Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

High-yield identification of pathogenic NF1 variants by skin fibroblast transcriptome screening after apparently normal diagnostic DNA testing.

Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.

A point mutation in the pre-ZRS disrupts sonic hedgehog expression in the limb bud and results in triphalangeal thumb-polysyndactyly syndrome.

PURPOSE: The zone of polarizing activity regulatory sequence (ZRS) is an enhancer that regulates sonic hedgehog during embryonic limb development. Recently, mutations in a noncoding evolutionary conserved sequence 500 bp upstream of the ZRS, termed the pre-ZRS (pZRS), have been associated with polydactyly in dogs and humans. Here, we report the first case of triphalangeal thumb-polysyndactyly syndrome (TPT-PS) to be associated with mutations in this region and show via mouse enhancer assays how this mutation leads to ectopic expression throughout the developing limb bud. METHODS: We used linkage analysis, whole-exome sequencing, Sanger sequencing, fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, single-nucleotide polymorphism array, and a mouse transgenic enhancer assay. RESULTS: Ten members of a TPT-PS family were included in this study. The mutation was linked to chromosome 7q36 (LOD score 3.0). No aberrations in the ZRS could be identified. A point mutation in the pZRS (chr7:156585476G>C; GRCh37/hg19) was detected in all affected family members. Functional characterization using a mouse transgenic enhancer essay showed extended ectopic expression dispersed throughout the entire limb bud (E11.5). CONCLUSION: Our work describes the first mutation in the pZRS to be associated with TPT-PS and provides functional evidence that this mutation leads to ectopic expression of this enhancer within the developing limb.

Aging of bone marrow- and umbilical cord-derived mesenchymal stromal cells during expansion.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are used as experimental immunotherapy. Extensive culture expansion is necessary to obtain clinically relevant cell numbers, although the impact on MSCs stability and function is unclear. This study investigated the effects of long-term in vitro expansion on the stability and function of MSCs. METHODS: Human bone marrow-derived (bmMSCs) and umbilical cord-derived (ucMSCs) MSCs were in vitro expanded. During expansion, their proliferative capacity was examined. At passages 4, 8 and 12, analyses were performed to investigate the ploidy, metabolic stability, telomere length and immunophenotype. In addition, their potential to suppress lymphocyte proliferation and susceptibility to natural killer cell lysis was examined. RESULTS: BmMSCs and ucMSCs showed decreasing proliferative capacity over time, while their telomere lengths and mitochondrial activity remained stable. Percentage of aneuploidy in cultures was unchanged after expansion. Furthermore, expression of MSC markers and markers associated with stress or aging remained unchanged. Reduced capacity to suppress CD4 and CD8 T-cell proliferation was observed for passage 8 and 12 bmMSCs and ucMSCs. Finally, susceptibility of bmMSCs and ucMSCs to NK-cell lysis remained stable. CONCLUSIONS: We showed that after long-term expansion, phenotype of bmMSCs and ucMSCs remains stable and cells exhibit similar immunogenic properties compared with lower passage cells. However, immunosuppressive properties of MSCs are reduced. These findings reveal the consequences of application of higher passage MSCs in the clinic, which will help increase the yield of therapeutic MSCs but may interfere with their efficacy.

Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Congenital diaphragmatic hernia (CDH) is a common life-threatening birth defect. Recessive mutations in the FRAS1-related extracellular matrix 1 (FREM1) gene have been shown to cause bifid nose with or without anorectal and renal anomalies (BNAR) syndrome and Manitoba oculotrichoanal (MOTA) syndrome, but have not been previously implicated in the development of CDH. We have identified a female child with an isolated left-sided posterolateral CDH covered by a membranous sac who had no features suggestive of BNAR or MOTA syndromes. This child carries a maternally-inherited ~86 kb FREM1 deletion that affects the expression of FREM1's full-length transcripts and a paternally-inherited splice site mutation that causes activation of a cryptic splice site, leading to a shift in the reading frame and premature termination of all forms of the FREM1 protein. This suggests that recessive FREM1 mutations can cause isolated CDH in humans. Further evidence for the role of FREM1 in the development of CDH comes from an N-ethyl-N-nitrosourea -derived mouse strain, eyes2, which has a homozygous truncating mutation in Frem1. Frem1(eyes2) mice have eye defects, renal agenesis and develop retrosternal diaphragmatic hernias which are covered by a membranous sac. We confirmed that Frem1 is expressed in the anterior portion of the developing diaphragm and found that Frem1(eyes2) embryos had decreased levels of cell proliferation in their developing diaphragms when compared to wild-type embryos. We conclude that FREM1 plays a critical role in the development of the diaphragm and that FREM1 deficiency can cause CDH in both humans and mice.

