Kennichi C. Dowdell

Somatic KRAS mutations associated with a human nonmalignant syndrome of autoimmunity and abnormal leukocyte homeostasis.

Somatic gain-of-function mutations in members of the RAS subfamily of small guanosine triphosphatases are found in > 30% of all human cancers. We recently described a syndrome of chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity associated with a mutation in NRAS affecting hematopoietic cells, and initially we classified the disease as a variant of the autoimmune lymphoproliferative syndrome. Here, we demonstrate that somatic mutations in the related KRAS gene can also be associated with a nonmalignant syndrome of autoimmunity and breakdown of leukocyte homeostasis. The activating KRAS mutation impaired cytokine withdrawal-induced T-cell apoptosis through the suppression of the proapoptotic protein BCL-2 interacting mediator of cell death and facilitated proliferation through p27(kip1) down-regulation. These defects could be corrected in vitro by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 or phosphatidyl inositol-3 kinase inhibition. We suggest the use of the term RAS-associated autoimmune leukoproliferative disease to differentiate this disorder from autoimmune lymphoproliferative syndrome.

Gender-dependent IL-12 secretion by APC is regulated by IL-10

Female SJL mice preferentially mount Th1-immune responses and are susceptible to the active induction of experimental allergic encephalomyelitis. By contrast, young adult male SJL are resistant to experimental allergic encephalomyelitis due to an APC-dependent induction of Th2 cells. The basis for this gender-dependent differential T cell induction was examined by analysis of macrophage APC cytokine secretion during T cell activation. APC derived from females secrete IL-12, but not IL-10, during T cell activation. By contrast, APC derived from males secrete IL-10, but not IL-12, during T cell activation. Activation of T cells with APC derived from the opposite sex demonstrated that these cytokines were derived from the respective APC populations. Furthermore, inhibition of IL-10, but not TGF-β, during T cell activation resulted in the secretion of IL-12 by male-derived APC. APC from naive male mice, in which IL-10 was reduced in vivo before isolation, also secrete IL-12, demonstrating altered APC cytokine secretion was due to an environment high in IL-10 before Ag encounter. Finally, APC derived from castrated male mice preferentially secrete IL-12 during T cell activation. These data demonstrate a link between gonadal hormones and APC activity and suggest that these hormones alter the APC, thereby influencing cytokine secretion during initial T cell activation.

Somatic FAS mutations are common in patients with genetically undefined autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by childhood onset of lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, elevated numbers of double-negative T (DNT) cells, and increased risk of lymphoma. Most cases of ALPS are associated with germline mutations of the FAS gene (type Ia), whereas some cases have been noted to have a somatic mutation of FAS primarily in their DNT cells. We sought to determine the proportion of patients with somatic FAS mutations among a group of our ALPS patients with no detectable germline mutation and to further characterize them. We found more than one-third (12 of 31) of the patients tested had somatic FAS mutations, primarily involving the intracellular domain of FAS resulting in loss of normal FAS signaling. Similar to ALPS type Ia patients, the somatic ALPS patients had increased DNT cell numbers and elevated levels of serum vitamin B(12), interleukin-10, and sFAS-L. These data support testing for somatic FAS mutations in DNT cells from ALPS patients with no detectable germline mutation and a similar clinical and laboratory phenotype to that of ALPS type Ia. These findings also highlight the potential role for somatic mutations in the pathogenesis of nonmalignant and/or autoimmune hematologic conditions in adults and children.

Using biomarkers to predict the presence of FAS mutations in patients with features of the autoimmune lymphoproliferative syndrome

