Influence of Vitamin D on Genome-Wide Expression in Natural Killer Cells

Introduction: Vitamin D deficiency has been associated with decreased overall survival in patients with diffuse large B-cell lymphoma treated with rituximab. Natural killer cell-mediated antibody-dependent cytotoxicity is one of the main mechanisms of action of rituximab, and it has been shown to be enhanced after in vivo vitamin D supplementation. This effect was more pronounced in females. We aimed to explore molecular mechanisms behind these findings using whole transcriptome analysis of natural killer cells after vitamin D supplementation. Methods: Natural killer cells were isolated by magnetic depletion from eight otherwise healthy subjects (four females and four males) with vitamin D deficiency, before and after vitamin D supplementation to a target serum level of 65 ng/ml. RNA was extracted followed by whole transcriptome microarray analysis. Results were verified by quantitative polymerase chain reaction. For differential expression analysis, the paired t-test and two-way ANOVA were used. Comparisons were made with regard to both vitamin D status and sex. In addition, pathway analysis using gene set enrichment analysis was performed. Results: After correction for multiple testing no genes showed statistically significant differential expression in the sex-independent comparison. For hypothesis generation, genes with an unadjusted p-value < 0.01 are reported (n = 505). An impact on known vitamin D-dependent genes was demonstrated. Genes involved in cytokine signaling like IFNL3 and IL-2RB were found to be upregulated, while others like IFNG and TLR genes were downregulated. In the sex-dependent analysis 28 transcripts with statistically significant sex- and vitamin D-dependent expression after adjustment were identified, like the JPX gene which was upregulated in females. Pathway analysis highlighted the role of interferon-α genes (IFNA2, -A4, -A6, -A8, -A10) in statistically significantly upregulated pathways. Other pathways involved in the immune response were found to be significantly downregulated, mainly by downregulation of the TLR2, -5, -7 and -8 genes. Finally, the ubiquitin ligase pathway was found to be downregulated. Conclusion: Vitamin D supplementation has only a slight effect on the natural killer cell transcriptome and the small sample size used in this study limits detection of such subtle changes. Our results implicate a role for vitamin D in gene expression involving the toll-like receptor and type I/III interferon axis and its regulation through the ubiquitin ligase system. IFN-γ downregulation may well be consistent with the observation of increased lymphoma killing of natural killer cells. JPX-dependent epigenetic regulation of X-chromosome deactivation in females could explain the downregulation of genes like TLR7 and -8 which are located on the X-chromosome. It is also possible, that vitamin D exerts its effects on natural killer cells indirectly through other, regulatory cells such as dendritic cells or macrophages

Management of Extranodal Marginal Zone Lymphoma: Present and Upcoming Perspectives

Extranodal marginal zone lymphoma (EMZL) encompasses a subgroup of non-Hodgkin lymphomas that often present with localized involvement and may manifest in a diversity of organs and tissues. EMZL pathogenesis is in some cases linked to chronic inflammation/infection, which may impose additional diagnostic and clinical challenges. The most studied and established connection is the presence of Helicobacter pylori in gastric EMZL. Due to its heterogeneity of presentation and intricate pathological features, treatment can be complex, and staging systems are decisive for the choice of therapy. Nevertheless, there is no consensus regarding the most suitable staging system, and recommendations vary among different countries. As a rule of thumb, in limited stages, a local therapy with surgery or radiation is the preferred option, and it is potentially curative. Of note, eradicating the causal agent may be an important step of treatment, especially in gastric EMZL, in which Helicobacter pylori eradication remains the first-line therapy for the majority of patients. In patients with more advanced stages, watch-and-wait is a valuable option, especially amongst those without clear indications for systemic therapy, and it may be carried on for several years. If watch-and-wait is not an option, systemic therapy may be needed. Even though several agents have been tested as monotherapy or in combination in recent years, there is no consensus regarding the first-line therapy, and decisions can vary depending on individual factors, such as age, clinical performance and stage. This review aims to discuss the several aspects of EMZL, including genetic milieu, pathogenesis and staging systems, that may influence the choice of therapy. In addition, we present a summary of evidence of several systemic therapies, compare different recommendations worldwide and discuss future perspectives and novelties in its therapy

Advances in Lymphoma Molecular Diagnostics

Lymphomas encompass a diverse group of malignant lymphoid neoplasms. Over recent years much scientific effort has been undertaken to identify and understand molecular changes in lymphomas, resulting in a wide range of genetic alterations that have been reported across all types of lymphomas. As many of these changes are now incorporated into the World Health Organization’s defined criteria for the diagnostic evaluation of patients with lymphoid neoplasms, their accurate identification is crucial. Even if many alterations are not routinely evaluated in daily clinical practice, they may still have implications in risk stratification, treatment, prognosis or disease monitoring. Moreover, some alterations can be used for targeted treatment. Therefore, these advances in lymphoma molecular diagnostics in some cases have led to changes in treatment algorithms. Here, we give an overview of and discuss advances in molecular techniques in current clinical practice, as well as highlight some of them in a clinical context

