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Multiple myeloma (MM) and plasmacytomas are cancers of antibody-secreting cells (ASCs). PRDM1/BLIMP1 is an essential regulator of ASC development. Histologic evidence shows that 100% of MM expresses PRDM1/BLIMP1, indicating that PRDM1/BLIMP1 is important for the development or persistence of MM. In contrast, some diffuse large B-cell lymphomas (DLBCLs) lose PRDM1 expression, suggesting that PRDM1 may act as a tumor suppressor in DLBCL. Thus, the role of PRDM1/BLIMP1 in transformation of mature B cells is unclear. We have used a plasmacytoma-prone transgenic mouse model to study the effect of Blimp1 loss on plasmacytoma prevalence, latency, and phenotype. Two possible outcomes could be envisaged: loss of Blimp1 might decrease plasmacytoma prevalence, through reduction of plasma cells, and so the number of susceptible transformation targets. Alternatively, Blimp1 may participate in the transformation process itself. Our results support the latter scenario, showing that decreasing Blimp1 dosage does not change plasma cell number in nontransgenic mice in vivo, but it significantly reduces plasmacytoma prevalence in transgenic mice. Loss of functional Blimp1 completely prevents plasmacytoma formation in this tumor model. These observations suggest that Blimp1 is limiting for plasma cell transformation and thus has potential as a target for new therapies to combat MM.

C/EBPalpha is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBPalpha in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBPalpha-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradiol reduced activated C/EBPalpha-ER to near the physiologic range. C/EBPalpha-ER increased Mac1(+)/Gr1(-)/MPO(-)/low monocytes 1.9-fold while reducing Mac1(+)/Gr1(+)/MPO(hi) granulocytes 2.5-fold at 48 hours, even in 0.01 microM estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transduced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in IL-3/IL-6/SCF or GM-CSF, but not G-CSF, even in 0.01 microM estradiol. C/EBPalpha-ER induced PU.1 mRNA, and PU.1-ER stimulated monocytic development, suggesting that transcriptional induction of PU.1 by C/EBPalpha contributes to monopoiesis. A C/EBPalpha variant incapable of zippering with c-Jun did not induce monopoiesis, and a variant unable to bind NF-kappaB p50 stimulated granulopoiesis, suggesting their cooperation with C/EBPalpha during monocytic commitment.

RUNX1 is an essential gene for mouse hematopoietic development. Mouse knockout models for either RUNX1 or its binding partner core binding factor(CBFβ) are embryonic lethal and lack definitive hematopoiesis. Dominant negative inhibitors of RUNX1 protein function CBFβ-SMMHC (INV), encoded by t(16;16) and AML1-ETO, encoded by t(8;21) are seen in approximately 8% and 15% of acute myeloid leukemia cases, respectively. Recently we reported that dominant inhibition of endogenous RUNX1 function by dual promoter lentivector-expressed INV inhibited proliferation and caused G1 arrest in both human and mouse myeloid progenitors. Conversely, exogenous expression of RUNX1 in mouse primary hematopoietic stem-progenitor cells resulted in increased proliferation [DCosta 2005]. In order to determine if exogenous expression of RUNX1 would enhance HESC-derived hematopoiesis, we used conditionally-regulated RUNX1 in human embryonic stem cells (HESC) as a model system. We transduced H1 (WA01) HESC with dual promoter lentivectors expressing modified estrogen receptor (ER) fusions: RUNX1-ER (EF-RUNX1-ER/PGK-GFP) or control GFP (Ub-GFP). Microscopically GFP+ HESC colonies were plucked and expanded on primary mouse embryonic fibroblasts to enrich for transduced cells. During growth under “pluripotent conditions,” no changes in HESC proliferation or differentiation (as assessed by staining for pluripotent surface markers SSEA-4, Tra-160 and CD9) were observed in cells induced to express RUNX1. Transduced HESC were allowed to undergo embryoid body (EB) formation. EB, cultured with or without 4-hydroxy-tamoxifen (4HT), were harvested at selected time points, dissociated with trypsin/EDTA, counted, and immunostained for CD34 and CD45 [Zambidis 2005; DCosta 2005]. Upon EB formation, there were 2–3-fold higher percentages and numbers of transduced (GFP+) cells in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture induced with 4HT than in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture without 4HT. In contrast, no 4HT-dependent difference was seen in the Ub.GFP control lentivector-transduced cell cultures. At these time points, 2–4% of the GFP+ cells in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture containing 4HT expressed CD34 and there were similar percentages of CD45+ cells, whereas there were lower numbers of cells expressing these markers in the control cultures. Thus, enforced expression of RUNX1 boosted the numbers of CD45+ and CD34+ cells in EB derived from HESC.

