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Splicing modulation is a promising treatment strategy pursued to date only in splicing-factor mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we show that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing-factor SF3B1 (H3B-8800 and E-7107). We identify drug-induced alterations in splicing and corresponding reduced gene expression of a large number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We find that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators given their long length and large number of exons per transcript. Furthermore, we demonstrate that treatment of cohesin-mutant cells with SF3B1 modulators not only results in impaired DNA damage response and accumulation of DNA damage, but it significantly sensitizes cells to subsequent killing by PARP inhibitors and chemotherapy, and leads to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML, and we propose this as a broader strategy for therapeutic targeting of other DNA damage-repair deficient cancers.One Sentence SummaryWe identify an unexpected effect of SF3B1 splicing inhibitors on regulation of DNA damage repair genes and show efficacy of combination treatment in cohesin-mutant MDS and AML.

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein’s E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase–binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

The IL-3, IL-5, and GM-CSF family of cytokines play an essential role in the growth, differentiation, and effector functions of multiple hematopoietic cell types. Receptors in this family are composed of cytokine-specific α chains and a common β chain (CSF2RB), responsible for the majority of downstream signaling. CSF2RB abundance and stability influence the magnitude of the cellular response to cytokine stimulation, but the exact mechanisms of regulation are not well understood. Here, we use genetic screens in multiple cellular contexts and cytokine conditions to identify STUB1, an E3 ubiquitin ligase, and CHIC2 as regulators of CSF2RB ubiquitination and protein stability. We demonstrate that Stub1 and Chic2 form a complex that binds Csf2rb and that genetic inactivation of either Stub1 or Chic2 leads to reduced ubiquitination of Csf2rb. The effects of Stub1 and Chic2 on Csf2rb were greatest at reduced cytokine concentrations, suggesting that Stub1/Chic2-mediated regulation of Csf2rb is a mechanism of reducing cell surface accumulation when cytokine levels are low. Our study uncovers a mechanism of CSF2RB regulation through ubiquitination and lysosomal degradation and describes a role for CHIC2 in the regulation of a cytokine receptor.

Refractoriness to platelet transfusion is a common clinical problem encountered by the transfusion medicine specialist. It is well recognized that most causes of refractoriness to platelet transfusion are not a consequence of alloimmunization to human leukocyte, platelet-specific, or ABO antigens, but are a consequence of platelet sequestration and consumption. This review summarizes the clinical factors that result in platelet refractoriness and highlights recent data describing novel biological mechanisms that contribute to this clinical problem., Highlights • Non-alloimmune pathophysiologic mechanisms lead to increased platelet consumption or sequestration in the majority of patients with thrombocytopenia and/or refractoriness to platelet transfusion. • Splenomegaly is associated with increased splenic platelet sequestration. • Sepsis can promote platelet consumption through effects on platelet activation, adhesion, apoptosis, and desialylation. • Neutrophil extracellular traps, endothelial activation, and decreased ADAMTS13 activity can also contribute to platelet consumption in sepsis. • There is an unmet need for effective strategies to improve hemostasis and the efficacy of platelet transfusion in patients with non-alloimmune causes of thrombocytopenia and/or refractoriness to platelet transfusion.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) is an inflammatory premalignant disorder resulting from acquired genetic mutations in hematopoietic stem cells. This condition is common in aging populations and associated with cardiovascular morbidity and overall mortality, but its role in CKD is unknown. METHODS: We performed targeted sequencing to detect CHIP mutations in two independent cohorts of 87 and 85 adults with an eGFR

Bleeding in patients with acute myeloid leukemia (AML) receiving intensive induction chemotherapy is multifactorial and contributes to early death. We sought to define incidence and risk factors of grade 4 bleeding to support strategies for risk mitigation. Bleeding events were assessed according to the WHO bleeding assessment scale, which includes grade 4 bleeding as fatal, life-threatening, retinal with visual impairment, or involving the central nervous system. Using multivariable competing-risk regression analysis with grade 4 bleeding as the primary outcome, we identified risk factors in the development cohort (n=341), which were tested in an independent cohort (n=143). Grade 4 bleeding occurred in 5.9% and 9.8% of patients in the development and validation cohort, respectively. Risk factors that were independently associated with grade 4 bleeding included baseline platelet count ≤40 ×109/L compared with >40 ×109/L, and baseline PT-INR >1.5 or >1.3-1.5 compared with ≤1.3. These variables were allocated points, which allowed for stratification of patients with low- and high-risk for grade 4 bleeding. Cumulative incidence of grade 4 bleeding at day+60 was significantly higher among patients with high- versus low-risk (development: 31±7% vs. 2±1%, PP=0.008). In both cohorts, high bleeding risk was associated with disseminated intravascular coagulation (DIC) and proliferative disease. We developed and validated a simple risk model for grade 4 bleeding, which enables development of rational risk mitigation strategies to improve early mortality of intensive induction treatment.KEY POINTSRisk factors predicting grade 4 bleeding were consistent with DIC-like coagulopathy, including prolonged PT-INR and thrombocytopenia.The grade 4 bleeding score was externally validated and allows for preventive strategies to improve early mortality in high-risk patients.

