Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner.

AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease.

Differential role of Id1 in MLL-AF9-driven leukemia based on cell of origin.

Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia.

The importance of regulatory T-cell heterogeneity in maintaining self-tolerance.

CD4(+) Forkhead box protein 3 (Foxp3)(+) regulatory T cells (Tregs) are the major cell type that mediates dominant tolerance in the periphery. Over the past decade, extensive study of Tregs has revealed that these cells express substantial heterogeneity to maintain tolerance and regulate immune responses. Tregs possess heterogeneity with respect to their origin and processes for development, functional activity, migratory pattern, and activation status. Some of the same environmental cues and molecular pathways utilized to generate specialized T-effector cells are also integrated by Tregs to colocalize and fine-tune suppressive mechanisms to optimally regulate and restrain distinctive self and antigen-specific T-cell responses. Here, we review our current understanding and significance of Treg heterogeneity in maintaining peripheral immune tolerance. We also highlight recent work from our laboratory that has studied the extent phenotypically distinct Treg subsets are related to each other and expand in an ordered fashion to give rise to highly activated short-lived Klrg1(+) suppressor cells to optimize immune regulation and maintain homeostasis of the Treg compartment.

IL-2R signaling is essential for functional maturation of regulatory T cells during thymic development.

CD4(+) Foxp3(+) regulatory T cells (Tregs) are an independent cell lineage, and their developmental progression during thymic development depends on IL-2R signaling. However, the role of IL-2R signaling during thymic Treg development remains only partially understood. The current study assessed the contribution of IL-2 to the expansion and functional programming of developing Tregs. In the absence of IL-2Rß signaling, predominantly CD4(+) CD25(-) Foxp3(lo) T cells were found, and these cells exhibited somewhat lower expression of the proliferative marker Ki67. These immature Tregs, which represent products of failed development, were also found in normal mice and were characterized by markedly lower expression of several Treg functional molecules. Therefore, IL-2R is required for the progression, functional programming, and expansion of Tregs during thymic development. An IL-2R-signaling mutant that lowers STAT5 activation readily supported Treg functional programming, but Treg proliferation remained somewhat impaired. The requirement for IL-2 during thymic Treg expansion was best illustrated in mixed chimeras where the Tregs with mutant IL-2Rs were forced to compete with wild-type Tregs during their development. Tregs with impaired IL-2R signaling were more prevalent in the thymus than spleen in these competitive experiments. The general effectiveness of mutant IL-2Rs to support thymic Treg development is partially accounted for by a heightened capacity of thymic Tregs to respond to IL-2. Overall, our data support a model in which limiting IL-2R signaling is amplified by thymic Tregs to readily support their development and functional programming, whereas these same conditions are not sufficient to support peripheral Treg homeostasis.

T-cell tolerance and the multi-functional role of IL-2R signaling in T-regulatory cells.

Signaling through the interleukin-2 receptor (IL-2R) contributes to T-cell tolerance by controlling three important aspects of regulatory T-cell (Treg) biology. IL-2 is essential for thymic Treg development and regulates Treg homeostasis and suppressive function. Analogous to activated conventional T lymphocytes, IL-2R signaling also plays an important part in Treg cell growth, survival, and effector differentiation. However, Treg cells somewhat distinctively assimilate IL-2R signaling. In particular, Treg cells require essentially only IL-2-dependent receptor proximal signal transducer and activator of transcription 5 (Stat5) activation, as they contain inhibitory pathways to minimize IL-2R-dependent activation of the phosphatidyinositol 3-kinase/Akt pathway. Moreover, many IL-2R-dependent activities, including full induction of Foxp3 expression, in Treg cells require minimal and transient Stat5 activation. Thus, Treg cells are equipped to sense and then develop and function within biological niches containing minimal IL-2. These distinguishing features of IL-2R signaling provide a mechanistic underpinning for using IL-2 as an agent to selectively target Treg cells in immunotherapy to induce tolerance in autoimmune diseases and in allogeneic transplant recipients.

IL-2 receptor signaling is essential for the development of Klrg1+ terminally differentiated T regulatory cells.

Thymic-derived natural T regulatory cells (Tregs) are characterized by functional and phenotypic heterogeneity. Recently, a small fraction of peripheral Tregs has been shown to express Klrg1, but it remains unclear as to what extent Klrg1 defines a unique Treg subset. In this study, we show that Klrg1(+) Tregs represent a terminally differentiated Treg subset derived from Klrg1(-) Tregs. This subset is a recent Ag-responsive and highly activated short-lived Treg population that expresses enhanced levels of Treg suppressive molecules and that preferentially resides within mucosal tissues. The development of Klrg1(+) Tregs also requires extensive IL-2R signaling. This activity represents a distinct function for IL-2, independent from its contribution to Treg homeostasis and competitive fitness. These and other properties are analogous to terminally differentiated short-lived CD8(+) T effector cells. Our findings suggest that an important pathway driving Ag-activated conventional T lymphocytes also operates for Tregs.