To the Editor: The autoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic lymphadenopathy, splenomegaly, autoimmune cytopenias, and expansion of T cell receptor (TCR) αβ+ CD3+CD4−CD8− (αβ-double-negative [DNT]) cells (see this article’s Table E1 in the Online Repository at www.jacionline.org). Approximately two thirds of the patients with ALPS symptoms are genetically characterized, and most have germline (ALPS Ia) or somatic (ALPS Ia-s) TNFRSF6 (FAS) mutations. A small number of patients have defects in genes encoding Fas ligand (ALPS Ib), caspase-10 (ALPS II), or neuroblatoma-RAS (NRAS) viral oncogene homolog (ALPS IV). In addition, a large group of patients with ALPS findings remain genetically uncharacterized (ALPS III), and yet another has an undefined ALPS-like syndrome (ALPS-phenotype; Table I).1,2 Given the clinical similarities among all these groups, we sought to develop a biomarkers-based algorithm to predict the presence or absence of FAS mutations in this setting. TABLE I Description of patients with ALPS and control groups included in the study To this end, we investigated 26 parameters including immunophenotyping, eosinophil and monocyte counts, serum or plasma vitamin B12 (B12), soluble FAS ligand (sFASL), immunoglobulins, and levels of 14 cytokines in 562 subjects classified into 6 categories (Tables I and E1). The number of measurements, medians, and first and third quartiles are presented in this article’s Tables E2 and E3 in the Online Repository at www.jacionline.org. A full description of the Methods can be found in the Online Repository at www.jacionline.org. Elevated αβ-DNT cells are a hallmark of ALPS, but their utility for predicting FAS mutations had not been previously evaluated. 3 Patients with ALPS Ia and Ia-s had a high percentage of αβ-DNT cells, with median values 5.1% and 7.7%, respectively, compared with 0.5% for control mutation-negative relatives (MNRs; P 4% found in 60% (90/152) of patients with type Ia and in the majority of patients with type Ia-s (7/9), but in only 13% (11/85) of patients with ALPS type III and ALPS-phenotype (Fig 1, A). This value was associated with a positive likelihood ratio (LR) of 5.0 and a posttest probability of 89.3% for harboring FAS mutations. Conversely, the presence of αβ-DNT cells in the 1% to 2% range decreased the posttest probability to 25%, with a LR of 0.19 (Fig 2, B and C; see this article’s Table E4 in the Online Repository at www.jacionline.org). FIG 1 Biomarkers in patients with ALPS and control groups. Dashed lines represent cut-off values used to calculate likelihood ratios. Bars denote median values. P values for the differences between groups were obtained by Mann-Whitney test and are shown above ... FIG 2 sFASL levels and combinations of biomarkers accurately predict FAS mutations. A, Scatter plot showing sFASL levels. Increasing (B) and decreasing (C) probabilities for having a FAS mutation according to the percentage of αβ-DNT cells, ... In line with previous reports, patients with ALPS, regardless of mutation status, had 16% made the diagnosis of ALPS very unlikely (LR = 0.17). Other described abnormalities including increased CD3+HLA-DR+ to CD3+CD25+ ratio and high number of B cells had no additional diagnostic utility.4 We also evaluated serum B12 levels in patients with ALPS and found very elevated median levels in ALPS Ia and Ia-s (2259 ng/L; 1653 ng/L) compared with control MNRs (474 ng/L; P 1500 ng/L was 4.0, with a posttest probability of 87%. In contrast, having B12 levels <1000 ng/L diminished the posttest probability to 35% (Fig 2, B and C; Table E4). Analysis of plasma cytokines revealed 2 additional biomarkers for ALPS: IL-18 and TNF-α. Median plasma IL-18 levels were elevated in patients with ALPS Ia and Ia-s compared with control MNRs (1041 pg/mL, 1526 pg/mL, and 208 pg/mL, respectively; P < .0001). Patients with ALPS III and ALPS-phenotype had median values of 521 pg/mL and 702 pg/mL, respectively (P < .001 compared with MNRs; Fig 1, D). Furthermore, IL-18 <500 pg/mL was rarely seen in patients with ALPS and FAS mutations (7/56), with an associated negative LR of 0.19. TNF-α levels were higher in all ALPS groups with median values of 5 pg/mL for ALPS Ia (P < .0001), 9 pg/mL for ALPS Ia-s (P < .05), 8 pg/mL for ALPS III (P < .0001), and 7 pg/mL for ALPS-phenotype (P < .0001) compared with 1.3 pg/mL for MNRs (Fig 1, E). As previously reported, IL-10 was markedly elevated in ALPS Ia and Ia-s compared with MNRs (P 40 pg/mL, contrasting with 26% (10/38) of patients with ALPS III and ALPS-phenotype. For levels of IL-10 >40 ng/mL, the positive LR was 3.8, with a posttest probability of 85% for having a FAS mutation. Notably, only 20% (29/141) of patients with ALPS Ia and no patients with ALPS Ia-s had IL-10 values <20 pg/mL, giving a negative LR of 0.31 and a posttest probability of 33% for FAS mutations (Fig 2, B and C; Table E4). A recent report documented high levels of sFASL in patients with ALPS.7We expanded these findings analyzing more than 200 patients and controls. Ninety-seven percent of patients with ALPS Ia (136/140) and all patients with ALPS Ia-s had plasma sFASL >200 pg/mL, with median values of 1114 pg/mL and 1329 pg/mL, respectively, compared with control MNR levels of 104 pg/mL (P <.0001 for both groups). Only modest elevations of sFASL were seen in patients with ALPS III and ALPS-phenotype, as well as healthy mutation-positive relatives, with median values of 208 pg/mL, 174 pg/mL, and 207 pg/mL, respectively (Fig 2, A). These findings make sFASL the most sensitive biomarker to rule out a FAS mutation, with values <200 pg/mL associated with a negative LR of 0.05 and a posttest probability of 7.7% (Fig 2, C; Table E4). Soluble FASL also showed a strong positive correlation with IL-10 (r = 0.8;P < .0001) and a moderate correlation with αβ-DNTcells (r = 0.6; P < .0001) and B12 levels (r = 0.69; P < .0001; see this article’s Fig E1, A, in the Online Repository at www.jacionline.org). The area under the ROC curve for sFASL, αβ-DNT cells, B12, and IL-10 levels were calculated to evaluate how well they discriminate patients with a FAS mutation from those without (Fig E1, B). The area under the curve for sFASL was 0.9 (defines an excellent test) and for αβ-DNT cells was 0.81. B12 and IL-10 exhibited areas significantly less than sFASL (P < .05), with values of 0.76 and 0.77. We next evaluated whether combinations of αβ-DNT cells, B12, IL-10, IL-18, and sFASL would have increased power to predict or exclude FAS mutations in patients suspected of ALPS (Fig 2, B; Table E4). The combination of αβ-DNT cells >4% with B12 >1500 ng/L or IL-10 >40 pg/mL or IL-18 >500 ng/mL or sFASL >300 pg/mL was associated with >95% probability of having a FAS mutation. Conversely, having αβ-DNT cells <2% in combination with IL-10 <20 pg/mL or B12 <1000 ng/L or IL-18 <500 ng/mL decreased the probability of a FAS mutation to less than 10% (Fig 2, C; Table E4). Finally, finding αβ-DNT cells <2% and sFASL <200 pg/mL resulted in <2% probability for a FAS mutation. In conclusion, the biomarkers described should aid in the selection of patients with findings of ALPS for further diagnostic workup. In addition, the presence of a combination of markers strongly suggestive of a FAS mutation in the setting of a negative genetic test should prompt a search for somatic mutations in sorted αβ-DNT cells.

