Formalin-Fixed, Paraffin-Embedded-Targeted Locus Capture: A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors.

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded-targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.

PRAME Staining in Sinonasal Mucosal Melanoma: A Single-Center Experience.

BACKGROUND: Sinonasal mucosal melanoma (MM) is a rare, aggressive melanoma subtype. Complete surgical excision, with or without adjuvant radiotherapy, remains the cornerstone of treatment and yields adequate locoregional control. Metastatic MM is managed similarly to metastatic cutaneous melanoma but with poorer survival. PReferentially expressed Antigen in MElanoma (PRAME) has been identified as a potential diagnostic marker and therapeutic target in the treatment of cutaneous melanoma. METHODS: Retrospective analysis of the clinical characteristics and immunohistochemical features of all sinonasal MM patients referred to the department of Head and Neck Surgical Oncology, UMC Utrecht Cancer Center, between 2011 and 2021 was performed. Single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) were performed in selected cases. RESULTS: A total of 26 patients with an MM were included. The median follow-up duration was 15 months. At the end of follow-up, 13 patients had died due to progression of their disease, and one patient died of intercurrent disease. PRAME immunohistochemistry was performed in 23 out of 26 cases, all displaying PRAME expression. In two cases PRAME expression was present both within the melanoma cells and in melanocytes in adjacent mucosa. SNP array showed ≥ 5 copy number variants (CNV) in all tested cases, with a median of 29.5 CNVs (IQR 23.25-40). The three most common mutations identified by NGS were NRAS (7 cases) and NF1 (2 cases). CONCLUSION: We show that expression of PRAME is common in sinonasal MM, making PRAME a useful ancillary diagnostic tool and a potential therapeutic target in sinonasal MM. The demonstrated occurrence of extensive presence of PRAME-positive melanocytes in the surrounding mucosa of sinonasal MM might explain the multifocal nature of melanoma in the (sinonasal) mucosa, and would be an extra argument for a PRAME targeting treatment in preventing local disease recurrence.

Towards diagnostic criteria for malignant deep penetrating melanocytic tumors using single nucleotide polymorphism array and next-generation sequencing.

Cutaneous deep penetrating melanocytic neoplasms frequently simulate melanoma and might occasionally progress to metastatic melanoma. Distinguishing deep penetrating nevi (DPN) and deep penetrating melanocytomas (DPM) from malignant deep penetrating tumors (MDPT) is difficult based on histopathology alone, and diagnostic criteria for MDPT are currently lacking. Using a molecular workup, we aimed to provide readily available diagnostic tools for classification of deep penetrating tumors. We used clinical follow-up and Single Nucleotide Polymorphism (SNP) array for tumor classification of 20 deep penetrating neoplasms to identify associations with histopathological, immunohistochemistry, and NGS findings. Ten neoplasms were classified as MDPT, four as DPM, and six as DPN. Two MDPT showed metastases. The following parameters were statistically significantly associated with MDPT: severe nuclear atypia (risk ratio [RR] 2.9, p < 0.05), absence of a nevus component (RR 10.0, p = 0.04), positive PRAME expression (RR 9.0, p = 0.02), complete loss of p16 expression (RR 3.5, p = 0.003), TERT-p and APC mutations (RR 11.0, p = 0.01 and RR 2.7, p = 0.002, respectively), and ≥1 additional pathogenic mutation (RR 9.0, p = 0.02). Ki-67 expression ≥ 5% was not significantly associated with MDPTs, although it was <5% in all DPNs. Three MDPT did not show nuclear ß-catenin expression despite having a CTNNB1 (n = 2) or an APC mutation (n = 1). Our findings suggest that complete loss of p16 and positive PRAME expression, a driver mutation in APC, ≥ 1 additional pathogenic mutation, especially in TERT-p, support an MDPT diagnosis in deep penetrating neoplasms. Besides severe nuclear atypia and possibly severe inflammation, we did not identify specific histopathological criteria for malignancy. Non-aberrant nuclear ß-catenin expression might not exclude a deep penetrating signature in MDPT.

Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Comel-Netherton syndrome: A local skin barrier defect in the absence of an underlying systemic immunodeficiency.

