Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Changing the phospholipid composition of lipid droplets alters localization of select lipid droplet proteins.

Our experiments aim to determine if decreasing the amount of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE) at the lipid droplet surface changes the localization of specific lipid droplet proteins. We manipulate lipid droplet phospholipids in both a cultured mouse hepatocyte (AML12) cell line and on synthetic lipid droplets. Decreasing the PC:PE ratio increases perilipin 2, decreases DGAT2, and does not change rab18 or lanosterol synthase levels on lipid droplets. These differences may be explained by the distinct structural motifs that mediate the protein-lipid droplet interactions.

APOBEC3B regulates R-loops and promotes transcription-associated mutagenesis in cancer.

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

Defective nuclear IKKα function in patients with ectodermal dysplasia with immune deficiency.

Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB-regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.

Partial immune reconstitution of X-linked hyper IgM syndrome with recombinant CD40 ligand.

X-linked hyper IgM syndrome (XHM) is a combined immune deficiency disorder caused by genetic alterations in CD40 ligand. The purpose of this study was to investigate the safety and efficacy of recombinant CD40 ligand (rCD40L) in the treatment of the disease. Three children were administered rCD40L subcutaneously 3 times per week at 0.03 mg/kg for 22 weeks, and after a 12-week drug-free interval, the dose was increased to 0.05 mg/kg for an additional 22 weeks of treatment. Although specific antibody responses to T cell-dependent antigens was lacking, administration of rCD40 resulted in acquisition of the capacity to mount cutaneous delayed type hypersensitivity reactions that disappeared during the drug-free interval as well as the postbiologic follow-up period. With rCD40L treatment, patient T cells developed a new capacity to respond to T-cell mitogens with synthesis of IFN-γ and TNF-α. Intracellular cytokine staining studies showed that both CD4(+) and CD8(+) T cells participated in this response. Finally, CD40L therapy was associated with changes in lymph node size and architecture based on comparison of biopsies taken before and after therapy. This clinical study showed that rCD40L is capable of improving T cell-immune function in patients with XHM.

Phase I study of recombinant human interleukin-7 administration in subjects with refractory malignancy.

PURPOSE: Interleukin-7 (IL-7) has critical and nonredundant roles in T-cell development, hematopoiesis, and postdevelopmental immune functions as a prototypic homeostatic cytokine. Based on a large body of preclinical evidence, it may have multiple therapeutic applications in immunodeficiency states, either physiologic (immunosenescence), pathologic (HIV), or iatrogenic (postchemotherapy and posthematopoietic stem cell transplant), and may have roles in immune reconstitution or enhancement of immunotherapy. We report here on the toxicity and biological activity of recombinant human IL-7 (rhIL-7) in humans. DESIGN: Subjects with incurable malignancy received rhIL-7 subcutaneously every other day for 2 weeks in a phase I interpatient dose escalation study (3, 10, 30, and 60 microg/kg/dose). The objectives were safety and dose-limiting toxicity determination, identification of a range of biologically active doses, and characterization of biological and, possibly, antitumor effects. RESULTS: Mild to moderate constitutional symptoms, reversible spleen and lymph node enlargement, and marked increase in peripheral CD3(+), CD4(+), and CD8(+) lymphocytes were seen in a dose-dependent and age-independent manner in all subjects receiving >or=10 microg/kg/dose, resulting in a rejuvenated circulating T-cell profile, resembling that seen earlier in life. In some subjects, rhIL-7 induced in the bone marrow a marked, transient polyclonal proliferation of pre-B cells showing a spectrum of maturation as well as an increase in circulating transitional B cells. CONCLUSION: This study shows the potent biological activity of rhIL-7 in humans over a well-tolerated dose range and allows further exploration of its possible therapeutic applications.

EBV-related lymphoproliferative disease complicating therapy with the anti-CD2 monoclonal antibody, siplizumab, in patients with T-cell malignancies.