Protection from herpes simplex virus (HSV)-2 infection with replication-defective HSV-2 or glycoprotein D2 vaccines in HSV-1-seropositive and HSV-1-seronegative guinea pigs.

BACKGROUND A herpes simplex virus (HSV)-2 candidate vaccine consisting of glycoprotein D (gD2) in alum and monophosphoryl lipid A (MPL) reduced genital herpes disease in HSV-1-seronegative women but not in men or HSV-1-seropositive women. METHODS To determine the effect of HSV-1 serostatus on effectiveness of different vaccines, we tested gD2 in alum/MPL, gD2 in Freunds adjuvant, and dl5-29 (a replication-defective HSV-2 mutant) in HSV-1-seropositive or HSV-1-seronegative guinea pigs. RESULTS In HSV-1-seronegative animals, dl5-29 induced the highest titers of neutralizing antibody, and after vaginal challenge with wild-type virus, dl5-29 resulted in lower rates of vaginal shedding, lower levels of HSV DNA in ganglia, and a trend for less acute and recurrent genital herpes, compared with the gD2 vaccines. In HSV-1-seropositive animals, all 3 vaccines induced similar titers of neutralizing antibodies and showed similar levels of protection against acute and recurrent genital herpes after vaginal challenge with wild-type virus, but dl5-29 reduced vaginal shedding after challenge more than did the gD2 vaccines. CONCLUSIONS dl5-29 Is an effective vaccine in both HSV-1-seropositive and HSV-1-seronegative guinea pigs and was superior to gD2 vaccines in reducing virus shedding after challenge in both groups of animals. dl5-29 Might reduce transmission of HSV-2.