BACKGROUND: Comel-Netherton syndrome (NS) is a rare autosomal disease, characterized by severe skin disease, hair shaft defects, atopic diathesis, and increased susceptibility for skin infections. Since patients with NS suffer from recurrent infections, it has been hypothesized that an underlying immunodeficiency attributes to this. Here, we studied clinical and immunological characteristics of the cohort of NS patients in the Netherlands in order to identify whether potential immunodeficiencies result in the increased risk of infectious complications. METHODS: Phenotypes were scored for severity of skin condition, specific hair shaft defects, atopy, and recurrent infections. Patients' blood samples were collected for quantification of serum immunoglobulin (Ig) levels, specific antibodies against Streptococcus pneumoniae, and allergen-specific IgE, as well as detailed immunophenotyping of blood leukocyte and lymphocyte subsets by flow cytometry. RESULTS: A total of 14 patients were included with age range 3-46 years and varying degrees of skin involvement. All patients presented with atopic symptoms (food allergy, n = 13; hay fever, n = 10; asthma, n = 7). Recurrent skin infections were common, particularly in childhood (n = 12). Low levels of specific antibodies against S pneumoniae were found in 10 of 11 evaluated patients. Detailed immunological analysis was performed on 9 adult patients. Absolute numbers of lymphocyte subsets and serum immunoglobulin levels were all within normal ranges. CONCLUSION: Multidisciplinary evaluation of our national cohort showed no evidence for a severe, clinically relevant systemic immunodeficiency. Therefore, we conclude that in Dutch NS patients the increased risk of infections most likely results from the skin barrier disruption and that increased allergen penetration predisposes to allergic sensitization.

Responsiveness of chronic lymphocytic leukemia cells to B-cell receptor stimulation is associated with low expression of regulatory molecules of the nuclear factor-κB pathway.

Chronic lymphocytic leukemia (CLL) is a disease with heterogeneous clinical and biological characteristics. Differences in Ca2+ levels among cases, both basal and upon B-cell receptor (BCR) stimulation, may reflect heterogeneity in the pathogenesis due to cell-intrinsic factors. Our aim was to elucidate cell-intrinsic differences between BCR-responsive and -unresponsive cases. We therefore determined BCR responsiveness ex vivo based on Ca2+ influx upon α-IgM stimulation of purified CLL cell fractions from 52 patients. Phosphorylation levels of various BCR signaling molecules, and expression of activation markers were assessed by flow cytometry. Transcription profiling of responsive (n=6) and unresponsive cases (n=6) was performed by RNA sequencing. Real-time quantitative polymerase chain reaction analysis was used to validate transcript level differences in a larger cohort. In 24 cases an α-IgM response was visible by Ca2+ influx which was accompanied by higher phosphorylation of PLCγ2 and Akt after α-IgM stimulation in combination with higher surface expression of IgM, IgD, CD19, CD38 and CD43 compared to the unresponsive cases (n=28). Based on RNA sequencing analysis several components of the canonical nuclear factor (NF)-κB pathway, especially those related to NF-κB inhibition, were expressed more highly in unresponsive cases. Moreover, upon α-IgM stimulation, the expression of these NF-κB pathway genes (especially genes coding for NF-κB pathway inhibitors but also NF-κB subunit REL) was upregulated in BCR-responsive cases while the level did not change, compared to basal level, in the unresponsive cases. These findings suggest that cells from CLL cases with enhanced NF-κB signaling have a lesser capacity to respond to BCR stimulation.

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEµ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEµ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEµ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEµ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.

One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides.

BACKGROUND: The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results.During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. RESULTS: In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. CONCLUSION: The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research.

Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.

One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.

Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency.