PURPOSE: We report an increased incidence of EBV-induced B-cell lymphoproliferative disease (LPD) in patients treated with siplizumab, an anti-CD2 antibody. The development of EBV-LPD has been associated with the use of immunosuppressive agents used in solid organ, bone marrow, and stem cell transplantation and in certain congenital immunodeficiencies. EXPERIMENTAL DESIGN: We conducted a single-institution phase I dose-escalation trial of siplizumab, a humanized monoclonal antibody to CD2, in 29 patients with T-cell malignancies. RESULTS: Although initial responses were encouraging, 4 (13.7%) patients developed EBV-LPD and the trial was stopped. Reductions in CD4(+) and CD8(+) cell count numbers in response to therapy were seen in all patients, but in those patients developing EBV-LPD a significantly greater reduction in natural killer (NK) cell number and CD2 expression on T cells was seen. These findings highlight the importance of NK-cell depletion and CD2 expression in addition to T-cell depletion in the etiology of EBV-LPD. CONCLUSIONS: The emergence of EBV-LPD may be associated with the ability of siplizumab to deplete both T and NK cells without affecting B cells. Agents that deplete T- and NK-cell populations without affecting B cell number should be screened for this potentially serious adverse event.

Pulmonary nontuberculous mycobacterial disease: prospective study of a distinct preexisting syndrome.

RATIONALE: Pulmonary nontuberculous mycobacterial (PNTM) disease is increasing, but predisposing features have been elusive. OBJECTIVES: To prospectively determine the morphotype, immunophenotype, and cystic fibrosis transmembrane conductance regulator genotype in a large cohort with PNTM. METHODS: We prospectively enrolled 63 patients with PNTM infection, each of whom had computerized tomography, echocardiogram, pulmonary function, and flow cytometry of peripheral blood. In vitro cytokine production in response to mitogen, LPS, and cytokines was performed. Anthropometric measurements were compared with National Health and Nutrition Examination Survey (NHANES) age- and ethnicity-matched female control subjects extracted from the NHANES 2001-2002 dataset. MEASUREMENTS AND MAIN RESULTS: Patients were 59.9 (+/-9.8 yr [SD]) old, and 5.4 (+/-7.9 yr) from diagnosis to enrollment. Patients were 95% female, 91% white, and 68% lifetime nonsmokers. A total of 46 were infected with Mycobacterium avium complex, M. xenopi, or M. kansasii; 17 were infected with rapidly growing mycobacteria. Female patients were significantly taller (164.7 vs. 161.0 cm; P < 0.001) and thinner (body mass index, 21.1 vs. 28.2; P < 0.001) than matched NHANES control subjects, and thinner (body mass index, 21.1 vs. 26.8; P = 0.002) than patients with disseminated nontuberculous mycobacterial infection. A total of 51% of patients had scoliosis, 11% pectus excavatum, and 9% mitral valve prolapse, all significantly more than reference populations. Stimulated cytokine production was similar to that of healthy control subjects, including the IFN-gamma/IL-12 pathway. CD4(+), CD8(+), B, and natural killer cell numbers were normal. A total of 36% of patients had mutations in the cystic fibrosis transmembrane conductance regulator gene. CONCLUSIONS: Patients with PNTM infection are taller and leaner than control subjects, with high rates of scoliosis, pectus excavatum, mitral valve prolapse, and cystic fibrosis transmembrane conductance regulator mutations, but without recognized immune defects.

Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets.