Varicella-Zoster Virus ORF12 Protein Triggers Phosphorylation of ERK1/2 and Inhibits Apoptosis

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases involved in many cellular processes, including cell proliferation, differentiation, inflammation, and cell death. Activation of several MAPKs, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), results in stimulation of activator protein 1 (AP-1), which promotes gene transcription. Previous studies have demonstrated that varicella-zoster virus (VZV) infection activates ERK1/2, p38, and JNK to promote viral replication, but the underlying mechanism(s) is unclear. To identify viral proteins responsible for the activation of MAPK, we used a proteomic approach to screen viral proteins for AP-1 promoter activation by an AP-1–luciferase reporter assay. We found that VZV ORF12 protein, located in the tegument of virions, enhances AP-1 reporter activity. This effect of ORF12 protein was markedly inhibited by a MAPK/ERK kinase 1 and 2 (MEK1/2) inhibitor (U0126), partially blocked by a p38 inhibitor (SB202190), but not inhibited by a JNK inhibitor (SP600125). Expression of VZV ORF12 protein in cells resulted in phosphorylation of ERK1/2 and p38 but not JNK. Infection of cells with a VZV ORF12 deletion mutant resulted in reduced levels of phosphorylated ERK1/2 (p-ERK1/2) compared to infection with wild-type VZV. Furthermore, deletion of ORF12 rendered VZV-infected cells more susceptible to staurosporine-induced apoptosis. In conclusion, VZV ORF12 protein activates the AP-1 pathway by selectively triggering the phosphorylation of ERK1/2 and p38. Cells infected with a VZV ORF12 deletion mutant have reduced levels of p-ERK1/2 and are more susceptible to apoptosis than cells infected with wild-type VZV.

Comparison of immunogenicity and protective efficacy of genital herpes vaccine candidates herpes simplex virus 2 dl5-29 and dl5-29-41L in mice and guinea pigs.

A replication-defective herpes simplex virus (HSV)-2 vaccine, dl5-29, which is deleted for two essential early genes, UL5 and UL29, is highly immunogenic and protective in mice and guinea pigs. In a prior study, a derivative of HSV-2 dl5-29 termed dl5-29-41L, which has an additional deletion in UL41 (that encodes the virion-host shut-off protein), was more immunogenic and protective against challenge with wild-type HSV-2 in mice when compared with dl5-29. To determine if deletion of UL41 improves the efficacy of dl5-29 in protecting guinea pigs from HSV-2, animals were immunized with dl5-29, dl5-29-41L, or PBS. The geometric mean neutralizing antibody titers from the dl5-29 and dl5-29-41L recipients were comparable (10(1.97) and 10(2.19), respectively, p=0.15). After intravaginal challenge with wild-type HSV-2, the dl5-29-41L and dl5-29 recipients shed similar titers of HSV-2 from the vagina. Mean acute disease severity scores, numbers of recurrences during 3 months after infection, and latent viral loads in sacral ganglia were similar for dl5-29 and dl5-29-41L (all p values >0.05). dl5-29 and dl5-29-41L completely protected mice from lethal challenge with HSV-2 and induced virus-specific CD8(+) T cells in the spleens of the animals. Thus, dl5-29 was as immunogenic and protective as dl5-29-41L under these conditions. dl5-29 was at least 250,000-fold less virulent than parental virus by intracranial inoculation in healthy mice, and caused no disease in SCID mice. Both dl5-29-41L and dl5-29 are equally effective and immunogenic in guinea pigs, and dl5-29 is very safe in immunocompromised animals.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, diminishes lymphoproliferation in the Fas -deficient MRL/lpr / murine model of autoimmune lymphoproliferative syndrome (ALPS)