Bleeding in patients with acute myeloid leukemia (AML) receiving intensive induction chemotherapy is multifactorial and contributes to early death. We sought to define the incidence and risk factors of grade 4 bleeding to support strategies for risk mitigation. Bleeding events were retrospectively assessed between day-14 and day +60 of induction treatment according to the World Health Organization (WHO) bleeding assessment scale, which includes grade 4 bleeding as fatal, life-threatening, retinal with visual impairment, or involving the central nervous system. Predictors were considered pretreatment or prior to grade 4 bleeding. Using multivariable competing-risk regression analysis with grade 4 bleeding as the primary outcome, we identified risk factors in the development cohort (n = 341), which were tested in an independent cohort (n = 143). Grade 4 bleeding occurred in 5.9% and 9.8% of patients in the development and validation cohort, respectively. Risk factors that were independently associated with grade 4 bleeding included baseline platelet count ≤40 × 109/L compared with >40 × 109/L, and baseline international normalized ratio of prothrombin time (PT-INR) >1.5 or 1.3 > 1.5 compared with ≤1.3. These variables were allocated points, which allowed for stratification of patients with low- and high-risk for grade 4 bleeding. Cumulative incidence of grade 4 bleeding at day+60 was significantly higher among patients with high- vs low-risk (development: 31 ± 7% vs 2 ± 1%; P < .001; validation: 25 ± 9% vs 7 ± 2%; P = .008). In both cohorts, high bleeding risk was associated with disseminated intravascular coagulation (DIC) and proliferative disease. We developed and validated a simple risk model for grade 4 bleeding, which enables the development of rational risk mitigation strategies to improve early mortality of intensive induction treatment.

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations occur in myeloid malignancies, but the mechanism by which these mutations drive oncogenesis remains incompletely understood. Here we performed a series of studies to define the phosphoproteome, CBL interactome and molecular mechanisms of signaling activation in cells expressing an allelic series of CBL mutants. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced PIK3R1 recruitment and downstream PI3K/AKT signaling in CBL-mutant cells. Furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary chronic myelomonocytic leukemia cells. Overall, our data provide rationale for exploring the therapeutic potential of LYN inhibition in patients with CBL-mutated myeloid malignancies.Statement of SignificanceWe investigated the oncogenic mechanisms of myeloid malignancy-associated CBL mutations by mass spectrometry-based proteomics and interactomics. Our findings indicate that increased LYN kinase activity in CBL-mutant cells stimulates PI3K/AKT signaling, revealing opportunities for the use of targeted inhibitors in CBL-mutated myeloid malignancies.