A multicenter study evaluation of the ClearLLab 10C panels.

Multiparameter flow cytometry plays an important role in the diagnosis, staging, and monitoring of patients with a suspected hematological malignancy. The ClearLLab 10C Panels consist of four reagent panels (B-Lineage Tube, T-Lineage Tube, and 2 Myeloid Lineage Tubes), each consisting of 10 color/10 antibody conjugates utilizing Beckman Coulters proprietary dry format optimized for investigating patients with suspected leukemia or lymphoma. A multicenter study was conducted to evaluate the performance of the ClearLLab 10C Panels for qualitative assessment of normal versus abnormal phenotype in peripheral blood, bone marrow, and lymph node samples with suspected hematological malignancies. ClearLLab 10C was compared to laboratory developed tests (LDTs) and final clinical diagnosis. Four clinical sites were used to enroll patient's spent specimens (n = 453); three laboratories in North America and one in Europe. Of the 453 specimens, 198 had no malignancy and 255 contained an abnormal population. The diagnostic accuracy of the ClearLLab 10C Panels was achieved with sensitivity of 96% and specificity of 95% with respect to patient final clinical diagnosis. The agreement of phenotyping between ClearLLab10C Panels and LDTs was 98%. Any differences noted between ClearLLab 10C and LDT were due to either the presence of populations below the level of detection, the lack of clinical information provided to the evaluators, or marker(s) not present in these panels. Overall, the ClearLLab 10C demonstrated excellent agreement to LDTs and diagnosis. These four reagent panels can be adopted by individual laboratories to assess the presence or absence of malignancy.

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure.

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP(393-408) (htNASP(393-408)), were investigated for their antifertility effect. Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice. Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice. Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP. There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide. The effect on fertility in the mice immunized with the synthesized peptide was reversible. Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody.

AIM: To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization. METHODS: A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 (anti-rhSMP-1) antibodies. Sperm acrosome reaction and sperm-zona pellucida (ZP) binding assay were carried out in 10-week-old BALB/c mice. RESULTS: Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 (P < 0.05). CONCLUSION: The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

[Cloning, expression and purification of human nuclear autoantigenic sperm protein (hNASP) and preparation of its polyclonal antibody].

OBJECTIVE: To acquire the purified human nuclear autoantigenic sperm protein (hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization. METHODS: The coding sequence of hNASP gene was amplified from human testis RNA with specific primers, and the PCR product was cloned first into pMD-18T and then into pET-28a ( + ) after restriction digestion with BamH I and Hind III. The fusion protein was expressed in E. coli BL21 (DE3) after induction with IPTG. The recombinant protein NASP was purified from the supernatant with Ni2 -NTA His-bind resin under native conditions. RESULTS: The results of DNA sequencing and SDS-PAGE analysis showed the protein to be what we had hoped to acquire. ELISA showed that we had acquired rabbit anti-hNASP serum with high titer. CONCLUSION: High purity recombinant hNASP protein could be obtained with the above-mentioned prokaryotic expression method, and so could the rabbit anti-hNASP serum with high titer and high specificity.

Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis.

Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells.

Screening of an antigen target for immunocontraceptives from cross-reactive antigens between human sperm and Ureaplasma urealyticum.

Epidemiologic studies indicated that some infertile men who were infected with Ureaplasma urealyticum displayed positive antisperm antibodies in their serum and/or semen. The purpose of this study was to investigate the possible mechanism of antisperm antibodies production after infection with U. urealyticum and to analyze the relationship between U. urealyticum and infertility. The existence of cross-reactive antigens (61, 50, and 25 kDa) between U. urealyticum and human sperm membrane proteins was confirmed. Among the cross-reactive antigens, the urease complex component UreG of U. urealyticum was determined. By searching the Swiss-Prot protein database, a pentapeptide identity (IERLT) between UreG and human nuclear autoantigenic sperm protein (NASP) was found. Furthermore, using Western blot analysis and enzyme-linked immunosorbent assay, the cross-reaction between the NASP and UreG was verified. Both anti-rUreG antibody and the antiserum against the synthetic peptide NASP393-408 containing the pentapeptide inhibited mouse sperm egg binding and fusion. After immunization by rUreG or the synthetic peptide, 81.2 and 75% female mice became sterile, respectively. The effect on fertility in mice immunized with the synthetic peptide was reversible. These findings proved for the first time that it was feasible to screen antigens for immunocontraceptives from cross-reactive antigens between sperm and microorganisms which induce infertility.