Potent high-avidity neutralizing antibodies and T cell responses after COVID-19 vaccination in individuals with B cell lymphoma and multiple myeloma.

Individuals with hematologic malignancies are at increased risk for severe coronavirus disease 2019 (COVID-19), yet profound analyses of COVID-19 vaccine-induced immunity are scarce. Here we present an observational study with expanded methodological analysis of a longitudinal, primarily BNT162b2 mRNA-vaccinated cohort of 60 infection-naive individuals with B cell lymphomas and multiple myeloma. We show that many of these individuals, despite markedly lower anti-spike IgG titers, rapidly develop potent infection neutralization capacities against several severe acute respiratory syndrome coronavirus 2 variants of concern (VoCs). The observed increased neutralization capacity per anti-spike antibody unit was paralleled by an early step increase in antibody avidity between the second and third vaccination. All individuals with hematologic malignancies, including those depleted of B cells and individuals with multiple myeloma, exhibited a robust T cell response to peptides derived from the spike protein of VoCs Delta and Omicron (BA.1). Consistently, breakthrough infections were mainly of mild to moderate severity. We conclude that COVID-19 vaccination can induce broad antiviral immunity including ultrapotent neutralizing antibodies with high avidity in different hematologic malignancies.

IFNL4 rs368234815 polymorphism does not predict risk of BK virus associated nephropathy after living-donor kidney transplant: A case-control study.

BACKGROUND: BK polyoma virus (BKPyV) associated nephropathy (BKPyVAN) is a major cause of kidney graft loss in renal transplant patients. Interferons (IFNs) are an important innate immune response against viral infections and genetic polymorphisms of the IFN-pathways can affect susceptibility and mortality during viral infection. Here, we investigated whether the dinucleotide polymorphism rs368234815 (ΔG/TT) in the IFNL4 gene contributed to BKPyV reactivation or BKPyVAN after living-donor kidney transplantation. METHODS: This retrospective case-control study determines the prevalence of IFNL4 variants in a Caucasian population of living-donor kidney transplant recipients and donors and explores its association with BKPyV infection and BKPyVAN development. We included 28 recipients with BKPyV reactivation, 10 of which developed BKPyVAN and 30 BKPyV negative controls. Targeted sequencing of the IFNL4 gene from both recipients and their respective donors was performed. RESULTS: We found IFNL4 rs368234815 ΔG allele frequencies of 41.7% in BKPyV negative and 39.3% in BKPyV positive recipients (P = .85), and 41.7% and 40.4% (P>.99) in their respective donors. IFNL4 rs368234815 ΔG allele frequencies in BKPyVAN developing recipients and their respective donors were 50% and 43.7% (P = .60 and P>.99). CONCLUSIONS: Our results indicate that the IFNL4 rs368234815 ΔG allele is not associated with BKPyV reactivation, nor the manifestation of BKPyVAN.

Pre-transplant HLA Antibodies and Delayed Graft Function in the Current Era of Kidney Transplantation.

Delayed graft function (DGF) occurs in a significant proportion of deceased donor kidney transplant recipients and was associated with graft injury and inferior clinical outcome. The aim of the present multi-center study was to identify the immunological and non-immunological predictors of DGF and to determine its influence on outcome in the presence and absence of human leukocyte antigen (HLA) antibodies. 1,724 patients who received a deceased donor kidney transplant during 2008-2017 and on whom a pre-transplant serum sample was available were studied. Graft survival during the first 3 post-transplant years was analyzed by multivariable Cox regression. Pre-transplant predictors of DGF and influence of DGF and pre-transplant HLA antibodies on biopsy-proven rejections in the first 3 post-transplant months were determined by multivariable logistic regression. Donor age ≥50 years, simultaneous pre-transplant presence of HLA class I and II antibodies, diabetes mellitus as cause of end-stage renal disease, cold ischemia time ≥18 h, and time on dialysis >5 years were associated with increased risk of DGF, while the risk was reduced if gender of donor or recipient was female or the reason for death of donor was trauma. DGF alone doubled the risk for graft loss, more due to impaired death-censored graft than patient survival. In DGF patients, the risk of death-censored graft loss increased further if HLA antibodies (hazard ratio HR=4.75, P < 0.001) or donor-specific HLA antibodies (DSA, HR=7.39, P < 0.001) were present pre-transplant. In the presence of HLA antibodies or DSA, the incidence of biopsy-proven rejections, including antibody-mediated rejections, increased significantly in patients with as well as without DGF. Recipients without DGF and without biopsy-proven rejections during the first 3 months had the highest fraction of patients with good kidney function at year 1, whereas patients with both DGF and rejection showed the lowest rate of good kidney function, especially when organs from ≥65-year-old donors were used. In this new era of transplantation, besides non-immunological factors, also the pre-transplant presence of HLA class I and II antibodies increase the risk of DGF. Measures to prevent the strong negative impact of DGF on outcome are necessary, especially during organ allocation for presensitized patients.