Background : Bleeding is a major complication of intensive AML induction therapy that has been linked to increased early mortality. Identifying patients with increased bleeding risk could improve outcomes by allowing development of preventative strategies. Prior studies have focused on clinical trial or registry cohorts to identify factors associated with bleeding outcomes, and may not reflect typical clinical practice or have adequately detailed clinical or genetic data. Therefore, we sought to define the incidence and distribution of bleeding in a real-world cohort of AML patients and determine which pretreatment clinical, laboratory and genetic factors were associated with bleeding. Patients and methods: We identified 341 consecutive adult patients with newly diagnosed AML who received anthracycline-based induction chemotherapy. The median age was 61 years (range 19-79). Diagnostic samples from all patients were sequenced for recurrently mutated myeloid genes. We collected pretreatment clinical and laboratory data, and annotated bleeding events during the first 60 days of treatment according to the WHO bleeding assessment scale. Bleeding was classified as clinically significant if WHO grade ≥2, or grade 1 if it prompted a change in platelet transfusion threshold. Disseminated intravascular coagulation (DIC) was assessed using a modified ISTH DIC-score including platelet count, PT-INR, and fibrinogen, where a score ≥3 points indicates high risk of DIC. D-dimer was excluded due to missing data. Results: A total of 87 patients (26%) had clinically significant bleeding events, most commonly involving gastrointestinal (n=35), genitourinary (n=31), and central nervous system [CNS (n=12)] sites. Patients with clinically significant bleeding events had worse survival within the first 60 days of induction treatment compared to patients without clinically significant bleeding (HR 5.24; 95% CI: 2.50-10.96, P < 0.001). The incidence of clinically significant bleeding was higher among patients with a high pretreatment modified DIC score compared to those with a low modified DIC score (44% vs. 24%, P = 0.002, Figure A-B). This difference was most prominent among patients with WHO grade 4 bleeds (22% vs. 3%, P < 0.001, Figure A). Most clinically significant bleeding events (n=57) occurred within the first two weeks of induction treatment (Figure B). Using multivariable logistic regression analysis, we found that pretreatment platelet count 1.3, and the presence of a DNMT3A mutation were independently associated with bleeding within the first 60 days of AML induction treatment (Table). Other factors previously linked with bleeding risk, including age, sex, hepatic and renal function, and leukocyte count, were not associated with an increased risk of bleeding in this cohort. Early mortality was particularly high (58%) in patients who experienced a CNS bleed, which events included intraparenchymal and/or subdural hemorrhage (n=9), and hemorrhagic transformation of ischemic stroke (n=3). Two thirds (n=8) of patients with CNS bleeds had high pretreatment modified DIC scores. Using univariate regression analysis, we found that the presence of DNMT3A or TET2 mutations had significantly increased odds ratios for CNS bleeds (3.84; 95% CI: 1.20-12.2 and 4.18; 95% CI: 1.27-13.71, respectively). The frequency of CNS bleeds was significantly higher amongst patients with DNMT3A and/or TET2 mutations compared to patients without these mutations (8% vs. 1%, P = 0.004). Conclusions: The incidence of bleeding in this real-world cohort of AML patients receiving intensive induction chemotherapy was higher than previously reported in selected clinical trial cohorts. Clinically significant bleeding during initial induction was associated with poor short-term survival. Low platelet count, elevated PT-INR and DNMT3A mutations were independently associated with clinically significant bleeding. The incidence of CNS bleeds was highest in patients with DNMT3A or TET2 mutations, and rare in patients without these mutations, raising the possibility that genetic events driving early leukemogenesis may exert pleiotropic effects that impact hemostasis. Together, these results identify a subset of AML patients who may benefit from additional supportive care measures to prevent early bleeding associated with morbidity and mortality of intensive induction treatment. Disclosures Wolach: Amgen: Other: Fees for lectures and Consultancy; Janssen: Other: Fees for lectures and Consultancy; Astellas: Consultancy, Honoraria, Other: Fees for lectures and Consultancy; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Fees for lectures and Consultancy, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Fees for lectures and Consultancy. Neuberg:Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding; Bluebird Bio: Consultancy.

Chronic graft-versus-host disease (cGVHD) affects >50% of hematopoietic stem cell transplant patients. Extracorporeal photopheresis (ECP), an immunomodulatory therapy, provides clinical benefit in steroid-refractory (SR) cGVHD, possibly via regulatory T (Treg) and natural killer (NK) cell expansion. We demonstrated that low-dose interleukin-2 (IL2) led to clinical improvement in SR-cGVHD and stimulated preferential Treg and NK-cell expansion with minimal effect on conventional T (Tcon) cells. We evaluated the effect of ECP (weeks 1-16) plus IL2 (1 × 106 IU/m2, weeks 9-16) in 25 adult patients with SR-cGVHD in a prospective phase 2 trial. Objective responses occurred in 29% and 62% of evaluable patients at weeks 8 (ECP alone) and 16 (ECP plus IL2), respectively. Eight weeks of ECP alone was associated with a marked decline in CD4+ Tcon (P = .03) and CD8+ T cells (P = .0002), with minimal change in Treg cells, Treg:Tcon cell ratio, or NK cells. Adding IL2 induced an increase in Treg cells (P < .05 at weeks 9-16 vs week 8), Treg:Tcon cell ratio (P < .0001 at weeks 9-16 vs week 8), and NK cells (P < .05 at weeks 9-16 vs week 8). Patients responding to ECP alone had significantly fewer CD4+ Tcon and CD8+ T cells at baseline compared with patients who responded after IL2 addition and patients who did not respond; neither Treg nor NK cells were associated with response to ECP alone. Altogether, ECP plus IL2 is safe and effective in patients with SR-cGVHD. ECP and IL2 have distinct immunologic effects, suggesting different therapeutic mechanisms of action. This trial was registered at www.clinicaltrials.gov as #NCT02340676.