Biallelic NDC1 variants that interfere with ALADIN binding are associated with neuropathy and Triple-A-like syndrome.

Nuclear pore complexes (NPC) regulate nucleocytoplasmic transport and are anchored in the nuclear envelope by the transmembrane nucleoporin NDC1. NDC1 is essential for post-mitotic NPC assembly and the recruitment of ALADIN to the nuclear envelope. While no human disorder has been associated to one of the three transmembrane nucleoporins, biallelic variants in AAAS, encoding ALADIN, cause Triple-A syndrome (Allgrove syndrome). Triple A syndrome, characterized by alacrima, achalasia and adrenal insufficiency, often includes progressive demyelinating polyneuropathy and other neurological complaints. In this report, diagnostic exome and/or RNA-sequencing was performed in 7 individuals from 4 unrelated consanguineous families with AAAS negative triple A syndrome. Molecular and clinical studies followed to elucidate the pathogenic mechanism. The affected individuals presented with intellectual disability, motor impairment, severe demyelinating with secondary axonal polyneuropathy, alacrima and achalasia.. None of the affected individuals has adrenal insufficiency. All individuals presented with biallelic NDC1 in-frame deletions or missense variants, that affect amino acids and protein domains required for ALADIN binding. No other significant variants associated with the phenotypic features were reported. Skin fibroblasts derived from affected individuals show decreased recruitment of ALADIN to the NE and decreased post-mitotic NPC insertion, confirming pathogenicity of the variants. Taken together, our results implicate biallelic NDC1 variants in the pathogenesis of polyneuropathy and a Triple-A-like disorder without adrenal insufficiency, by interfering with physiological NDC1 functions, including the recruitment of ALADIN to the NPC.

Culture expansion induces non-tumorigenic aneuploidy in adipose tissue-derived mesenchymal stromal cells.

BACKGROUND AIMS: Adipose tissue-derived mesenchymal stromal cells (ASCs) are of interest as a cell therapeutic agent for immunologic and degenerative diseases. During in vitro expansion, ASCs may be at risk for genetic alterations, and genetic screening is a prerequisite. We examined the presence of aneuploidy in ASCs and its origin and development during culture and evaluated the implications of aneuploidy for therapeutic use of ASCs. METHODS: Adipose tissue of healthy individuals was used for isolation and expansion of ASCs. Chromosome copy numbers were studied using fluorescence in situ hybridization analysis. Aneuploidy was studied in freshly isolated ASCs, in ASCs cultured for 0-16 passages and in senescent cultures. To evaluate the plasticity of ploidy, ASCs were cloned, and the variation of ploidy in the clones was examined. Tumorigenicity was studied by subcutaneous injection of aneuploid ASCs in immunodeficient NOD/SCID mice. RESULTS: No aneuploidy was detected in freshly isolated ASCs. In low passages (passages 0-4), aneuploidy was detected in 3.4% of ASCs. Prolonged culture expansion of ASCs (passages 5-16) resulted in a significant increase of aneuploidy to 7.1%. With senescence, aneuploidy increased further to 19.8%. Aneuploidy was observed in clones of diploid ASCs, demonstrating the de novo development of aneuploidy. No transformation of ASCs was observed, and in contrast to cancer cell lines, aneuploid ASCs were incapable of tumor formation in immunodeficient mice. CONCLUSIONS: ASC cultures contain a stable percentage of aneuploid cells. Aneuploidy was not a predecessor of transformation or tumor formation. This finding indicates that aneuploidy is culture-induced but unlikely to compromise clinical application of ASCs.

Mapping of homozygous deletions in verified esophageal adenocarcinoma cell lines and xenografts.

