Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome

Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells

Preparation of cytokine-activated NK cells for use in adoptive cell therapy in cancer patients: Protocol optimization and therapeutic potential

Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine- activated NK-cell product. NK cells were isolated either by double depletion (CD3-, CD19-) or by sequential depletion and enrichment (CD3-, CD56+) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3-CD56+ procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3-CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3-CD56+ products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3-CD56+ products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell-based immunotherapeutic strategies in a clinical setting

Applicability of Dickson Charge Pump in Energy Harvesting Systems: Experimental Validation of Energy Harvesting Charge Pump Model

Energy harvesting methods provide very low instantaneous power. Accordingly, available voltage levels are low and must be increased so that an energy harvesting method can be used as a power supply. One approach uses charge pumps to boost low AC voltage from energy harvester to a higher DC voltage. Characterized by very low output current and a wide span of operating frequencies, energy harvesting methods introduce a number of limitations to charge pump operation. This paper describes and models behavior of Dickson charge pump in energy harvesting applications. Proposed Energy Harvesting model is evaluated and compared with Standard and Tanzawa charge pump models and with measurement results. Based on the proposed model, the conditions that need to be satisfied so that a charge pump can reach maximum power point of energy harvesting system are defined. Parameter selection method optimized for maximum power point is presented and is experimentally validated

Impact of Serotherapy on Immune Reconstitution and Survival Outcomes After Stem Cell Transplantations in Children : Thymoglobulin Versus Alemtuzumab

The outcome of allogeneic hematopoietic stem cell transplantation (HSCT) is strongly affected by the kinetics of reconstitution of the immune system. This study compared the effects of antithymocyte globulin (ATG) and alemtuzumab on various outcome parameters after HSCT. The study cohort consisted of 148 children, with a median age of 9.6 years (range, .4 to 19.0), who underwent HSCT for malignant and benign hematological disorders in a single HSCT unit. Conditioning included ATG (n= 110) or alemtuzumab (n= 38). Cox proportional hazard regression analysis showed that alemtuzumab significantly delayed the recovery of CD3+ T cells and CD4+as well as CD8+ T cell subsets (P ≤ .001) and natural killer (NK) cells (P= .008) compared with ATG. In both ATG- and alemtuzumab-treated patients, shorter drug exposure lead to significantly faster recoveryof T cells. Alemtuzumab was associated with lower donor chimerism 3 and 6 months after transplantation and a higher risk of disease relapse (P= .001). The overall survival and event-free survival risks were significantly lower for alemtuzumab-treated patients (P= .020 and P <.001, respectively). Patients who received alemtuzumab showed a trend to lower risk of acute graft-versus-host disease, more human adenovirus, and less Epstein-Barr virus reactivations compared with patients who received ATG. These data indicate that children treated with alemtuzumab as part of the conditioning regimen have a slower T cell and NK cell reconstitution compared with those treated with ATG, which compromises the overall and event-free survival. Prolonged length of lympholytic drug exposure delayed the T cell recovery in both ATG- andalemtuzumab-treated patients. Therefore, we recommend detailed pharmacokinetic/pharmacodynamic (PK/PD) analyses in a larger cohort of patients to develop an algorithm aiming at optimization of the serotherapy containing conditioning regimen

T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vβ antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vβ repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27− and/or CD28− memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.Pattern Recognition and Bioinformatic

Risk Factors, Treatment, and Immune Dysregulation in Autoimmune Cytopenia after Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients

Autoimmune or alloimmune cytopenia (AIC) is a known rare complication of hematopoietic stem cell transplantation (SCT). AIC after SCT is considered difficult to treat and is associated with high morbidity and mortality. In this retrospective study in pediatric patients we evaluated incidence, outcome, potential risk factors, and current treatment strategies. A nested matched case-control study was performed to search for biomarkers associated with AIC. Of 531 consecutive SCTs at our center between 2000 and 2016, 26 were complicated by the development of AIC (cumulative incidence, 5.0%) after a median of 5 months post-SCT. Autoimmune hemolytic anemia was the most common AIC with 12 patients (46%). We identified nonmalignant disease, alemtuzumab serotherapy pre-SCT, and cytomegalovirus (CMV) reactivation as independently associated risk factors. The cytokine profile of patients at the time of AIC diagnosis appeared to skew toward a more pronounced Th 2 response compared with control subjects at the corresponding time point post-SCT. Corticosteroids and intravenous immunoglobulin as first-line treatment or a wait-and-see approach led to resolution of AIC in 35% of cases. Addition of step-up therapies rituximab (n = 15), bortezomib (n = 7), or sirolimus (n = 3) was associated with AIC resolution in 40%, 57%, and 100% of cases, respectively. In summary, we identified CMV reactivation post-SCT as a new clinical risk factor for the development of AIC in children. The cytokine profile during AIC appears to favor a Th 2 response. Rituximab, bortezomib, and sirolimus are promising step-up treatment modalities

T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vβ antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vβ repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27− and/or CD28− memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT

Mutations in ZBTB24 Are Associated with Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome Type 2

Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general