BACKGROUND Alloantibodies recognizing human leukocyte antigens (HLA) can cause immune-mediated refractoriness to platelet transfusion. An association between HLA alloimmunization and red blood cell (RBC) alloimmunization has been suggested but remains uncertain. STUDY DESIGN AND METHODS We tested for HLA alloantibodies in 660 patients with and without RBC alloantibodies. Calculated panel reactive antibody (cPRA) values were determined for HLA alloimmunized patients. Current and historical diagnoses and blood product exposure were catalogued. Variables associated with high-level HLA alloimmunization (cPRA ≥ 90%) were evaluated. RESULTS The cohort included 366 women and 294 men with median age of 66 years (interquartile range [IQR], 53-76). The number of patients with and without RBC alloantibodies was 447 and 213, respectively. Among patients with and without RBC alloantibodies, 20.6% and 8.5% had a cPRA ≥ 90%, respectively (p

BACKGROUND Collection of hematopoietic progenitor cells by apheresis (HPC-A) requires separation of cells by density. Previous studies highlighted the challenges of HPC-A collection from patients with abnormal red blood cells (RBCs). TEMPI syndrome is a recently described condition defined by teleangiectasias, elevated erythropoietin and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting. Patients with TEMPI syndrome have responded to therapies used to treat plasma cell dyscrasias and may benefit from autologous HPC transplantation. We report HPC-A collection from a patient with TEMPI syndrome that was complicated by severe iron deficiency. STUDY DESIGN AND METHODS The patient received granulocyte–colony-stimulating factor (G-CSF) and plerixafor for HPC mobilization and underwent 3 days of HPC-A collection. RESULTS The patient presented for collection with a microcytic erythrocytosis. Over 3 days, approximately 50 L of whole blood was processed, and 2 × 108 CD34+ cells were collected (2.8 × 106 CD34+ cells/kg). The mean collection efficiency (CE), percentage of mononuclear cells, hematocrit (Hct), and RBC count were 18%, 90%, 14%, and 9 × 1011, respectively. Altering collection variables to avoid RBC contamination reduced CE. Ficoll preparations of the products after freeze-thaw showed RBC contamination and hemolysis. Postthaw viability exceeded 95%. The products were not RBC reduced or washed. There were no adverse reactions during or after infusion. CONCLUSIONS HPC-A collection from a patient with TEMPI syndrome was complicated by microcytic erythrocytosis, leading to RBC contamination and hemolysis in the product. Adequate HPCs were collected and the patient tolerated infusion without RBC depletion or washing. Our report highlights difficulties of HPC-A collection from iron-deficient patients.

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.

Summary CD8α + dendritic cells (DCs) prime cytotoxic T lymphocytes during viral infections and produce interleukin-12 in response to pathogens. Although the loss of CD8α + DCs in Batf3 −/− mice increases their susceptibility to several pathogens, we observed that Batf3 −/− mice exhibited enhanced resistance to the intracellular bacterium Listeria monocytogenes . In wild-type mice, Listeria organisms, initially located in the splenic marginal zone, migrated to the periarteriolar lymphoid sheath (PALS) where they grew exponentially and induced widespread lymphocyte apoptosis. In Batf3 −/− mice, however, Listeria organisms remain trapped in the marginal zone, failed to traffic into the PALS, and were rapidly cleared by phagocytes. In addition, Batf3 −/− mice, which lacked the normal population of hepatic CD103 + peripheral DCs, also showed protection from liver infection. These results suggest that Batf3 -dependent CD8α + and CD103 + DCs provide initial cellular entry points within the reticuloendothelial system by which Listeria establishes productive infection.

Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56bright CD16- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4+FoxP3+Helios+ regulatory T cells and CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright NK cells expanded by low-dose IL-2 has not previously been studied. Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x106 IU/M2/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56bright and CD56dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets. Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment. Cytolytic activity was measured in CD56bright and CD56dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim NK cells during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant. Disclosures Nikiforow: Kite Pharma: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Immature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)–containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca2+-mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse-specific functions. To determine if SV2A, -B, and -C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse-containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse-specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse-specific roles during synaptogenesis. J. Comp. Neurol. 460:106–122, 2003. © 2003 Wiley-Liss, Inc.

