, 31 (31%) were certain that patients purchased kidneys. Sixty-five (65%) had suspicions that patients had bought kidneys. The majority reported a conflict of duties. Eighty percent reported a need for guidelines. Professionals can help prevent organ purchase by disclosing information about organ trafficking networks to law enforcement. Such disclosure can support the investigation and prosecution of networks. We offer key components for guidelines on disclosure of these networks.

Mental health among living kidney donors: a prospective comparison with matched controls from the general population.

The impact of living kidney donation on donors' mental health has not been sufficiently nor comprehensively studied. Earlier studies demonstrated that mental health did not change in the majority of donors, however they often lacked a suitable control group and/or had other methodological limitations. Consequently, it remains unclear whether changes in mental health found among a minority of donors reflect normal fluctuations. In this study we matched 135 donors with individuals from the general Dutch population on gender and baseline mental health and compared changes in mental health over time. Mental health was measured using the Brief Symptom Inventory and Mental Health Continuum Short Form. Primary analyses compared baseline and 6 months follow-up. Secondary analyses compared baseline and 9 (controls) or 15 months (donors) follow-up. Primary multilevel regression analyses showed that there was no change in psychological complaints (p = 0.20) and wellbeing (p = 0.10) over time and donors and controls did not differ from one another in changes in psychological complaints (p = 0.48) and wellbeing (p = 0.85). Secondary analyses also revealed no difference in changes between the groups. We concluded that changes in mental health in the short term after donation do not significantly differ from normal fluctuations found in the Dutch general population.

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.

Follicular T helper cells and humoral reactivity in kidney transplant patients.

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 (CD4(POS) CXCR5(POS) 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.

Home-based family intervention increases knowledge, communication and living donation rates: a randomized controlled trial.

Our aim was to develop and test an educational program to support well-informed decision making among patients and their social network regarding living donor kidney transplantation (LDKT). One hundred sixty-three patients who were unable to find a living donor were randomized to standard care or standard care plus home-based education. In the education condition, patients and members of their social network participated in home-based educational meetings and discussed renal replacement therapy options. Patients and invitees completed pre-post self-report questionnaires measuring knowledge, risk perception, communication, self-efficacy and subjective norm. LDKT activities were observed for 6 months postintervention. Patients in the experimental group showed significantly more improvements in knowledge (p < 0.001) and communication (p = 0.012) compared with the control group. The invitees showed pre-post increases in knowledge (p < 0.001), attitude toward discussing renal replacement therapies (p = 0.020), attitude toward donating a kidney (p = 0.023) and willingness to donate a kidney (p = 0.039) and a decrease in risk perception (p = 0.003). Finally, there were significantly more inquiries (29/39 vs. 13/41, p < 0.001), evaluations (25/39 vs. 7/41, p < 0.001) and actual LDKTs (17/39 vs. 4/41, p = 0.003) in the experimental group compared with the control group. Home-based family education supports well-informed decision making and promotes access to LDKT.

The protein kinase C inhibitor sotrastaurin allows regulatory T cell function.

The novel immunosuppressant sotrastaurin is a selective inhibitor of protein kinase C isoforms that are critical in signalling pathways downstream of the T cell receptor. Sotrastaurin inhibits nuclear factor (NF)-κB, which directly promotes the transcription of forkhead box protein 3 (FoxP3), the key regulator for the development and function of regulatory T cells (Tregs). Our center participated in a randomized trial comparing sotrastaurin (n = 14) and the calcineurin inhibitor Neoral (n = 7) in renal transplant recipients. We conducted ex vivo mixed lymphocyte reaction (MLR) and flow cytometry studies on these patient samples, as well as in vitro studies on samples of blood bank volunteers (n = 38). Treg numbers remained stable after transplantation and correlated with higher trough levels of sotrastaurin (r = 0·68, P = 0·03). A dose-dependent effect of sotrastaurin on alloresponsiveness was observed: the half maximal inhibitory concentration (IC50 ) to inhibit alloactivated T cell proliferation was 45 ng/ml (90 nM). In contrast, Treg function was not affected by sotrastaurin: in the presence of in vitro-added sotrastaurin (50 ng/ml) Tregs suppressed the proliferation of alloactivated T effector cells at a 1:5 ratio by 35 versus 47% in the absence of the drug (P = 0·33). Signal transducer and activator of transcription 5 (STAT)-5 phosphorylation in Tregs remained intact after incubation with sotrastaurin. This potent Treg function was also found in cells of patients treated with sotrastaurin: Tregs inhibited the anti-donor response in MLR by 67% at month 6, which was comparable to pretransplantation (82%). Sotrastaurin is a potent inhibitor of alloreactivity in vitro, while it did not affect Treg function in patients after kidney transplantation.

Treatment of steroid-resistant acute renal allograft rejection with alemtuzumab.