Inefficient induction of circulating TAA-specific CD8+ T-cell responses in hepatocellular carcinoma.

Background & Aims: In hepatocellular carcinoma (HCC), CD8+ T-cell responses targeting tumor-associated antigens (TAA) are considered to be beneficial. However, the molecular profile of TAA-specific CD8+ T cells in HCC is not well defined due to their low frequency. Results: In this study, we demonstrate that TAA-specific CD8+ T-cell responses are not efficiently induced in the peripheral blood of HCC patients as supported by the following observations: First, in HCC patients, frequencies of TAA-specific CD8+ T cells were not increased compared to healthy donors (HD) or patients with liver cirrhosis. Second, a remarkable proportion of TAA-specific CD8+ T cells were naïve despite the presence of antigen within the tumor tissue. Third, antigen-experienced TAA-specific CD8+ T cells lack the characteristic transcriptional regulation of exhausted CD8+ T cells, namely EomeshiTbetdim, and express inhibitory receptors only on a minor proportion of cells. This suggests restricted antigen recognition and further supports the hypothesis of inefficient induction and activation. Methods: By applying peptide/MHCI tetramer-based enrichment, a method of high sensitivity, we now could define the heterogeneity of circulating TAA-specific CD8+ T cells targeting glypican-3, NY-ESO-1, MAGE-A1 and MAGE-A3. We focused on therapy-naïve HCC patients of which the majority underwent transarterial chemoembolization (TACE). Conclusion: Our analysis reveals that circulating TAA-specific CD8+ T cells targeting 4 different immunodominant epitopes are not properly induced in therapy-naïve HCC patients thereby unravelling new and unexpected insights into TAA-specific CD8+ T-cell biology in HCC. This clearly highlights severe limitations of these potentially anti-tumoral T cells that may hamper their biological and clinical relevance in HCC.

OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection.

BACKGROUND & AIMS: Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways. METHODS: Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls. RESULTS: Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells. CONCLUSIONS: Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses. LAY SUMMARY: CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway.

Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8+ T Cells and Evolve at the Population Level.

BACKGROUND & AIMS: Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8+ T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8+ T-cell-mediated response. METHODS: We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8+ T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection. RESULTS: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8+ T cells; we confirmed that CD8+ T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8+ T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms. CONCLUSIONS: We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8+ T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8+ T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles.

Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load.

OBJECTIVE: A hallmark of chronic HBV (cHBV) infection is the presence of impaired HBV-specific CD8+ T cell responses. Functional T cell exhaustion induced by persistent antigen stimulation is considered a major mechanism underlying this impairment. However, due to their low frequencies in chronic infection, it is currently unknown whether HBV-specific CD8+ T cells targeting different epitopes are similarly impaired and share molecular profiles indicative of T cell exhaustion. DESIGN: By applying peptide-loaded MHC I tetramer-based enrichment, we could detect HBV-specific CD8+ T cells targeting epitopes in the HBV core and the polymerase proteins in the majority of 85 tested cHBV patients with low viral loads. Lower detection rates were obtained for envelope-specific CD8+ T cells. Subsequently, we performed phenotypic and functional in-depth analyses. RESULTS: HBV-specific CD8+ T cells are not terminally exhausted but rather exhibit a memory-like phenotype in patients with low viral load possibly reflecting weak ongoing cognate antigen recognition. Moreover, HBV-specific CD8+ T cells targeting core versus polymerase epitopes significantly differed in frequency, phenotype and function. In particular, in comparison with core-specific CD8+ T cells, a higher frequency of polymerase-specific CD8+ T cells expressed CD38, KLRG1 and Eomes accompanied by low T-bet expression and downregulated CD127 indicative of a more severe T cell exhaustion. In addition, polymerase-specific CD8+ T cells exhibited a reduced expansion capacity that was linked to a dysbalanced TCF1/BCL2 expression. CONCLUSIONS: Overall, the molecular mechanisms underlying impaired T cell responses differ with respect to the targeted HBV antigens. These results have potential implications for immunotherapeutic approaches in HBV cure.

Phenotype, penetrance, and treatment of 133 cytotoxic T-lymphocyte antigen 4-insufficient subjects.

BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative immune regulator. Heterozygous CTLA4 germline mutations can cause a complex immune dysregulation syndrome in human subjects. OBJECTIVE: We sought to characterize the penetrance, clinical features, and best treatment options in 133 CTLA4 mutation carriers. METHODS: Genetics, clinical features, laboratory values, and outcomes of treatment options were assessed in a worldwide cohort of CTLA4 mutation carriers. RESULTS: We identified 133 subjects from 54 unrelated families carrying 45 different heterozygous CTLA4 mutations, including 28 previously undescribed mutations. Ninety mutation carriers were considered affected, suggesting a clinical penetrance of at least 67%; median age of onset was 11 years, and the mortality rate within affected mutation carriers was 16% (n = 15). Main clinical manifestations included hypogammaglobulinemia (84%), lymphoproliferation (73%), autoimmune cytopenia (62%), and respiratory (68%), gastrointestinal (59%), or neurological features (29%). Eight affected mutation carriers had lymphoma, and 3 had gastric cancer. An EBV association was found in 6 patients with malignancies. CTLA4 mutations were associated with lymphopenia and decreased T-, B-, and natural killer (NK) cell counts. Successful targeted therapies included application of CTLA-4 fusion proteins, mechanistic target of rapamycin inhibitors, and hematopoietic stem cell transplantation. EBV reactivation occurred in 2 affected mutation carriers after immunosuppression. CONCLUSIONS: Affected mutation carriers with CTLA-4 insufficiency can present in any medical specialty. Family members should be counseled because disease manifestation can occur as late as 50 years of age. EBV- and cytomegalovirus-associated complications must be closely monitored. Treatment interventions should be coordinated in clinical trials.

Activation of human macrophages by human corneal allogen in vitro.

PURPOSE: To study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection. METHODS: Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFNγ), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively. RESULTS: Corneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1α and MIP-1ß, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation. CONCLUSIONS: Human macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.

B-cell signaling in persistent polyclonal B lymphocytosis (PPBL).

Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)-like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cells in vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF-κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF-κB signaling after CD40 stimulation and both B-cell receptor- and Toll-like receptor 9-mediated activation remained unaffected. Reduced canonical NF-κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression of Bcl-xL in MZ-like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL-derived B cells that were observed in vitro. The B-cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ-like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF-κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.

One hundred ABO-incompatible kidney transplantations between 2004 and 2014: a single-centre experience.

BACKGROUND: ABO-incompatible kidney transplantation (ABOi KTx) expands the living donor transplantation options. However, long-term outcome data, especially in comparison with ABO-compatible kidney transplantation (ABOc KTx), remain limited. Since the first ABOi KTx in Germany on 1 April 2004 at our centre, we have followed 100 ABOi KTx over up to 10 years. METHODS: One hundred ABOi KTx and 248 ABOc KTx from 1 April 2004 until 28 October 2014 were analysed in this observational, single-centre study. Three ABOi KTx and 141 ABOc KTx were excluded because of cyclosporine A-based immunosuppression, and 1 ABOc KTx was lost to follow-up. RESULTS: Median estimated 10-year patient and graft survival in ABOi KTx was 99 and 94%, respectively, and surpassed ABOc-KTx patient and graft survival of 80 and 88%, respectively. The incidence rate of antibody-mediated rejections was 10 and 8%, and that of T-cell-mediated rejections was 17 and 20% in ABOi KTx and ABOc KTx, respectively. Infectious and malignant complications in ABOi KTx were not more common than in ABOc KTx. However, postoperative lymphoceles occurred more frequently in ABOi KTx. Subgroup analysis of ABOi-KTx patients revealed that patients with high-titre isohaemagglutinins before transplantation had equal long-term results compared with low-titre isohaemagglutinin patients. CONCLUSION: Taken together, long-term outcome of ABOi KTx is not inferior to ABOc KTx. Incidences of rejection episodes, infectious complications and malignancies are not increased, despite the more vigorous immunosuppression in ABOi KTx. Our data provide further evidence that ABOi KTx with living donation is a safe, successful and reasonable option to reduce the organ shortage.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Genome-wide profiling in AML patients relapsing after allogeneic hematopoietic cell transplantation.

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.

Long-term outcome of ABO-incompatible living donor kidney transplantation based on antigen-specific desensitization. An observational comparative analysis.

