Autoimmune Cytopenia as an Early and Initial Presenting Manifestation in Activated PI3 Kinase Delta Syndrome: Case Report and Review.

Activated PI3 kinase delta syndrome (APDS) is a combined immunodeficiency characterized by recurrent sinopulmonary infections, increased risk of herpesvirus infections, lymphoproliferation, autoimmunity, and increased risk of lymphoid malignancies. Gain-of-function mutations in PIK3CD and PIK3R1 result in increased phosphoinositide-3-kinase-delta activity which causes hyperactivation of lymphocytes and abnormal development and activation of T and B cells. Cytopenias are the most common autoimmune process occurring in patients with APDS and typically occur as a later manifestation of the disease. Here we present a female patient with an early autoimmune hemolytic anemia, hepatosplenomegaly, and frequent infections presenting in infancy, followed by development of significant lymphadenopathy before her diagnosis with APDS type 1. She had significant improvement in her infectious history with immunoglobulin replacement, and control of autoimmune hemolytic anemia with initiation of sirolimus after her diagnosis with APDS type 1. We utilize this case to review the literature on APDS and present the novel finding of early-onset autoimmune disease in the setting of APDS. Autoimmune cytopenias are seen in many primary immunodeficiencies, and workup of autoimmune cytopenias in young patients should include evaluation for underlying immune disorder.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia.

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

TSLP or IL-7 provide an IL-7Rα signal that is critical for human B lymphopoiesis.

Thymic stromal lymphopoietin (TSLP) and IL-7 are cytokines that signal via the IL-7 receptor alpha (IL-7Rα) to exert both overlapping and unique functions during early stages of mouse B-cell development. In human B lymphopoiesis, the requirement for IL-7Rα signaling is controversial and the roles of IL-7 and TSLP are less clear. Here, we evaluated human B-cell production using novel in vitro and xenograft models of human B-cell development that provide selective IL-7 and human TSLP (hTSLP) stimulation. We show that in vitro human B-cell production is almost completely blocked in the absence of IL-7Rα stimulation, and that either TSLP or IL-7 can provide a signal critical for the production and proliferation of human CD19(+) PAX5(+) pro-B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL-7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro-B cells under the influence of mouse IL-7 in a xenograft scenario is reduced by anti-IL-7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL-7Rα mediated signals for normal human B-cell production.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis.

Thymic stromal lymphopoietin (TSLP) stimulates in-vitro proliferation of human fetal B-cell precursors. However, its in-vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in-vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (-T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in -T mice. Patient-derived xenografts generated from +T as compared to -T mice showed a 3-6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from -T mice. +T/-T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

Differences in mouse and human nonmemory B cell pools.

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19(+)IgM(+) B cells, the CD21/CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ∼ 8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans.

Subcutaneous Immunoglobulin Therapy with IgPro20 in Patients with Stiff Person Syndrome and Primary Immunodeficiency Disease.

Stiff Person Syndrome (SPS), a rare autoimmune neurologic disorder characterized by fluctuating muscle spasms and rigidity, is mediated by autoantibodies to glutamic acid decarboxylase (GAD) antibodies. Symptoms of SPS have been shown to improve after administration of intravenous immunoglobulin (IVIG) however, there is a paucity of information regarding use of SCIg in SPS. Four patients with Stiff Person Syndrome were treated with SCIgPro20 for a period between 31 to 101 months. Most reactions were local and mild. All patients reported improvement in spasticity, and 2 patients reported improvement in seizure frequency. SCIgPro20 was well tolerated in patients with SPS and was associated with improvement in symptoms.

Common food allergens and cross-reactivity.

The most clinically relevant food allergens are cow's milk, hen's egg, peanut, tree nuts, wheat, soy, fish, shellfish, and seeds. Heat-stable food allergens have molecular characteristics that enhance protein stability and gastrointestinal absorption and thus are more likely to cause systemic reactions on ingestion. In contrast, heat-labile food allergens lack these characteristics and do not typically elicit reactions if sufficiently altered by heat or acid. Immunologic cross-sensitization between food allergens is more common than clinical cross-reactivity. However, certain groups of food allergens, such as tree nuts, fish, and shellfish, are associated with high rates of clinical cross-reactivity. Knowing the rates of clinical cross-reactivity is important when providing guidance to patients with food allergy and families on what foods can be safely added to the diet and what foods should be avoided.

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line.

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Fine-tuning patient-derived xenograft models for precision medicine approaches in leukemia.

Many leukemias are characterized by well-known mutations that drive oncogenesis. Mice engineered with these mutations provide a foundation for understanding leukemogenesis and identifying therapies. However, data from whole genome studies provide evidence that malignancies are characterized by multiple genetic alterations that vary between patients, as well as inherited genetic variation that can also contribute to oncogenesis. Improved outcomes will require precision medicine approaches-targeted therapies tailored to malignancies in each patient. Preclinical models that reflect the range of mutations and the genetic background present in patient populations are required to develop and test the combinations of therapies that will be used to provide precision medicine therapeutic strategies. Patient-derived xenografts (PDX) produced by transplanting leukemia cells from patients into immune deficient mice provide preclinical models where disease mechanisms and therapeutic efficacy can be studied in vivo in context of the genetic variability present in patient tumors. PDX models are possible because many elements in the bone marrow microenvironment show cross-species activity between mice and humans. However, several cytokines likely to impact leukemia cells are species-specific with limited activity on transplanted human leukemia cells. In this review we discuss the importance of PDX models for developing precision medicine approaches to leukemia treatment. We illustrate how PDX models can be optimized to overcome a lack of cross-species cytokine activity by reviewing a recent strategy developed for use with a high-risk form of B-cell acute lymphoblastic leukemia (B-ALL) that is characterized by overexpression of CRLF2, a receptor component for the cytokine, TSLP.

Regulator of myeloid differentiation and function: The secret life of Ikaros.

Ikaros (also known as Lyf-1) was initially described as a lymphoid-specific transcription factor. Although Ikaros has been shown to regulate hematopoietic stem cell renewal, as well as the development and function of cells from multiple hematopoietic lineages, including the myeloid lineage, Ikaros has primarily been studied in context of lymphoid development and malignancy. This review focuses on the role of Ikaros in myeloid cells. We address the importance of post-transcriptional regulation of Ikaros function; the emerging role of Ikaros in myeloid malignancy; Ikaros as a regulator of myeloid differentiation and function; and the selective expression of Ikaros isoform-x in cells with myeloid potential. We highlight the challenges of dissecting Ikaros function in lineage commitment decisions among lymphoid-myeloid progenitors that have emerged as a major myeloid differentiation pathway in recent studies, which leads to reconstruction of the traditional map of murine and human hematopoiesis.