Parkinson's disease (PD) is characterized pathologically by preferential degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). Nigral cell death is accompanied by the accumulation of a wide range of poorly degraded proteins and the formation of proteinaceous inclusions (Lewy bodies) in dopaminergic neurons. Mutations in the genes encoding alpha-synuclein and two enzymes of the ubiquitin-proteasome system, parkin and ubiquitin C-terminal hydrolase L1, are associated with neurodegeneration in some familial forms of PD. We now show that, in comparison to age-matched controls, alpha-subunits (but not beta-subunits) of 26/20S proteasomes are lost within dopaminergic neurons and 20S proteasomal enzymatic activities are impaired in the SNc in sporadic PD. In addition, while the levels of the PA700 proteasome activator are reduced in the SNc in PD, PA700 expression is increased in other brain regions such as the frontal cortex and striatum. We also found that levels of the PA28 proteasome activator are very low to almost undetectable in the SNc compared to other brain areas in both normal and PD subjects. These findings suggest that failure of the ubiquitin-proteasome system to adequately clear unwanted proteins may underlie vulnerability and degeneration of the SNc in both sporadic and familial PD.

Summary Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.

Collection of hematopoietic progenitor cells by apheresis (HPC-A) requires separation of cells by density. Previous studies highlighted the challenges of HPC-A collection from patients with abnormal red blood cells (RBCs). TEMPI syndrome is a recently described condition defined by teleangiectasias, elevated erythropoietin and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting. Patients with TEMPI syndrome have responded to therapies used to treat plasma cell dyscrasias and may benefit from autologous HPC transplantation. We report HPC-A collection from a patient with TEMPI syndrome that was complicated by severe iron deficiency.The patient received granulocyte-colony-stimulating factor (G-CSF) and plerixafor for HPC mobilization and underwent 3 days of HPC-A collection.The patient presented for collection with a microcytic erythrocytosis. Over 3 days, approximately 50 L of whole blood was processed, and 2 × 10(8) CD34+ cells were collected (2.8 × 10(6) CD34+ cells/kg). The mean collection efficiency (CE), percentage of mononuclear cells, hematocrit (Hct), and RBC count were 18%, 90%, 14%, and 9 × 10(11) , respectively. Altering collection variables to avoid RBC contamination reduced CE. Ficoll preparations of the products after freeze-thaw showed RBC contamination and hemolysis. Postthaw viability exceeded 95%. The products were not RBC reduced or washed. There were no adverse reactions during or after infusion.HPC-A collection from a patient with TEMPI syndrome was complicated by microcytic erythrocytosis, leading to RBC contamination and hemolysis in the product. Adequate HPCs were collected and the patient tolerated infusion without RBC depletion or washing. Our report highlights difficulties of HPC-A collection from iron-deficient patients.

A high-salt diet (HSD) in humans is linked to a number of complications, including hypertension and cardiovascular events. Whether a HSD affects the immune response in transplantation is unknown. Using a murine transplantation model, we investigated the effect of NaCl on the alloimmune response in vitro and in vivo. Incremental NaCl concentrations in vitro augmented T cell proliferation in the settings of both polyclonal and allospecific stimulation. Feeding a HSD to C57BL/6 wild-type recipients of bm12 allografts led to accelerated cardiac allograft rejection, despite similar mean BP and serum sodium levels in HSD and normal salt diet (NSD) groups. The accelerated rejection was associated with a reduction in the proportion of CD4(+)Foxp3(+) regulatory T cells (Tregs) and a significant decrease in Treg proliferation, leading to an increased ratio of antigen-experienced CD4(+) T cells to Tregs in mice recipients of a HSD compared with mice recipients of a NSD. Because serum- and glucocorticoid-regulated kinase-1 (SGK1) has been proposed as a potential target of salt in immune cells, we fed a HSD to CD4(Cre)SGK1(fl/fl) B6-transplanted recipients and observed abrogation of the deleterious effect of a HSD in the absence of SGK1 on CD4(+) cells. In summary, we show that NaCl negatively affects the regulatory balance of T cells in transplantation and precipitates rejection in an SGK1-dependent manner.

Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes[superscript 1], but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing[superscript 2, 3, 4] to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease., National Institutes of Health (U.S.) (P01 CA108631), Leukemia & Lymphoma Society of America (Scholar Award), trategic Pharma-Academic Research Consortium for Translational Medicine (SPARC), National Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (Grant 5P50HG006193-02), Broad Institute of MIT and Harvard

Peptides derived from exogenous proteins are presented by both MHC class I and II. Despite extensive study, the features of the endocytic pathway that mediate cross-presentation of exogenous antigens on MHC class I are not entirely understood and difficult to generalize to all proteins. Here, we used dendritic cells and macrophages to examine MHC class I and II presentation of hen egg-white lysozyme (HEL) in different forms, soluble and liposome encapsulated. Soluble HEL or HEL targeted to a late endosomal compartment only allowed for MHC class II presentation, in a process that was blocked by chloroquine and a cathepsin S (CatS) inhibitor; brefeldin A (BFA) also blocked presentation, indicating a requirement for nascent MHC class II. In contrast, liposome-encapsulated HEL targeted to early endosomes entered the MHC class I and II presentation pathways. Cross-presentation of HEL in early endosomal liposomes had several unique features: it was markedly increased by BFA and by blockade of the proteasome or CatS activity, it occurred independently of the transporter associated with antigen processing but required an MHC class I surface-stabilizing peptide, and it was inhibited by chloroquine. Remarkably, chloroquine facilitated MHC class I cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, MHC class I and II presentation of HEL occurred through pathways having distinct molecular and proteolytic requirements. Moreover, MHC class I sampled antigenic peptides from various points along the endocytic route.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.

Synaptogyrins comprise a family of tyrosine-phosphorylated proteins with two neuronal (synaptogyrins 1 and 3) and one ubiquitous (cellugyrin) isoform. Previous studies have indicated that synaptogyrins are involved in the regulation of neurotransmitter release. Synaptogyrin 1 is a synaptic vesicle protein; cellugyrin, by contrast, is absent from synaptic vesicles. In an effort to further characterize the synaptogyrin family, we studied the distribution of the synaptogyrin 3 protein in the nervous system. Subcellular fractionation and immunoprecipitation of synaptic vesicles from mouse brain showed that synaptogyrin 3 is associated with synaptic vesicles and that synaptogyrins 1 and 3 can reside on the same synaptic vesicle. Immunofluorescent staining of cultured hippocampal neurons confirmed the synaptic localization of synaptogyrin 3. Analysis of the relative distributions of synaptogyrins 1 and 3 in mouse brain revealed a more restricted expression pattern for synaptogyrin 3 compared to the ubiquitous distribution of synaptogyrin 1. Strong synaptogyrin 3 labeling was observed in the mossy fiber region of the hippocampus, substantia nigra pars reticulata, pallidum, and deep cerebellar nuclei. By comparison, the striatum and reticular and ventral posterolateral thalamic nuclei, which all showed synaptogyrin 1 labeling, contained significantly less synaptogyrin 3. Finally, we used in situ hybridization experiments to correlate synaptogyrin 3 mRNA in cell bodies with synaptogyrin 3 protein at synapses. Altogether, our data indicate that neuronal synaptogyrins are differentially expressed protein isoforms that may represent functionally distinct populations of synapses and/or synaptic vesicles.

To define potential mechanisms of cell death during neural cell transplantation, we investigated the role of intracellular caspase activation in combination with the activation of serum complement. We demonstrated that ventral mesencephalic (VM) cells are susceptible to complement-mediated cell lysis that can be blocked with an anti-C5 complement inhibitor (18A10). We also determined that incubating freshly isolated allogenic VM cells with the caspase inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), followed by immediate striatal implantation, led to a 2.5-fold increase in tyrosine hydroxylase (TH) cell survival 12 weeks postimplantation (P < 0.05). In contrast, overnight incubation with BAF followed by striatal implantation led to a 2-fold reduction in TH cell survival at 12 weeks (P < 0.05). Using the optimal BAF treatment and complement inhibition, we tested the hypothesis that these treatments would lead to increased cell survival in both allogeneic and xenogeneic transplantation models. We transplanted cell suspensions of (a) rat E14 VM or VM treated with (b) BAF alone, (c) anti-C5, or (d) a combination of BAF and anti-C5. There was a significant increase in the relative number of TH-positive cells in the BAF/anti-C5 group versus control at 12 weeks posttransplantation. Similar results were achieved in a pig to rat xenotransplant paradigm. A neuronal xenograft marker (70-kDa neurofilament) also demonstrated relative increases in graft volume in the BAF/anti-C5 treatment group. These studies indicate that more than one mechanism can mediate cell death during neural cell transplantation and that a combined treatment using caspase and complement inhibition can significantly improve cell survival.