OBJECTIVE Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of apoptosis, often presenting in childhood. Similarly, MRL/lpr(-/-) mice homozygous for Fas mutations develop an ALPS-like disease with autoimmunity, lymphadenopathy, splenomegaly, and expansion of double-negative T cells. Currently, there are no proven therapies with adequate safety margins for sustained abolition of the lymphoproliferation associated with ALPS. We sought to test the ability of valproic acid (VPA), a histone deacetylase inhibitor, to induce apoptosis and inhibit lymphoproliferation. MATERIALS AND METHODS Human peripheral blood mononuclear cells from patients with ALPS and normal controls were tested in vitro to determine the efficacy of VPA at inducing cell death. VPA was used in vivo to control lymphoproliferation in MRL/lpr(-/-) mice, a model for ALPS. RESULTS VPA induced cell death in vitro, and was partially inhibited by the pan caspase inhibitor, Z-VAD-FMK. MRL/lpr(-/-) mice treated with VPA for 8 weeks showed significant reductions in spleen and lymph node weights and cellularity compared to controls. A concomitant decrease in double-negative T cells was observed in the spleen, lymph nodes, and peripheral blood. Serum levels of VPA peaked 1 hour after injection, and a 2.5-fold increase in histone acetylation was observed in the spleen at 4 hours after injection. CONCLUSION Based on our data, VPA is effective at reducing lymphoproliferation in mice, and is currently being studied in a clinical trial as a lympholytic agent in patients with ALPS.

Autoimmune lymphoproliferative syndrome due to FAS mutations outside the signal-transducing death domain: molecular mechanisms and clinical penetrance

Purpose:Autoimmune lymphoproliferative syndrome is a disorder of lymphocyte apoptosis. Although FAS molecules bearing mutations in the signal-transducing intracellular death domain exhibit dominant-negative interference with FAS-mediated apoptosis, mechanisms for pathology of non-death domain FAS mutations causing autoimmune lymphoproliferative syndrome are poorly defined.Methods:RNA stability, protein expression, ligand binding, and ability to transmit apoptosis signals by anti-FAS antibody or FAS ligand were determined for a cohort of 39 patients with non-death domain autoimmune lymphoproliferative syndrome. Correlations between mutation type and disease penetrance were established in mutation-positive family members.Results:Frameshifts or transcriptional stop mutations before exon 7 resulted in messenger RNA haploinsufficiency, whereas an amino-terminal signal sequence mutation and certain intracellular truncations prevented cell surface localization of FAS. All resulted in decreased FAS localization, inability to bind FAS ligand, and reduced FAS ligand-induced apoptosis. Extracellular missense mutations and in-frame deletions expressed defective FAS protein, failed to bind FAS ligand, and exhibited dominant-negative interference with FAS-mediated apoptosis. Mutation-positive relatives with haploinsufficient or extracellular mutations had lower penetrance of autoimmune lymphoproliferative syndrome clinical phenotypes than did relatives with death domain mutations.Conclusion:We have defined molecular mechanisms by which non-death domain FAS mutations result in reduced lymphocyte apoptosis, established a hierarchy of genotype-phenotype correlation among mutation-positive relatives of patients with autoimmune lymphoproliferative syndrome, and demonstrated that FAS haploinsufficiency can lead to autoimmune lymphoproliferative syndrome.Genet Med 2012:14(1):81–89

Association of GATA2 Deficiency With Severe Primary Epstein-Barr Virus (EBV) Infection and EBV-associated Cancers

BACKGROUND Most patients infected with Epstein-Barr virus (EBV) are asymptomatic, have nonspecific symptoms, or have self-limiting infectious mononucleosis. EBV, however, may result in severe primary disease or cancer. METHODS We report EBV diseases associated with GATA2 deficiency at one institution and describe the hematology, virology, and cytokine findings. RESULTS Seven patients with GATA2 deficiency developed severe EBV disease. Three presented with EBV infectious mononucleosis requiring hospitalization, 1 had chronic active EBV disease (B-cell type), 1 had EBV-associated hydroa vacciniforme-like lymphoma with hemophagocytic lymphohistiocytosis, and 2 had EBV-positive smooth muscle tumors. Four of the 7 patients had severe warts and 3 had disseminated nontuberculous mycobacterial infections. All of the patients had low numbers of monocytes, B cells, CD4 T cells, and natural killer cells. All had elevated levels of EBV in the blood; 2 of 3 patients tested had expression of the EBV major immediate-early gene in the blood indicative of active EBV lytic infection. Mean plasma levels of tumor necrosis factor α, interferon γ, and interferon gamma-induced protein 10 were higher in patients with GATA2 deficiency than in controls. CONCLUSIONS GATA2 is the first gene associated with EBV hydroa vacciniforme-like lymphoma. GATA2 deficiency should be considered in patients with severe primary EBV infection or EBV-associated cancer, especially in those with disseminated nontuberculous mycobacterial disease and warts.