Human esophageal adenocarcinoma (EAC) cell lines and xenografts are powerful tools in the search for genetic alterations because these models are composed of pure human cancer cell populations without admixture of normal human cells. In particular detection of homozygous deletions (HDs) is easier using these pure populations of cancer cells. Identification of HDs could potentially lead to the subsequent identification of new tumor suppressor genes (TSGs) involved in esophageal adenocarcinogenesis. Genome wide single nucleotide polymorphism (SNP) arrays were used to identify HDs in 10 verified EAC cell lines and nine EAC xenografts. In total, 61 HDs (range 1-6 per sample) were detected and confirmed by polymerase chain reaction. Besides HDs observed in common fragile genomic regions (n = 26), and gene deserts (n = 8), 27 HDs were located in gene-containing regions. HDs were noted for known TSGs, including CDKN2A, SMAD4 and CDH3/CDH1. Twenty-two new chromosomal regions were detected harboring potentially new TSGs involved in EAC carcinogenesis. Two of these regions of homozygous loss, encompassing the ITGAV and RUNX1 gene, were detected in multiple samples indicating a potential role in the carcinogenesis of EAC. To exclude culturing artifacts, these last two deletions were confirmed by fluorescent in situ hybridization in the primary tumors of which the involved cell lines and xenografts were derived. In summary, in this report we describe the identification of HDs in a series of verified EAC cell lines and xenografts. The deletions documented here are a step forward identifying the key genes involved in EAC development.

Mesenchymal stem cells derived from adipose tissue are not affected by renal disease.

Mesenchymal stem cells are a potential therapeutic agent in renal disease and kidney transplantation. Autologous cell use in kidney transplantation is preferred to avoid anti-HLA reactivity; however, the influence of renal disease on mesenchymal stem cells is unknown. To investigate the feasibility of autologous cell therapy in patients with renal disease, we isolated these cells from subcutaneous adipose tissue of healthy controls and patients with renal disease and compared them phenotypically and functionally. The mesenchymal stem cells from both groups showed similar morphology and differentiation capacity, and were both over 90% positive for CD73, CD105, and CD166, and negative for CD31 and CD45. They demonstrated comparable population doubling times, rates of apoptosis, and were both capable of inhibiting allo-antigen- and anti-CD3/CD28-activated peripheral blood mononuclear cell proliferation. In response to immune activation they both increased the expression of pro-inflammatory and anti-inflammatory factors. These mesenchymal stem cells were genetically stable after extensive expansion and, importantly, were not affected by uremic serum. Thus, mesenchymal stem cells of patients with renal disease have similar characteristics and functionality as those from healthy controls. Hence, our results indicate the feasibility of their use in autologous cell therapy in patients with renal disease.

Comparable levels of folate-induced aneusomy in B-lymphoblasts from oral-cleft patients and controls.

BACKGROUND: Peri-conceptional use of folic acid contributes to protection against congenital malformations, such as neural tube defects and cleft lip with or without cleft palate (CL/P). Previous studies showed that low folate levels cause DNA damage, leading to chromosomal instability and aneusomy. This study seeks to confirm this finding and investigates whether the in vitro sensitivity towards aneusomy of chromosome 17 and 21 in the folate-deficient state differs between CL/P patients and controls. METHODS: Epstein-Barr virus-immortalized B-lymphoblasts derived from 15 CL/P children and 15 controls, were cultured in medium with high and low concentrations - approximately 40nM and 5nM - of 5-methyltetrahydrofolate, respectively. Fluorescence in situ hybridization was used to detect specific fluorescence signals for chromosomes 17 and 21. RESULTS: A significant increase in aneusomy of chromosomes 17 (2.3% vs 7.6%; p ≤ 0.001) and 21 (2.5% vs 7.0%; p ≤ 0.001) was observed after 10 days of culturing in low folate. These results were comparable in cell lines from patients and controls. Interestingly, for chromosome 17 the folate deficiency mainly resulted in an increase of monosomy (6%, p ≤ 0.001), while for chromosome 21 the increase of trisomy was larger (4.9%, p ≤ 0.001). CONCLUSIONS: These data suggest that folate deficiency is a significant risk factor in the development of aneusomy and may affect the distribution of chromosomes during cell division. The comparable aneusomy frequencies in CL/P and in controls suggest that other folate-related processes are involved in the pathogenesis of CL/P, and additional investigations are needed to identify the causal mechanisms.