Interleukin-7 (IL-7) is a homeostatic cytokine for resting T cells with increasing serum and tissue levels during T cell depletion. In preclinical studies, IL-7 therapy exerts marked stimulating effects on T cell immune reconstitution in mice and primates. First-in-human clinical studies of recombinant human IL-7 (rhIL-7) provided the opportunity to investigate the effects of IL-7 therapy on lymphocytes in vivo. rhIL-7 induced in vivo T cell cycling, bcl-2 up-regulation, and a sustained increase in peripheral blood CD4(+) and CD8(+) T cells. This T cell expansion caused a significant broadening of circulating T cell receptor (TCR) repertoire diversity independent of the subjects' age as naive T cells, including recent thymic emigrants (RTEs), expanded preferentially, whereas the proportions of regulatory T (T reg) cells and senescent CD8(+) effectors diminished. The resulting composition of the circulating T cell pool more closely resembled that seen earlier in life. This profile, distinctive among cytokines under clinical development, suggests that rhIL-7 therapy could enhance and broaden immune responses, particularly in individuals with limited naive T cells and diminished TCR repertoire diversity, as occurs after physiological (age), pathological (human immunodeficiency virus), or iatrogenic (chemotherapy) lymphocyte depletion.

Eosinophilia is associated with a higher mortality rate among patients with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder associated with heritable defects in lymphocyte apoptosis that result in chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity. To examine the prevalence, mechanisms, and potential implications of eosinophilia in ALPS, we reviewed data retrospectively from 187 consecutive ALPS patients and their family members studied at the National Institutes of Health. ALPS patients with eosinophilia were compared with ALPS patients without eosinophilia with respect to their clinical and immunologic phenotype. Potential mechanisms for the eosinophilia, including abnormal Fas-mediated eosinophil apoptosis, increased production of eosinophilopoietic cytokines, and presence of anti-eosinophilic autoantibodies were also explored in a small number of patients from whom samples were available. Analysis of data from 68 ALPS patients and 119 of their relatives identified a distinct subgroup of patients with prominent and persisting eosinophilia that proved to be associated with increased numbers of peripheral blood leukocytes (PBL) of multiple lineages and a trend towards increased serum IgE levels. Eosinophilic ALPS patients also had a significantly higher risk of death due to infectious complications. Although the specific etiology of the eosinophilia in these patients remains uncertain, it does not appear to be associated with an altered serum cytokine profile, increased survival responsiveness of eosinophils to IL-5, defective Fas-mediated eosinophil apoptosis, or anti-eosinophil antibodies. Eosinophilia defines a distinct subgroup of ALPS patients with increased serum IgE levels, increased numbers of PBL of multiple lineages, and higher mortality from infectious complications.

Patients with chronic granulomatous disease have a reduced peripheral blood memory B cell compartment.

In this study, we have identified an altered B cell compartment in patients with chronic granulomatous disease (CGD), a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH activity. This is characterized by an expansion of CD5-expressing B cells, and profound reduction in B cells expressing the memory B cell marker, CD27. Both findings were independent of the age, genotype, and clinical status of the patients, and were not accompanied by altered CD5 and CD27 expression on T cells. Focusing on CD27-positive B cells, considered to be memory cells based on somatically mutated Ig genes, we found that the reduction was not caused by CD27 shedding or abnormal retention of CD27 protein inside the cell. Rather, it was determined that CD27-negative B cells were, appropriately, CD27 mRNA negative, consistent with a naive phenotype, whereas CD27-positive B cells contained abundant CD27 mRNA and displayed somatic mutations, consistent with a memory B cell phenotype. Thus, it appears that CGD is associated with a significant reduction in the peripheral blood memory B cell compartment, but that the basic processes of somatic mutation and expression of CD27 are intact. X-linked carriers of CGD revealed a significant correlation between the percentage of CD27-positive B cells and the percentage of neutrophils with normal NADPH activity, reflective of the degree of X chromosome lyonization. These results suggest a role for NADPH in the process of memory B cell formation, inviting further exploration of secondary Ab responses in CGD patients.

Unique abnormalities of CD4(+) and CD8(+) central memory cells associated with chronic graft-versus-host disease improve after extracorporeal photopheresis.