Vitamin D Enhances Immune Effector Pathways of NK Cells Thus Providing a Mechanistic Explanation for the Increased Effectiveness of Therapeutic Monoclonal Antibodies

Patients with diffuse large cell lymphoma who have an adequate vitamin D supply derive significantly more benefit from immuno-chemotherapy with rituximab than patients with vitamin D deficiency; this is especially true for female patients. We have already been able to show that vitamin D increases the antibody-dependent cytotoxicity (ADCC) of NK cells in a sex-dependent manner, but it is unclear how vitamin D makes NK cells more efficient. Methods: Healthy individuals with vitamin D deficiency were supplemented with vitamin D to sufficient levels. NK cells were isolated from blood samples before and after vitamin D saturation. For transcriptome analysis, we used the Affymetrix Gene-Chip 2.0™. Gene expression analysis as well as supervised and unsupervised pathway analysis were performed. Results: Among others the “NK cell-associated cytotoxicity pathway” increased after vitamin D substitution. Five IFN-α subtypes (2, 4, 6, 7 and 10) and IFN-κ were more highly expressed and are mainly responsible in these pathways. In contrast, the pathway “interferon-gamma response”, as well as other sets in cytokine production and chemotaxis showed a reduction. Toll-like receptor genes (TLR-8, TLR-7, TLR-2) were downregulated and, therefore, are responsible for the decline of these pathways. The same could be shown for the “ubiquitin-ligase” pathway. Conclusions: Increased expression of several IFN-α subtypes may explain the increased ADCC of NK cells in vitamin D-replenished and otherwise healthy subjects. Other regulators of interferon production and ADCC are compensatory upregulated in compensation, such as Toll-like receptors and those of the ubiquitin ligase, and normalize after vitamin D substitution

IgG seroprevalence of COVID-19 among people living with HIV or at high risk of HIV in south-west Germany: A seroprevalence study

Objectives: Seroprevalence studies of SARS-CoV-2 have shown that there is a high number of undiagnosed missing cases. Seroprevalence of SARS-CoV-2 in people living with HIV (PLWH) is lacking. Therefore, we conducted a prospective cross-sectional study to estimate the seroprevalence of SARS-CoV-2 among PLWH without known diagnosis of COVID-19 in the south-west of Germany. Methods: Serological testing for SARS-CoV-2 immunoglobulin G (IgG) antibodies based on two assays was performed in PLWH who visited the outpatient HIV centre of two hospitals from April to June 2020. Additionally, patients had to answer questionnaires about possible COVID-19-related symptoms and predefined risk factors. Moreover, we tested 50 non-HIV-infected patients receiving post- or pre-exposure (PEP/PrEP) HIV prophylaxis. Results: In all, 594 (488 male, 106 female) PLWH (median age 51 years) and 50 PEP/PrEP-users were included in the study. The estimated seroprevalence of the PLWH cohort was 1.85% (11/594), with 11 positive tested cases in the cohort. Among all patients, only five had COVID-19-related symptoms. One PCRpositive patient did not show any antibody response in repeatedly carried out tests. None of the patients was hospitalized due to COVID-19. Three PrEP users were tested positive. Three patients had been previously diagnosed with SARSCOV-2 infection before inclusion. The used questionnaire did not help to detect SARS-CoV-2 positive patients. Conclusions: Despite the limitation of being only a snapshot in time because of the ongoing pandemic, to our knowledge this is the largest study so far on seroprevalence of SARS-CoV-2 in PLWH in Germany. Our study suggests that the seroprevalence of SARS-CoV-2 in PLWH is comparable to those previously reported for parts of the general German population and that the questionnaire used here might not be the best tool to predict COVID-19 diagnosis

Characterization of an HLA-restricted and human cytomegalovirus-specific antibody repertoire with therapeutic potential

With an infection rate of 60–90%, the human cytomegalovirus (HCMV) is very common among adults but normally causes no symptoms. When T cell-mediated immunity is compromised, HCMV reactivation can lead to increased morbidity and mortality. HCMV antigens are processed and presented as peptides on the cell surface via HLA I complexes to the T cell receptor (TCR) of T cells. The generation of antibodies against HCMV peptides presented on HLA complexes (TCR-like antibodies) has been described, but is without therapeutic applications to date due to the polygenic and polymorphic nature of HLA genes. We set out to obtain antibodies specific for HLA/HCMV-peptides, covering the majority of HLA alleles present in European populations. Using phage display technology, we selected 10 Fabs, able to bind to HCMV-peptides presented in the 6 different HLA class I alleles A*0101, A*0201, A*2402, B*0702, B*0801 and B*3501. We demonstrate specific binding of all selected Fabs to HLA-typed lymphoblastoid cell lines (EBV-transformed B cells) and lymphocytes loaded with HCMV-peptides. After infection with HCMV, 4/10 tetramerized Fabs restricted to the alleles HLA-A*0101, HLA-A*0201 and HLA-B*0702 showed binding to infected primary fibroblasts. When linked to the pseudomonas exotoxin A, these Fab antibodies induce highly specific cytotoxicity in HLA matched cell lines loaded with HCMV peptides. TCR-like antibody repertoires therefore represent a promising new treatment modality for viral infections and may also have applications in the treatment of cancers