Chronic lymphocytic leukemia (CLL) can be divided into prognostically distinct subsets with stereotyped or non-stereotyped, mutated or unmutated B cell receptors (BCRs). Individual subsets vary in antigen specificity and origin, but the impact of antigenic pressure on the CLL BCR repertoire remains unknown. Here, we employed IgH.TEµ mice that spontaneously develop CLL, expressing mostly unmutated BCRs of which ~35% harbor VH11-2/Vκ14-126 and recognize phosphatidylcholine. Proportions of VH11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEµ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of VH11/Vκ14-expressing CLL. Furthermore, CLL onset was accelerated by enhanced BCR signaling in Siglec-G-/- mice or in mice expressing constitutively active Bruton's tyrosine kinase. Transcriptional profiling revealed that VH11 and non-VH11 CLL differed in the upregulation of specific pathways implicated in cell signaling and metabolism. Interestingly, principal component analyses using the 148 differentially expressed genes revealed that VH11 and non-VH11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively. We identified an expression signature consisting of 13 genes that were differentially expressed in a larger panel of T cell-dependent non-VH11 CLL compared with T cell-independent VH11/Vκ14 or mutated IgH.TEµ CLL. Parallel differences in the expression of these 13 signature genes were observed between heterogeneous and stereotypic human unmutated CLL. Our findings provide evidence for two distinct unmutated CLL subsets with a specific transcriptional signature: one is T cell-independent and B-1 cell-derived while the other arises upon antigen stimulation in the context of T-cell help.

Correction to: Circulating T Cells of Patients with Nijmegen Breakage Syndrome Show Signs of Senescence.

The original version of the article, "Circulating T Cells of Patients with Nijmegen Breakage Syndrome Show Signs of Senescence" incorrectly listed the affiliation of the fourth author, Iwona Solarska. The correct affiliation is "Molecular Biology Laboratory, Institute of Hematology and Transfusion Medicine.

Protective Cytomegalovirus (CMV)-Specific T-Cell Immunity Is Frequent in Kidney Transplant Patients without Serum Anti-CMV Antibodies.

The absence of anti-cytomegalovirus (CMV) immunoglobulin G (IgG) is used to classify pretransplant patients as naïve for CMV infection (CMVneg patients). This study assessed whether pretransplant CMV-specific T-cell immunity exists in CMVneg patients and whether it protects against CMV infection after kidney transplantation. The results show that CMV-specific CD137+IFNγ+CD4+ and CD137+IFNγ+CD8+ memory T cells were present in 46 and 39% of CMVneg patients (n = 28) although at much lower frequencies compared to CMVpos patients (median 0.01 versus 0.58% for CD4+ and 0.05 versus 0.64% for CD8+ T cells) with a less differentiated CD28-expressing phenotype. In line with these data, CMV-specific proliferative CD4+ and CD8+ T cells were observed in CMVneg patients, which significantly correlated with the frequency of CMV-specific T cells. CMV-specific IgG antibody-secreting cells (ASC) could be detected at low frequency in 36% of CMVneg patients (1 versus 45 ASC/105 cells in CMVpos patients). CMVneg patients with pretransplant CMV-specific CD137+IFNγ+CD4+ T cells had a lower risk to develop CMV viremia after transplantation with a CMVpos donor kidney (relative risk: 0.43, P = 0.03). In conclusion, a solitary CMV-specific T-cell response without detectable anti-CMV antibodies is frequent and clinically relevant as it is associated with protection to CMV infection following transplantation with a kidney from a CMVpos donor.

Circulating T Cells of Patients with Nijmegen Breakage Syndrome Show Signs of Senescence.

PURPOSE: The Nijmegen breakage syndrome (NBS) is an inherited genetic disorder characterized by a typical facial appearance, microcephaly, growth retardation, immunodeficiency, and a strong predisposition to malignancies, especially of lymphoid origin. NBS patients have a mutation in the NBN gene which involves the repair of DNA double-strand breaks (DSBs). Here we studied the peripheral T cell compartment of NBS patients with a focus on immunological senescence. METHODS: The absolute numbers and frequencies of the different T cell subsets were determined in NBS patients from young age till adulthood and compared to age-matched healthy individuals (HI). In addition, we determined the expression of senescent T cell markers and the signal joint T cell receptor excision circles (sjTRECs) content. RESULTS: Our results demonstrate that NBS patients have reduced T cell numbers. NBS patients showed lower numbers of αß+ T cells, but normal γδ+ T cell numbers compared to HI. Concerning the αß+ T cells, both CD4+ as well as CD8+ T cells were excessively reduced in numbers compared to aged-matched HI. In addition, NBS patients showed higher frequencies of the more differentiated T cells expressing the senescent cell marker CD57 and did not express co-stimulatory molecule CD28. These effects were already present in the youngest age group. Furthermore, NBS patients showed lower sjTREC content in their T cells possibly indicative of a lower thymic output. CONCLUSIONS: We conclude that circulating T cells from NBS patients show signs of a senescent phenotype which is already present from young age on and which might explain their T cell immune deficiency.