Structural and functional defects in 26/20S proteasomes occur in the substantia nigra pars compacta and may underlie protein accumulation, Lewy body formation and dopaminergic neuronal death in Parkinson's disease. We therefore determined the pathogenicity of proteasomal impairment following stereotaxic unilateral infusion of lactacystin, a selective proteasome inhibitor, into the substantia nigra pars compacta of rats. These animals became progressively bradykinetic, adopted a stooped posture and displayed contralateral head tilting. Administration of apomorphine to lactacystin-treated rats reversed behavioral abnormalities and induced contralateral rotations. Lactacystin caused dose-dependent degeneration of dopaminergic cell bodies and processes with the cytoplasmic accumulation and aggregation of alpha-synuclein to form inclusion bodies. These findings support the notion that failure of the ubiquitin-proteasome system to degrade and clear unwanted proteins is an important etiopathogenic factor in Parkinson's disease.

The proteolytic activities of 26/20S proteasomes are impaired in the substantia nigra pars compacta (SNc) in sporadic Parkinson's disease (PD). In the present study, we examined the structural integrity of the proteasome by determining the levels of the beta- and alpha-subunits which together normally constitute the catalytic core of 26/20S proteasomes. Western blot analyzes and immunohistochemical staining revealed a major and selective loss of alpha-subunits in dopaminergic neurons of the SNc but not in other brain regions in sporadic PD. This defect is known to cause the proteasome to become unstable and prevents its assembly with resultant impairment of enzymatic activity. Thus, structural and function defects in 26/20S proteasomes may underlie protein accumulation, formation of proteinaceous Lewy bodies and dopaminergic neuronal death in the SNc in sporadic PD.

Flow cytometry for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) has been widely used in pediatric patients to quantify therapeutic response and to assess the risk of relapse. Flow cytometry for MRD provides roughly the same level of sensitivity (0.01%) as molecular methods but at lower cost and with faster turnaround time. MRD assessment in ALL currently requires an evaluation of 20 or more parameters divided among multiple tubes. In part due to the assessment complexity, the use of flow cytometry for MRD detection in adult ALL patients has been relatively limited. We developed a 6-color, single-tube, flow cytometry assay to detect MRD in bone marrow (BM) aspirate specimens from adult ALL patients. The 73 patients included 52 patients with B-ALL (71%), 19 patients with T-ALL (26%) and 2 patients with T/myeloid leukemia (3%) and were treated with one of several standard chemotherapeutic regimens or targeted therapies. Patients were tested for MRD by flow cytometry after induction or re-induction therapy and serially thereafter. The 6-marker MRD panel was customized for each patient based on the 18-20-marker diagnostic immunophenotype. Sixty-three percent of B-ALL patients (n=33) had lymphoblasts with an aberrant immunophenotype; expression of a myeloid marker (e.g., CD13, CD15 or CD33) was the most common aberrancy. The remaining 37% of B-ALL patients (n=19) had disease with a hematogone immunophenotype, which comprised surface expression of CD10, CD19, CD20, CD34, CD38 and CD45; in the majority of these cases, leukemic cells were distinguishable from normal hematogones based on the intensity of surface marker expression. Forty-seven percent of T-ALL patients (n=9) had an aberrant immunophenotype, most often characterized by CD33 expression. One-hundred forty-six consecutive specimens analyzed for MRD by flow cytometry were classified as positive (23%), negative (72%) or uncertain (5%). Of the 34 samples classified as positive, 14 (41%) showed morphologic (i.e., BM aspirate or biopsy) evidence of disease; nineteen (65%) samples did not show morphologic evidence of disease and 1 sample did not have a concurrent morphologic assessment. Of the 105 samples classified as negative by flow cytometry, 103 (98%) were also negative by morphology and 1 sample did not have a concurrent morphologic assessment. One sample that was negative by flow cytometry had morphologic evidence of disease in the biopsy (10-20% blasts) but not the aspirate, suggesting that aspirate sampling artifact was responsible for the discrepancy. None of the 7 samples classified as uncertain by flow cytometry had morphologic evidence of disease; five out of 7 uncertain classifications were in B-ALL patients with hematogone immunophenotypes. Overall, MRD flow cytometry showed 86% concordance with the results of morphologic assessment. We evaluated outcomes in all patients with negative morphologic results and any positive MRD flow cytometry result(s). Of the 73 patients in this study, 61 had morphology-negative results that were either MRD-negative (n=45) or MRD-positive (n=16). Patients in this group were at various points of treatment post-induction or re-induction. Four out of 45 patients (9%) with MRD-negative results relapsed during a median follow-up period of 22 months, and 8 out of 16 patients (50%) with an MRD-positive result relapsed during a median follow-up period of 15 months (odds ratio for relapse 10.3, 95% confidence interval 2.5-42.4, P=0.001). In addition, relapse-related and overall mortality (Figure 1) were higher in patients with MRD-positive results (P=0.0023 and P=0.0016, respectively, by the log-rank test). In summary, we present a simplified, single-tube, flow cytometry assay that can be used to detect MRD in adult ALL at relatively low cost with rapid turnaround time; our approach was applicable to cases with either hematogone or aberrant immunophenotype, yielding a definitive result in 95% of cases. Notably, the presence of MRD was associated with relapse and mortality, suggesting that our method of MRD assessment could be used to guide treatment of adult ALL. Further analysis of the correlations between MRD results, clinical management and patient outcomes is ongoing. Disclosures: No relevant conflicts of interest to declare.