Proliferation is regarded as a sensitive parameter for assessing immune response to infectious agents, autoimmune conditions, and organ engraftment in transplantation. Proliferation of peripheral blood mononuclear (PBMC) cells has been measured by a number of flow cytometric methods including propidium iodide (PI), incorporation of Bromo-deoxy-uridine (BrdUr), and dilution of Carboxy-fluorescein diacetate, succinimidyl ester (CFSE) intensity. Standardization of these methods however, has been hampered by their technical complexity. The restricted expression of CyclinA2 protein in the cytoplasm during the S and G2 cell cycle phases has provided the basis for a simplified intracellular flow-based proliferation assay. In these studies, ex vivo stimulated human PBMC were tested for the validation of CyclinA2 as a proliferation marker for T cell subsets. Freshly isolated or cryopreserved PBMCs were incubated in vitro with SEB/CD28 for periods up to 72 hours and processed using different methodologies for lymphocyte cell cycle measurement. These included BrdUr /7AAD, PI/CyclinA2; cylinA2 with additional T cell phenotyping markers and a combined CyclinA2 /BrdUr proliferation method. Results for each cell subset were scored for the percentage of positive proliferating cells by each methodology. PBMC co-stained for CyclinA2 and DNA content by PI demonstrated that all cells expressing Cyclin A2 corresponded to cells at S or G2 phase. Furthermore, the time course assays combining BrdUr labeling with CvclinA2 confirmed that CyclinA2 expression corresponded selectively to cells incorporating BrdUr. When PMBC were surface phenotyped for CD3 and CD4/CD8, reproducible populations of CD4 and CD8 T cell subsets incorporating BrdUr were measured for expression of Cyclin A2. In our assay model 11 to 15 % positively stained for Cyclin A2 corresponded to approximately 10% and 5% of CD3/8 and CD3/4 CyclinA2 cells, respectively. The percentage of total BrdUr and total CyclinA2 positive cells that were detected in the dual-labeled specimens corresponded to the percentage detected by each individual method. In conclusion, measurement of in vitro lymphocytes proliferation using expression of CyclinA2 protein expression is highly correlated with BrdUr uptake demonstrating that CyclinA2 is an accurate marker of cell proliferation. Preparation of cells for measuring Cyclin A2 takes only 60 minutes as compared with a total 12 to 16 hours for BrdUr pulsing and staining time. The simple CyclinA2 preparation method described here makes it a practical, fast, and reliable proliferation flow assay that can be easily standardized for enumeration of proliferating lymphocyte subsets.

Understanding complex responses in biological systems is important to the development of new biomarkers, diagnostic assays, drugs and biologics required for early disease detection, prognosis, monitoring, and treatment. Although several cutting edge technologies have been introduced in the last decade, they have not significantly impacted the speed and economics of new drug applications and introduction of relevant diagnostic tests. In order to overcome this predicament, a Systems Biology approach is necessary to interrogate complex biological responses. This requires that assays that have so far been carried out in isolation, for e.g. gene expression, protein synthesis, phenotype and function, etc. need to be integrated and evaluated in a complementary fashion. In the current study, the authors have attempted such an evaluation by integrating well-known techniques of flow cytometry-based phenotypic analysis and cell sorting with protein fractionation and analysis to evaluate immune cellular activation profiles. To evaluate changes in protein profiles associated with the activation of an immune response, peripheral blood mononuclear cells (PBMCs) were stimulated with the superantigen, Staphylococcus enterotoxin B (SEB) and anti-CD28 or left un-treated for 24 hours. Lysates of PBMC populations from each culture were directly fractionated by two-dimensional gel-free liquid chromatography using isoelectric point (pI) and hydrophobicity. Alternatively, flow-based sorting techniques were 1st utilized to isolate T cells of interest from activated and control cultures and then fractionated as described above. Cell free supernatants from activated and non activated cultures were also fractionated and analyzed. The 1st dimension fractions obtained by separation based on pI alone did not reveal significant differences in activated and non activated lysates. The majority of the proteins in both the sorted and non-sorted lysates eluted in the acidic fractions of the pH gradient. However, using advanced differential display software, the hydrophobicity-dependent second dimension separations of the 1st dimension pI fractions did reveal significant quantitative and qualitative differences for several peaks in both the acidic and basic regions of the gradient in the activated and non activated fractions. Further combination of sorting of T cell populations with two-dimensional gel-free liquid chromatography permitted the characterization of an increased number of proteins that were up or down regulated by activation as compared to non sorted cell cultures. Thus the cumulative approach of sorting and 2-dimensional analysis was the most sensitive in discovering changes associated with activation. Such profiles could potentially be used as surrogate markers of efficacy in vaccine development and immunotoxicity in drug development. Further combinations of genomic, proteomic and cytomic profiling will further advance and ultimately simplify the discovery process and enable the mapping of biological pathways to accomplish a unified evaluation of relevant biomarkers in this response and other clinical models.

The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized. However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis. Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.