Loss of CD28 on Peripheral T Cells Decreases the Risk for Early Acute Rejection after Kidney Transplantation.

BACKGROUND: End-stage renal disease patients have a dysfunctional, prematurely aged peripheral T-cell system. Here we hypothesized that the degree of premature T-cell ageing before kidney transplantation predicts the risk for early acute allograft rejection (EAR). METHODS: 222 living donor kidney transplant recipients were prospectively analyzed. EAR was defined as biopsy proven acute allograft rejection within 3 months after kidney transplantation. The differentiation status of circulating T cells, the relative telomere length and the number of CD31+ naive T cells were determined as T-cell ageing parameters. RESULTS: Of the 222 patients analyzed, 30 (14%) developed an EAR. The donor age and the historical panel reactive antibody score were significantly higher (p = 0.024 and p = 0.039 respectively) and the number of related donor kidney transplantation was significantly lower (p = 0.018) in the EAR group. EAR-patients showed lower CD4+CD28null T-cell numbers (p<0.01) and the same trend was observed for CD8+CD28null T-cell numbers (p = 0.08). No differences regarding the other ageing parameters were found. A multivariate Cox regression analysis showed that higher CD4+CD28null T-cell numbers was associated with a lower risk for EAR (HR: 0.65, p = 0.028). In vitro, a significant lower percentage of alloreactive T cells was observed within CD28null T cells (p<0.001). CONCLUSION: Immunological ageing-related expansion of highly differentiated CD28null T cells is associated with a lower risk for EAR.

End stage renal disease patients have a skewed T cell receptor Vß repertoire.

BACKGROUND: End stage renal disease (ESRD) is associated with defective T-cell mediated immunity. A diverse T-cell receptor (TCR) Vß repertoire is central to effective T-cell mediated immune responses to foreign antigens. In this study, the effect of ESRD on TCR Vß repertoire was assessed. RESULTS: A higher proportion of ESRD patients (68.9 %) had a skewed TCR Vß repertoire compared to age and cytomegalovirus (CMV) - IgG serostatus matched healthy individuals (31.4 %, P < 0.001). Age, CMV serostatus and ESRD were independently associated with an increase in shifting of the TCR Vß repertoire. More differentiated CD8(+) T cells were observed in young ESRD patients with a shifted TCR Vß repertoire. CD31-expressing naive T cells and relative telomere length of T cells were not significantly related to TCR Vß skewing. CONCLUSIONS: ESRD significantly skewed the TCR Vß repertoire particularly in the elderly population, which may contribute to the uremia-associated defect in T-cell mediated immunity.

Uremia-associated immunological aging is stably imprinted in the T-cell system and not reversed by kidney transplantation.

The uremia-induced inflammatory environment in end-stage renal disease (ESRD) patients is associated with premature T-cell aging resulting in a defective T-cell immunity. As kidney transplantation (KTx) reduces the pro-inflammatory environment, we hypothesized that KTx would rejuvenate the aged T-cell system. As aging parameters, we determined in 70 KTx recipients the differentiation status by immunophenotyping, thymic output by the T-cell receptor excision circle (TREC) content together with CD31(+) naïve T-cell numbers and the relative telomere length (RTL) as a measure for proliferative history at pre-KTx, 3, 6 and 12 months post-KTx. In addition, T-cell function was determined by measuring the proliferative capacity and percentages of cytokine-producing cells. Directly post-KTx, memory T-cell numbers were diminished but restored to pre-KTx values at 12 months, except for CD4(+) EM T cells. The RTL of (memory) CD4(+) and CD8(+) T cells did not change. In contrast, TREC content and CD31(+) naïve T-cell numbers were stable post-KTx although the RTL of naïve CD4(+) and CD8(+) T cells decreased implying homeostatic proliferation of naïve cells, in response to a temporary decrease in memory cells. The T-cell function was not improved post-KTx. Our findings demonstrate that the uremia-associated aged phenotype is stably imprinted in the T-cell system and not reversed by KTx.