Steroid-resistant renal allograft rejections are commonly treated with rabbit antithymocyte globulin (RATG), but alemtuzumab could be an effective, safe and more convenient alternative. Adult patients with steroid-resistant renal allograft rejection treated with alemtuzumab (15-30 mg s.c. on 2 subsequent days) from 2008 to 2012 (n = 11) were compared to patients treated with RATG (2.5-4.0 mg/kg bodyweight i.v. for 10-14 days; n = 20). We assessed treatment-failure (graft loss, lack of improvement of graft function or need for additional anti-rejection treatment), infections during the first 3 months after treatment and infusion-related side effects. In both groups, the median time-interval between rejection and transplantation was 2 weeks, and approximately 75% of rejections were classified as Banff-IIA or higher. Three alemtuzumab-treated patients (27%) experienced treatment failure, compared to eight RATG treated patients (40%, p = 0.70). There was no difference in the incidence of infections. There were mild infusion-related side-effects in three alemtuzumab-treated patients (27%), and more severe infusion-related side effects in 17 RATG-treated patients (85%, p = 0.013). Drug related costs of alemtuzumab-treatment were lower than of RATG-treatment (€1050 vs. €2024; p < 0.01). Alemtuzumab might be an effective therapy for steroid-resistant renal allograft rejections. In contrast to RATG, alemtuzumab is nearly devoid of infusion-related side-effects. These data warrant a prospective trial.

Tacrolimus predose concentrations do not predict the risk of acute rejection after renal transplantation: a pooled analysis from three randomized-controlled clinical trials(†).

Therapeutic drug monitoring (TDM) for tacrolimus (Tac) is universally applied. However, the concentration-effect relationship for Tac is poorly defined. This study investigated whether Tac concentrations are associated with acute rejection in kidney transplant recipients. Data from three large trials were pooled. We used univariate and multivariate analysis to investigate the relationship between biopsy-proven acute rejection (BPAR) and Tac predose concentration at five time points (day 3, 10 and 14, and month 1 and 6 after transplantation). A total of 136/1304 patients experienced BPAR, giving an overall incidence of 10.4%. We did not find any significant correlations between Tac predose concentrations and the incidence of BPAR at the different time points. In the multivariate analysis, only delayed graft function (DGF) and the use of induction therapy were independently correlated with BPAR, with an odds ratio of 2.7 [95% CI: 1.8-4.0; p < 0.001] for DGF and 0.66 [95% CI: 0.44-0.99; p = 0.049] for induction therapy. The other variables, including the Tac predose concentrations, were not statistically significantly associated with BPAR. We did not find an association between the Tac predose concentrations measured at five time points after kidney transplantation and the incidence of acute rejection occurring thereafter. Based on this study it is not possible to define the optimal target concentrations for Tac.

Mesenchymal stem cells control alloreactive CD8(+) CD28(-) T cells.

CD28/B7 co-stimulation blockade with belatacept prevents alloreactivity in kidney transplant patients. However, cells lacking CD28 are not susceptible to belatacept treatment. As CD8(+) CD28(-) T-cells have cytotoxic and pathogenic properties, we investigated whether mesenchymal stem cells (MSC) are effective in controlling these cells. In mixed lymphocyte reactions (MLR), MSC and belatacept inhibited peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent manner. MSC at MSC/effector cell ratios of 1:160 and 1:2·5 reduced proliferation by 38·8 and 92·2%, respectively. Belatacept concentrations of 0·1 µg/ml and 10 µg/ml suppressed proliferation by 20·7 and 80·6%, respectively. Both treatments in combination did not inhibit each other's function. Allostimulated CD8(+) CD28(-) T cells were able to proliferate and expressed the cytolytic and cytotoxic effector molecules granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. While belatacept did not affect the proliferation of CD8(+) CD28(-) T cells, MSC reduced the percentage of CD28(-) T cells in the proliferating CD8(+) T cell fraction by 45·9% (P = 0·009). CD8(+) CD28(-) T cells as effector cells in MLR in the presence of CD4(+) T cell help gained CD28 expression, an effect independent of MSC. In contrast, allostimulated CD28(+) T cells did not lose CD28 expression in MLR-MSC co-culture, suggesting that MSC control pre-existing CD28(-) T cells and not newly induced CD28(-) T cells. In conclusion, alloreactive CD8(+) CD28(-) T cells that remain unaffected by belatacept treatment are inhibited by MSC. This study indicates the potential of an MSC-belatacept combination therapy to control alloreactivity.

Human adipose-tissue derived mesenchymal stem cells induce functional de-novo regulatory T cells with methylated FOXP3 gene DNA.