Heritability and De Novo Mutations in Oesophageal Atresia and Tracheoesophageal Fistula Aetiology.

Tracheoesophageal Fistula (TOF) is a congenital anomaly for which the cause is unknown in the majority of patients. OA/TOF is a variable feature in many (often mono-) genetic syndromes. Research using animal models targeting genes involved in candidate pathways often result in tracheoesophageal phenotypes. However, there is limited overlap in the genes implicated by animal models and those found in OA/TOF-related syndromic anomalies. Knowledge on affected pathways in animal models is accumulating, but our understanding on these pathways in patients lags behind. If an affected pathway is associated with both animals and patients, the mechanisms linking the genetic mutation, affected cell types or cellular defect, and the phenotype are often not well understood. The locus heterogeneity and the uncertainty of the exact heritability of OA/TOF results in a relative low diagnostic yield. OA/TOF is a sporadic finding with a low familial recurrence rate. As parents are usually unaffected, de novo dominant mutations seems to be a plausible explanation. The survival rates of patients born with OA/TOF have increased substantially and these patients start families; thus, the detection and a proper interpretation of these dominant inherited pathogenic variants are of great importance for these patients and for our understanding of OA/TOF aetiology.

Unbalanced der(5)t(5;20) translocation associated with megalencephaly, perisylvian polymicrogyria, polydactyly and hydrocephalus.

The combination of megalencephaly, perisylvian polymicrogyria, polydactyly and hydrocephalus (MPPH) is a rare syndrome of unknown cause. We observed two first cousins affected by an MPPH-like phenotype with a submicroscopic chromosome 5q35 deletion as a result of an unbalanced der(5)t(5;20)(q35.2;q13.3) translocation, including the NSD1 Sotos syndrome locus. We describe the phenotype and the deletion breakpoints of the two MPPH-like patients and compare these with five unrelated MPPH and Sotos patients harboring a 5q35 microdeletion. Mapping of the breakpoints in the two cousins was performed by MLPA, FISH, high density SNP-arrays and Q-PCR for the 5q35 deletion and 20q13 duplication. The 5q35 deletion area of the two cousins almost completely overlaps with earlier described patients with an atypical Sotos microdeletion, except for the DRD1 gene. The five unrelated MPPH patients neither showed submicroscopic chromosomal aberrations nor DRD1 mutations. We reviewed the brain MRI of 10 Sotos patients and did not detect polymicrogyria in any of them. In our two cousins, the MPPH-like phenotype is probably caused by the contribution of genes on both chromosome 5q35 and 20q13. Some patients with MPPH may harbor a submicroscopic chromosomal aberration and therefore high-resolution array analysis should be part of the diagnostic workup.

Novel GAA Variants and Mosaicism in Pompe Disease Identified by Extended Analyses of Patients with an Incomplete DNA Diagnosis.

Pompe disease is a metabolic disorder caused by a deficiency of the glycogen-hydrolyzing lysosomal enzyme acid α-glucosidase (GAA), which leads to progressive muscle wasting. This autosomal-recessive disorder is the result of disease-associated variants located in the GAA gene. In the present study, we performed extended molecular diagnostic analysis to identify novel disease-associated variants in six suspected Pompe patients from four different families for which conventional diagnostic assays were insufficient. Additional assays, such as a generic-splicing assay, minigene analysis, SNP array analysis, and targeted Sanger sequencing, allowed the identification of an exonic deletion, a promoter deletion, and a novel splicing variant located in the 5' UTR. Furthermore, we describe the diagnostic process for an infantile patient with an atypical phenotype, consisting of left ventricular hypertrophy but no signs of muscle weakness or motor problems. This led to the identification of a genetic mosaicism for a very severe GAA variant caused by a segmental uniparental isodisomy (UPD). With this study, we aim to emphasize the need for additional analyses to detect new disease-associated GAA variants and non-Mendelian genotypes in Pompe disease where conventional DNA diagnostic assays are insufficient.