Loss of renal function causes premature aging of the immune system.

BACKGROUND: Uremia-associated immune deficiency is a well-known complication of loss of renal function and contributes significantly to the overall mortality and morbidity of patients with end-stage renal disease. Chronic inflammation and increased oxidative stress are underlying the uremia-associated immune deficiency. SUMMARY: In this review, the differential impact of uremia on the cellular immune system is summarized. Virtually all immune cells studied show a combination of an activated status and loss of function. However, uremia preferentially decreases the number and function of lymphoid cells while myeloid cells show normal and/or elevated cell numbers with increased production of inflammatory cytokines and reactive oxygen species. These particular changes are compatible with immunological aging, which is characterized by loss of thymic function, attrition of telomeres and an expanded memory T cell population. Similar to aging in healthy individuals, the proinflammatory and potential cardiotoxic subsets of CD28(null) T cells and CD16(+) monocytes are increased. Epigenetically changed hematopoietic stem cells may be involved in immunological aging as specific DNA regions become hypermethylated. Proinflammatory T cells and monocytes persist after kidney transplantation, which constitutes a persistent cardiovascular risk factor. Possible therapeutic options to reverse or halt uremia-associated immunological aging are discussed. KEY MESSAGES: Premature aging of the immune system is a dominant feature in patients with end-stage renal failure, which corresponds to immunological aging in elderly healthy individuals, which is characterized by preferential loss of cells belonging to the lymphoid cell lineage, inflammation and expansion of proinflammatory immune cells.

Terminally differentiated CD8+ Temra cells are associated with the risk for acute kidney allograft rejection.

BACKGROUND: End-stage renal disease (ESRD) is associated with T-cell dysregulation, leading to a variable degree of lymphopenia and increased T-cell differentiation. This may cause a relevant reduction in T-cell immunity, yielding a lowered risk for acute rejection (AR) of kidney allografts. METHODS: Before kidney transplantation, circulating CD4 and CD8 T-cell differentiation was established by determining the frequency of naive T cells, central memory and effector memory T cells, and the highly differentiated CD8 Temra cells. In addition, the frequency of differentiated T cells without expression of the costimulatory molecule CD28 was measured. RESULTS: In 47 patients of the 185 patients included, a biopsy-proven AR occurred. Compared with healthy controls, T cells of patients with ESRD were significantly more differentiated. Patients with AR showed the least signs of T-cell dysregulation with significantly more T cells, more naive T cells, and less terminal differentiation of memory T cells compared with nonrejecting patients. After multivariate analysis, only the frequency of terminally differentiated CD8 Temra cells (per percent, 4% decrease of risk [P=0.006]; per tertile, 34% decrease in risk [P=0.002]) and the number of human leukocyte antigen mismatches (per mismatch, 33% [P=0.005]) predicted the risk for AR. Functional analysis showed that CD8 Temra cells have a highly proinflammatory and cytotoxic profile. In vitro T-cell proliferation assays did not reveal a suppressor function of these cells. CONCLUSIONS: Advanced ESRD-related T-cell dysregulation that is associated with a relative increase of terminally differentiated CD8 Temra cells protects against AR after kidney transplantation.

A killer on the road: circulating CD4(+)CD28null T cells as cardiovascular risk factor in ESRD patients.

Chronic kidney disease (CKD) is associated with a sharp increase in the risk for cardiovascular disease, which can only be partially explained by known classical risk factors. However, there is a well-established association with increased systemic inflammation. In the last decade, an unique cytotoxic CD4(+) T cell population has been identified, which can be recognized by the loss of the costimulatory cell surface marker CD28, hence their name CD4(+)CD28null T cells. These cells are highly proinflammatory, have the functional features of professional killer lymphocytes and can expand from less than 1% to over 50% of the total CD4(+) T cell population. In this review, we show that these cells probably play an important role in destabilizing atherosclerotic plaques and could explain, at least in part, the association of cardiovascular disease with an increased inflammatory milieu in CKD patients.