BACKGROUND: ABO-incompatible living donor kidney transplantation based on specific conditioning has been successfully adopted by transplant centres worldwide. Excellent short-term results have been reported in small cohorts. However, long-term data and comparative analyses are still sparse. We report on the outcome of 40 consecutive ABO-incompatible living donor kidney transplant recipients and compare their clinical course to a control group of 43 ABO-compatible living donor transplant patients transplanted during the same time period. METHODS: This is an observational single-centre analysis of 40 consecutive patients undergoing ABO-incompatible kidney grafting between April 2004 and April 2009, using a protocol of rituximab, antigen-specific immunoadsorption, intravenous immunoglobulin, basiliximab induction and oral triple immunosuppression with tacrolimus, mycophenolic acid and prednisone. Forty-three ABO-compatible kidney transplant recipients served as controls. The control group had also received basiliximab induction and an identical initial maintenance immunosuppression. The two groups were observed for an average of 39 and 19 months, respectively. RESULTS: There was a significantly higher incidence of lymphoceles requiring surgical revisions in the ABO-incompatible group. However, this surgical complication did not affect patient or graft survival. Mean serum creatinine, estimated glomerular filtration rate and proteinuria did not differ between the two groups. Furthermore, ABO-incompatible and ABO-compatible patients had the same incidence of humoral and cellular rejections. Despite a more aggressive induction therapy, no differences in infectious or malignant complications were observed. CONCLUSIONS: ABO-incompatible living donor kidney transplantation utilizing a combination of rituximab and antigen-specific immunoadsorption yielded results identical to ABO-compatible transplantation despite a significantly higher number of human leukocyte antigen mismatches.

Role of the spleen in peripheral memory B-cell homeostasis in patients with autoimmune thrombocytopenia purpura.

The effect of splenectomy on circulating memory B cells in autoimmune thrombocytopenia purpura (AITP) patients has not yet been addressed. We therefore analyzed the distribution and phenotypic characteristics of B-cell subsets in non-splenectomized and splenectomized AITP patients and controls, as well as CD95 expression after B cell activation. Decreased frequencies of memory B cells in splenectomized individuals were observed, with a rapid decline of CD27+IgD+ and a slower decrease of CD27+IgD- and CD27-/IgD- cells. Similar results were noted following splenectomy in healthy donors (HD). CD95+ B cells were substantially increased in all subsets in patients with active AITP, indicating their enhanced activation status. After splenectomy, the percentage of CD95+ B cells were further increased in the CD27+IgD- post-switch memory population in AITP, but not in HD. CD95+CD27+ memory B cells largely reside in the region in the human spleen analogous to the murine marginal zone. Thus, the spleen plays a fundamental role in controlling peripheral memory B cell homeostasis in both AITP and HD and regulates activated CD95+ B cells in patients with AITP.

High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets are opsonised by autoantibodies and destroyed by macrophages. Therefore, ITP represents a prototype of a B-cell-mediated autoimmune disorder. B-cell activating factor (BAFF) is a member of the tumour necrosis factor family and an important regulator of B-cell development. BAFF levels were determined in serum samples from 53 patients with ITP. Serum BAFF levels in patients with an active ITP were increased when compared with the healthy control group (median 1620 pg/ml vs. 977 pg/ml; P < 0.001). Moreover, immunosuppressive treatment was associated with strongly suppressed BAFF levels (median 629 pg/ml; P < 0.01). In addition, a polymorphic site was detected in the BAFF promoter region (-871) that appeared to occur more frequently in ITP patients than in healthy persons. This promoter variant was associated with very high BAFF levels in ITP patients. Our data suggest that BAFF is an important pathogenetic factor in the development of ITP.

Inactivating I kappa B epsilon mutations in Hodgkin/Reed-Sternberg cells.

The pathogenesis of Hodgkin lymphoma (HL) is still unclear. Previous investigations have demonstrated constitutive nuclear activity of the transcription factor NF kappa B (NF-kappaB) in Hodgkin/Reed-Sternberg (HRS) cells as an important prerequisite in protecting these cells from apoptosis. As a molecular mechanism leading to constitutive NF-kappaB activity in HRS cells, mutations of the NF-kappaB inhibitor I kappa B alpha (IkappaBalpha) have recently been identified in classical (c) HL-derived cell lines in a patient with cHL. In the present study, the NF-kappaB inhibitor I kappa B epsilon (IkappaBepsilon) has been analysed for somatic mutations in the same group of six patients already studied for IkappaBalpha mutations, as well as in cHL-derived cell lines. In one cHL-derived cell line (L428), a hemizygous frame-shift mutation generating a pre-terminal stop codon resulting in a severely truncated protein was found. Moreover, in the HRS cells of one patient, a hemizygous mutation affecting the 5'-splicing site of intron 1 of the IkappaBepsilon gene was found. These results, in combination with recently described IkappaBalpha mutations, indicate that defective NF-kappaB inhibitors appear more frequent than previously thought and might explain the constitutive nuclear activity of NF-kappaB in a significant proportion of cHL cases.