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft-versus-host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC-induced CD4(+) CD25(+) CD127(-) forkhead box P3 (FoxP3)(+) T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT(reg)). Perirenal adipose-tissue derived MSC (ASC) obtained from kidney donors induced a 2·1-fold increase in the percentage of CD25(+) CD127(-) FoxP3(+) cells within the CD4(+) T cell population from allostimulated CD25(-/dim) cells. Interleukin (IL)-2 receptor blocking prevented this induction. The ASC-induced T cells (iT(reg)) inhibited effector cell proliferation as effectively as nT(reg). The vast majority of cells within the iT(reg) fraction had a methylated FOXP3 gene T(reg)-specific demethylated region (TSDR) indicating that they were not of nT(reg) origin. In conclusion, ASC induce T(reg) from effector T cells. These iT(reg) have immunosuppressive capacities comparable to those of nT(reg). Their induction is IL-2 pathway-dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de-novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.

Cytomegalovirus contributes partly to uraemia-associated premature immunological ageing of the T cell compartment.

Cytomegalovirus (CMV) infection has been implicated in accelerated T cell ageing. End-stage renal disease (ESRD) patients have a severely immunologically aged T cell compartment but also a high prevalence of CMV infection. We investigated whether CMV infection contributes to T cell ageing in ESRD patients. We determined the thymic output by the T cell receptor excision circle (TREC) content and percentage of CD31+ naïve T cells. The proliferative history of the T cell compartment by determination of the relative telomere length (RTL) and the T cell differentiation status was determined by immunophenotyping. It appeared that CMV infection did not affect thymic output but reduced RTL of CD8+ T cells in ESRD patients. Moreover, increased T cell differentiation was observed with higher percentages of CD57+ and CD28null CD4+ and CD8+ memory T cells. These CD28null T cells had significantly shorter telomeres compared to CD28+ T cells. Therefore we concluded that CMV infection does not affect the decreased thymic output but increases T cell differentiation as observed in ESRD-related premature T cell ageing.

Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

A criminological perspective: why prohibition of organ trade is not effective and how the Declaration of Istanbul can move forward.

The Declaration of Istanbul is the first document that has been established by the international transplant community that defines and prohibits transplant commercialism and organ trafficking. Its Custodian Group has successfully led various countries to implement legislation against trafficking and commercialism. The question arises, however, whether efforts to prohibit organ trade are realistic and effective. The Declaration differentiates trafficking from commercialism, yet it does not mention how both acts should be approached by policy. Policies that address transplant commercialism work differently from policies that tackle organ trafficking. There is considerable room for improvement in the current prohibitive approach to commercialism and organ trafficking. The Custodian Group and World Health Organization (WHO) should address commercialism by encouraging the expansion of living donation in the same manner as they encourage deceased donation. Furthermore, the Custodian Group and the WHO can improve their strategy to combat organ trafficking by raising awareness for enforcement. To achieve a consistent and effective prohibition of trafficking, legislation and law enforcement must go hand in hand. Ideally, this can best be achieved by close collaboration between the medical field and (international) criminal justice agencies.

Randomized phase 2b trial of tofacitinib (CP-690,550) in de novo kidney transplant patients: efficacy, renal function and safety at 1 year.

In this Phase 2b study, 331 low-to-moderate risk de novo kidney transplant patients (approximately 60% deceased donors) were randomized to a more intensive (MI) or less intensive (LI) regimen of tofacitinib (CP-690, 550), an oral Janus kinase inhibitor or cyclosporine (CsA). All patients received basiliximab induction, mycophenolic acid and corticosteroids. Primary endpoints were: incidence of biopsy-proven acute rejection (BPAR) with a serum creatinine increase of ≥0.3 mg/dL and ≥20% (clinical BPAR) at Month 6 and measured GFR at Month 12. Similar 6-month incidences of clinical BPAR (11%, 7% and 9%) were observed for MI, LI and CsA. Measured GFRs were higher (p < 0.01) at Month 12 for MI and LI versus CsA (65 mL/min, 65 mL/min vs. 54 mL/min). Fewer (p < 0.05) patients in MI or LI developed chronic allograft nephropathy at Month 12 compared with CsA (25%, 24% vs. 48%). Serious infections developed in 45%, 37% and 25% of patients in MI, LI and CsA, respectively. Anemia, neutropenia and posttransplant lymphoproliferative disorder occurred more frequently in MI and LI compared with CsA. Tofacitinib was equivalent to CsA in preventing acute rejection, was associated with improved renal function and less chronic allograft histological injury, but had side-effects at the doses evaluated.

A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.

Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B-cell hybridomas producing HLA antibodies. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non-HLA-immunized individuals and transplant patients with serum HLA antibodies. B-cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA-immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while nonimmunized individuals had none. Patients who were immunized by an HLA-A2-mismatched graft had up to 143 HLA-A2-specific B cells per million total B cells. In conclusion, we have developed and validated a highly specific and sensitive HLA-specific B-cell ELISPOT assay, which needs further validation in a larger series of transplant patients. This technique constitutes a new tool for quantifying humoral immune responses.