Killer immunoglobulin-like receptor 2DS5 is associated with recovery from coronavirus disease 2019

Background Despite numerous advances in the identification of risk factors for the development of severe coronavirus disease 2019 (COVID-19), factors that promote recovery from COVID-19 remain unknown. Natural killer (NK) cells provide innate immune defense against viral infections and are known to be activated during moderate and severe COVID-19. Killer immunoglobulin-like receptors (KIR) mediate NK cell cytotoxicity through recognition of an altered MHC-I expression on infected target cells. However, the influence of KIR genotype on outcome of patients with COVID-19 has not been investigated so far. We retrospectively analyzed the outcome associations of NK cell count and KIR genotype of patients with COVID-19 related severe ARDS treated on our tertiary intensive care unit (ICU) between February and June 2020 and validated our findings in an independent validation cohort of patients with moderate COVID-19 admitted to our tertiary medical center. Results Median age of all patients in the discovery cohort (n = 16) was 61 years (range 50–71 years). All patients received invasive mechanical ventilation; 11 patients (68%) required extracorporeal membrane oxygenation (ECMO). Patients who recovered from COVID-19 had significantly higher median NK cell counts during the whole observational period compared to patients who died (121 cells/µL, range 16–602 cells/µL vs 81 cells/µL, range 6–227 cells/µL, p-value = 0.01). KIR2DS5 positivity was significantly associated with shorter time to recovery (21.6 ± 2.8 days vs. 44.6 ± 2.2 days, p-value = 0.01). KIR2DS5 positivity was significantly associated with freedom from transfer to ICU (0% vs 9%, p-value = 0.04) in the validation cohort which consisted of 65 patients with moderate COVID-19. Conclusion NK cells and KIR genotype might have an impact on recovery from COVID-19

IgG seroprevalence of COVID-19 among people living with HIV or at high risk of HIV in south-west Germany: A seroprevalence study.

Objectives: Seroprevalence studies of SARS-CoV-2 have shown that there is a high number of undiagnosed missing cases. Seroprevalence of SARS-CoV-2 in people living with HIV (PLWH) is lacking. Therefore, we conducted a prospective cross-sectional study to estimate the seroprevalence of SARS-CoV-2 among PLWH without known diagnosis of COVID-19 in the south-west of Germany. Methods: Serological testing for SARS-CoV-2 immunoglobulin G (IgG) antibodies based on two assays was performed in PLWH who visited the outpatient HIV centre of two hospitals from April to June 2020. Additionally, patients had to answer questionnaires about possible COVID-19-related symptoms and predefined risk factors. Moreover, we tested 50 non-HIV-infected patients receiving post- or pre-exposure (PEP/PrEP) HIV prophylaxis. Results: In all, 594 (488 male, 106 female) PLWH (median age 51 years) and 50 PEP/PrEP-users were included in the study. The estimated seroprevalence of the PLWH cohort was 1.85% (11/594), with 11 positive tested cases in the cohort. Among all patients, only five had COVID-19-related symptoms. One PCR-positive patient did not show any antibody response in repeatedly carried out tests. None of the patients was hospitalized due to COVID-19. Three PrEP users were tested positive. Three patients had been previously diagnosed with SARS-COV-2 infection before inclusion. The used questionnaire did not help to detect SARS-CoV-2 positive patients

Ars2‐containing bispecific, Fab‐ and IgG1‐format BAR‐bodies to target DLBCL cells

Abstract Despite recent advances in the therapy of diffuse large B‐cell lymphoma, not otherwise specified (DLBCL), around 30% of patients develop refractory disease or relapse after first‐line treatment. Recently, Ars2 was reported as the auto‐antigenic target of the B‐cell receptor (BCR) in approximately 25% of activated B‐cell DLBCL cases. Ars2 could be used to specifically target B cells expressing Ars2‐reactive BCRs. However, the optimal therapeutic format to integrate Ars2 into has yet to be determined. To mimic therapeutic antibody formats, Ars2‐containing bispecific and IgG1‐like constructs (BCR antigens for reverse [BAR]‐bodies) were developed. Two bispecific BAR‐bodies connecting single‐chain antibodies against CD16 or CD3 to the BCR‐binding epitope of Ars2 were constructed. Both constructs showed strong binding to U2932 cells and induced effector cell‐dependent and selective cytotoxicity against U2932 cells of up to 44% at concentrations of 20 μg/ml. Additionally, IgG1‐format Ars2 BAR‐bodies were constructed by replacing the variable heavy‐ and light‐chain regions of a full‐length antibody with the Ars2 epitope. IgG1‐format Ars2 BAR‐bodies also bound selectively to U2932 and OCI‐Ly3 cells and induced selective cytotoxicity of up to 60% at 10 μg/ml. In conclusion, Ars2‐containing bispecific and IgG1‐format BAR‐bodies both are new therapeutic formats to target DLBCL cells