Odell et al. reported in Aging that murine embryo fibroblasts (MEFs) undergo p53 mutations and subsequent immortalizations in culture. This “release from cell cycle arrest” occurs in murine fibroblasts with a humanized or murine WT p53 [1]. The authors also noted that the cultured cells had frequently sustained a mutation matching a human tumor p53 mutation. Jang, et al. also reported spontaneous “immortalization and tumorigenic transformation” of human keratinocytes associated with p53 inactivating mutations [2]. Odell's remarkable findings are of great interest to us and support a recently proposed hypothesis for tumorigenesis. We and others have proposed that cells seek to survive, in a Darwinian sense [3, 4, 5]. Specifically we have presented evidence to suggest that while WT p53 might primarily function as the “guardian of the genome” (inducing cells to undergo apoptosis or cell cycle arrest under stress), the WT p53 gene sequence is evolutionarily maintained such that it is susceptible to inactivating mutations and consequent cell survival (with the untoward result of tumorigenesis). The facts that these mutations occur spontaneously and frequently are the exact mutations seen in human cancers lends key support to this notion. Taken together, spontaneous mutations allowing cell survival and tumorigenesis might be referred to as the “Selfish Cell Theory” in recognition of Dawkin's The Selfish Gene published in 1976.

Peptides derived from exogenous proteins are displayed by MHC class I via a pathway termed cross-presentation. Despite extensive study, the endocytic compartment(s) that mediate cross-presentation are poorly understood. Immunization of mice with hen egg-white lysozyme (HEL) protein cross-primes CD8 T cells recognizing HEL 23-31 presented by Db. Here we used liposome-encapsulated HEL to examine the cross-presentation capacity of specific subcellular compartments. Liposome-encapsulated HEL targeted to early endosomes, but not late endosomes/lysosomes, entered the MHC class I pathway. Cross-presentation of early endosomal HEL liposomes apparently required the transporter associated with antigen processing (TAP). However, additional experiments indicated no requirement for proteasome-dependent processing or nascent Db. To test the possibility that early endosomal HEL liposomes were presented by endosomal Db independently of TAP, we incubated TAP-deficient antigen presenting cells (APCs) with early endosomal HEL liposomes and a peptide to promote Db surface-stabilization and eventual entry into the endosomal pathway. Addition of Db surface-stabilizing peptide rescued cross-presentation of early endosomal HEL liposomes by TAP-deficient APCs, implying that Db mis-localization accounted for the cross-presentation defect observed previously. Cross-presentation of early endosomal HEL liposomes was inhibited when endolysosomal acidification was blocked by chloroquine. Surprisingly, chloroquine treatment facilitated cross-presentation of both late endosomal/lysosomal HEL liposomes and soluble HEL. Altogether, these data indicate that exogenous protein antigen can enter the MHC class I pathway through multiple routes within the APC.

A case of documented significant arsenic exposure followed by the development of dermatofibrosarcoma protuberans is reported. Exposure to arsenic is associated with an increased chance of the subsequent development of a variety of neoplasms. It is possible that dermatofibrosarcoma protuberans may be one such tumor.