MN1 affects expression of genes involved in hematopoiesis and can enhance as well as inhibit RAR/RXR-induced gene expression.

The oncoprotein meningioma 1 (MN1) is overexpressed in several subtypes of acute myeloid leukemia (AML) and overexpression was associated with a poor response to chemotherapy. MN1 is a cofactor of retinoic acid receptor/retinoic x receptor (RAR/RXR)-mediated transcription and this study identified genes in the promonocytic cell line U937 that were regulated by MN1. We found that MN1 can both stimulate and inhibit transcription. Combining MN1 expression with all-trans retinoic acid (ATRA), the ligand of the RAR/RXR dimer, showed that MN1 could both enhance and repress ATRA effects. Many of the identified genes are key players in hematopoiesis and leukemogenesis (e.g. MEIS1 and BMI1). Another interesting target is DHRS9. DHRS9 is involved in the synthesis of ATRA from vitamin A. MN1 inhibited DHRS9 expression and completely abolished its induction by ATRA. MN1 is also the target of a rare AML-causing translocation encoding the MN1-TEL protein. MN1-TEL induces expression of only a few genes and its most pronounced effect is inhibition of a large group of ATRA-induced genes including DHRS9. In conclusion, both MN1 and MN1-TEL interfere with the ATRA pathway and this might explain the differentiation block in leukemias in which these genes are involved.

Molecular cytogenetic analysis of archival uveal melanoma with known clinical outcome.

Uveal melanoma (UM) is the most common primary intraocular tumor in the Western world. Cytogenetically, this tumor is characterized by typical chromosomal aberrations such as loss of 1p, 3, and 6q, and gain of 6p and 8q. Routinely, karyotyping and fluorescent in situ hybridization (FISH) on fresh tumor-biopsies are used to identify chromosomal changes. In addition, archival UM samples can be examined using comparative genomic hybridization (CGH). In the presented study, we used CGH on a series of 46 archival uveal melanomas to identify chromosomal changes. In 44 tumors aberrations were present and classic prognostic markers as loss of 1p (12 tumors, 26.1%), monosomy 3 (26 tumors, 56.5%), loss of 6q (10 tumors, 21.7%), and gain of chromosome arm 8q (27 tumors, 58.7%) were observed. Gain of chromosome arms 18q or 21q was found in three UMs. Multiplex ligation-dependent probe amplification (MLPA), a novel technique in UM, was performed to verify this low number of chromosome 18 and 21 abnormalities, but we could not confirm the previously reported gain of 18q11.2 and 21q11.2 as poor prognostic factors in UM.

Variants in members of the cadherin-catenin complex, CDH1 and CTNND1, cause blepharocheilodontic syndrome.

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

A familial inverted duplication 2q33-q34 identified and delineated by multiple cytogenetic techniques.

We describe a unique family with two children having a delay in psychomotor development. In both children we identified an interstitial duplication dup(2)(q34q33) using multiple, complementary molecular cytogenetic techniques. Comparative genomic hybridisation (CGH) and array-CGH were used to determine the size and the location of the duplicated region, the orientation of the duplicated region was identified with fluorescence in situ hybridisation (FISH). Both parents demonstrated a normal karyotype and normal CGH and array-CGH-profiles. However, FISH on peripheral blood cells from the mother showed the inv dup(2) in 9% of metaphases and 19% of interphase nuclei. To our knowledge this is the first report of a mosaic carrier of duplication in the long arm of chromosome 2. The finding of chromosomal mosaicism of at least 19% in the mother increases the recurrence risk. The exact characterisation of the inv dup(2) with FISH probes enabled us to offer a reliable prenatal FISH test. Comparison of the clinical features of the two children with those of previously described cases supports the hypothesis that the characteristic facial phenotype is linked to the distal part of the 2q33-q37 region. This report illustrates that in case of two sibs with an identical structural chromosomal abnormality the possibility of parental chromosomal mosaicism must be thoroughly investigated.

An Acute Cellular Rejection With Detrimental Outcome Occurring Under Belatacept-Based Immunosuppressive Therapy: An Immunological Analysis.

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