Chronic graft-versus-host disease (cGVHD) remains a problematic complication of allogeneic hematopoietic stem cell transplantation. Laboratory parameters correlated with cGVHD have not been fully defined, although changes in CD4/CD8 ratios occur and a decrease in CD4(+) central memory T cells has been noted. Extracorporeal photopheresis (ECP) is an effective therapy for steroid-refractory cGVHD. We have noted changes in lymphocyte subsets after ECP. CD4(+) and CD8(+) T-cell central and effector memory populations were enumerated by flow cytometry in a cohort of 37 patients postallogeneic transplantation with symptomatic cGVHD. Of the patients with symptomatic cGVHD, 7 were treated with ECP over 6 months and prospectively assessed for changes in lymphocyte subsets. There was a highly significant correlation of an increase in CD8(+) central memory cells and a concomitant decrease in CD4(+) central memory cells in patients with symptomatic cGVHD. These changes were not detected in patients without cGVHD posttransplantation. In all, 7 patients with cGVHD followed up prospectively during ECP treatment showed a statistically significant normalization of the pattern of CD4(+) and a trend toward normalization of CD8(+) central memory T cells coincident with improvement of cGVHD. These data indicate a high correlation between disturbances in the balance of central and effector memory populations and cGVHD suggesting use in following up responses to therapy. The normalization of central and effector memory populations in response to ECP coincident with clinical improvement of cGVHD support a correlation between these laboratory parameters and cGVHD. Further studies are needed to demonstrate whether laboratory measurements of the magnitude of changes in central and effector memory populations are useful prognostically or can be used to guide response to therapy. The contrasting change in central memory cells (CD8(+) increased versus CD4(+) decreased) in cGVHD provide support for recent reports suggesting unique differences in the differentiation pathways for CD8(+) versus CD4(+) T cells.

Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness.

The IFN-gammaR complex is composed of two IFN-gammaR1 and two IFN-gammaR2 polypeptide chains. Although IFN-gammaR1 is constitutively expressed on all nucleated cells, IFN-gammaR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-gammaR2 expression constructs to follow the molecule's cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255-256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-gammaR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Delta2) or alanine substitution (LI255-256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Delta2), but not downstream (257Delta2), of Leu-Ile 255-256 also led to overaccumulation. A truncation within the IFN-gammaR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-gammaR2 signaling. We have created a series of novel mutants of IFN-gammaR2 that have facilitated the identification of intracellular domains that control IFN-gammaR2 accumulation and IFN-gamma responsiveness. In contrast to IFN-gammaR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-gammaR2 Leu-Ile 255-256. These IFN-gammaR2 mutants will allow fine dissection of the role of IFN-gamma signaling in immunity.

Persistent Mycobacterium avium infection following nonmyeloablative allogeneic peripheral blood stem cell transplantation for interferon-gamma receptor-1 deficiency.

Interferon-gamma receptor-1 (IFNgammaR1) deficiency is a rare inherited immunodeficiency. We performed a nonmyeloablative allogeneic stem cell transplantation on a boy with complete IFNgammaR1 deficiency and refractory disseminated Myco- bacterium avium infection. Despite the patient's profound immune defect, early donor stem cell engraftment was low. Full donor engraftment was accomplished only following multiple donor lymphocyte infusions. Detection of IFNgammaR1 expression on peripheral blood monocytes and neutrophils corresponded with establishment of stable, complete donor hematopoietic chimerism. However, expression of, and signaling through IFNgammaR1 disappeared shortly thereafter. Disseminated Mycobacterium avium infection persisted and the patient died. Coculture of Myco- bacterium avium with normal myeloid cells resulted in an IFNgamma signaling defect similar to that observed in vivo. Active disseminated Mycobacterium avium infection may significantly compromise normal immune reconstitution following allogeneic stem cell transplantation. Patients with IFNgammaR1 deficiency should receive transplants before developing refractory mycobacterial infections.

Diagnostic paradigm for evaluation of male patients with chronic granulomatous disease, based on the dihydrorhodamine 123 assay.

BACKGROUND: Chronic granulomatous disease (CGD) is a phagocyte disorder caused by mutations in nicotinamide dinucleotide phosphate (NADPH) oxidase subunits. The dihydrorhodamine 123 (DHR) assay is an effective test for CGD that for most patients also might help to differentiate between the 2 most common forms, X-linked (X) gp91(phox) defect CGD and autosomal recessive (AR) p47(phox) defect CGD. However, some male patients with X-CGD have DHR patterns that overlap the AR-CGD pattern. OBJECTIVE: The purpose of this investigation was to develop a diagnostic paradigm to delineate male patients with X-CGD expressing a DHR pattern suggestive of p47(phox) deficiency. METHODS: The DHR assay measured change in fluorescence of DHR-loaded granulocytes after phorbol myristate acetate (PMA) stimulation. Western blot analysis measured the presence of NADPH oxidase subunits gp91(phox), p47(phox), p67(phox), and p22(phox). CYBB exonic sequencing was performed on PCR-amplified genomic DNA through use of intronic flanking primers. Ferricytochrome-c assay evaluated specific superoxide production by PMA-stimulated granulocytes. RESULTS: Although 83% of patients with X-CGD have virtually no neutrophil DHR activity, we found that 17% of patients, proven to have X-CGD by other criteria, have modest DHR activity that is most consistent with p47(phox) deficiency. We describe a diagnostic paradigm to deal with such patients, and we present 2 cases, along with results of additional studies, including carrier evaluation, protein assessment, and mutation analysis, that are useful in establishing the genotype under these circumstances. DHR assays from the 2 patients described showed a fluorescence shift most characteristic of p47(phox)-AR-CGD; however, each of the patients' mothers showed mosaicism with a bimodal DHR pattern. Patient 1 had some gp91(phox) protein with a Y41D mutation and modest superoxide activity. Patient 2 had a normal level of gp91(phox) protein with a C537R mutation without detectable superoxide activity, as determined by ferricytochrome-c assay, despite the modest DHR activity. CONCLUSIONS: Evaluation of male patients with CGD with modest DHR activity should initially include evaluation of potential female carriers for mosaicism with the use of the DHR assay. In circumstances in which this is uninformative, patients should be referred to centers capable of additional testing, including Western blot analysis and CYBB mutation analysis, to clarify the disease genotype.

A composite picture of TcR alpha/beta(+) CD4(-)CD8(-) T Cells (alpha/beta-DNTCs) in humans with autoimmune lymphoproliferative syndrome.

The discovery of an unusual T-cell subset characterized by the expression of the alpha/beta T-cell receptor without expression of either CD4 or CD8 [alpha/beta-double-negative T cells (alpha/beta-DNTCs)] provided critical insights in the evaluation of a "new" lymphoproliferative disorder known as autoimmune lymphoproliferative syndrome (ALPS). ALPS is a disorder of defective Fas-mediated lymphocyte apoptosis, manifested by accumulation of alpha/beta-DNTCs and other lymphocyte subsets, leading to lymphadenopathy and splenomegaly, autoimmunity, and an increased risk of lymphoma. The expanded population of alpha/beta-DNTCs from ALPS patients has a remarkable uniform phenotype that is for the most part similar to alpha/beta-DNTCs from mice with defective Fas (lpr) or Fas ligand (gld). This is in contrast to the minor alpha/beta-DNTC compartment in healthy individuals that contains multiple, immunophenotypically distinct subpopulations. Current data indicate that alpha/beta-DNTCs from ALPS patients are derived from cytotoxic CD8(+) T cells, chronically activated in vivo but anergic in vitro. Their anergic state may be related to persistent modifications of O-linked carbohydrates on cell surface molecules, such as CD43 and CD45, as well as to the increased presence of interleukin-10. Although largely consistent with a model of (linear) CD8(+) cytotoxic T-cell differentiation, the expression patterns of certain surface molecules, such as CD27 and CD28, are not consistent with this model. This may be the result of the perturbed homeostasis of lymphocytes in ALPS, thereby revealing pathways of differentiation and immunophenotypes, including phenotypes pertaining to cell surface glycosylation that are hidden from view in healthy individuals.