C/EBPα (−/−) cells do not effectively generate the granulocyte-monocyte progenitor (GMP) from the common myeloid progenitor (CMP). However, the role of C/EBPα in the further commitment of the GMP remains uncertain. Transduction of murine marrow or fetal liver cells with KRAB-C/EBPα-ER, an estradiol-regulated, dominant inhibitor of C/EBPs, suppresses formation of CFU-M, CFU-G, and CFU-GM without affecting formation of BFU-E, suggesting a role for C/EBPα in both myeloid lineages. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells and several other cell lines, but induces monocytic development from transduced B cell progenitors. We have now assessed the effect of C/EBPα on the myeloid commitment and maturation of murine myeloid progenitors. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPα-ER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient and only then subjected to lineage-depletion, just prior to culture +/− estradiol. This protocol, combined with rapid assessment of lineage maturation aims to avoid biases due to effects on proliferation or differentiation during transduction. C/EBPα-ER was expressed approximately 4-fold above endogeous levels. By 48 hrs, 80% of the lin- cells had acquired myeloid surface markers. Mac1+/Gr1+ or Mac1+/MPO+ cells, assessed by FACS, were granulocytes and Mac1+/Gr1− or Mac1+/MPO− cells were monocytes, as confirmed by staining cytospins. Activation of C/EBPα-ER with estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold at 48 hrs in five assessments (P < 0.002). A similar effect was seen when the estradiol dose was reduced from 1.0 to 0.3, 0.1, 0.03, or 0.01 μM, suggesting that observed effects on myeloid maturation occur near a physiologic activity level. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68, a 2-fold increase in M-CSFR and PU.1, and a 4-fold decrease in NE by 48 hrs. Estradiol had little effect on the myeloid development of vector-transduced cells or those expressing a leucine zipper mutant. Interestingly, transduction with the BR3 basic region mutant favored granulopoiesis in 5 independent experiments (P < 0.005), potentially reflecting dominant inhibition of endogenous C/EBPα or the the inability of this variant to interact with NF-κB p50. To assess effects on more immature progenitors, transduced cells were plated in methylcellulose. If estradiol was included, a 3- to 5-fold reduction in colony number precluded assessment of lineage commitment. However, if transduced progenitors were cultured with reduced concentrations of estradiol for 24 hrs and then plated without estradiol, C/EBPα-ER only reduced CFU numbers 1.2 to 2-fold. In two independent experiments, culture in IL3/IL6/SCF or in GM-CSF after transient activation of C/EBPα-ER resulted in a 2.6- to 5-fold increase in the ratio of CFU-M to CFU-G, indicating an effect on monocyte commitment at an early progenitor stage. For example, in one experiment no estradiol gave an average of 111 CFU per dish in IL3/IL6/SCF, culture for 24 hrs with 0.1 μM estradiol prior to plating gave 93 CFU, and pretreatment with estradiol increased the ratio of CFU-M to CFU-G from 1.1 to 3.9. We suggest a model of myelopoiesis in which C/EBPα first directs commitment of stem cells to the GMP by inducing PU.1 transcription. C/EBPα then has the capacity to direct monocytic commitment and maturation, likely dependent on cytokine conditions and availability of cooperating factors such as AP-1 or NF-κB.

CBFβ complexes with RUNX1/AML1 to form Core Binding Factor. CBFβ-SMMHC is expressed from the inv(16) or t(16;16) chromosome in 8% of AML cases. This fusion protein contains the majority of CBFβ linked to the α-helical rod domain of smooth muscle myosin heavy chain. CBFβ-SMMHC is thought to contribute to leukemogensis by dominantly inhibiting RUNX1/AML1. Inhibition of AML1 depends upon the integrity of a 28 amino acid region near the C-terminus of the SMMHC segment termed the Assembly Competence Domain (ACD). A homologous region is present in multiple myosins and is required for optimal multimerization of their respective rod domains. The ACD is located within a 63 residue "extended" ACD, which includes 12 residues N-terminal and 23 residues C-terminal to the ACD. The extended ACD was noted to have a more neutral charge than other segments of myosin rods. We have now carried out a mutagenic analysis of individual α-helices within or near the extended ACD and have assessed the effect of these mutations on the ability of CBFβ-SMMHC to multimerize in vitro and to inhibit endogenous AML1 activities in the Ba/F3 cell line and in normal murine myeloid progenitors. The 7 amino acids constituting a single turn of the rod domain α-helix are designated abcdefg. The a and d residues form a hydrophobic surface that mediates coiled-coil dimerization, the e and g residues often form salt bridges that stabilize the dimer, and the b, c and f residues are on the outer surface of the helix and are the best candidates for mediating multimerization. We have therefore mutated the bcf residues as a group in ten helices, N3, N1, A, B, C, D, E, F, G, and H. A–D constitutes the core, 28 residue ACD. N3 and N1 are three or one helix N-terminal to helix A. Mutation of N3 or N1 did not affect multimerization in low ionic strength or the ability of CBFβ-SMMHC to inhibit AML1-mediated G1 to S cell cycle progression in Ba/F3 cells. In contrast, mutation of helices A, B, C, D, E, F, G, or H both impaired multimerization in vitro and prevented cell cycle slowing in Ba/F3 cells. Mutants A–E are each located predominantly in the cell nucleus. In transduced murine myeloid progenitors, mutant N3 again behaved similar to intact CBFβ-SMMHC, mutant A also markedly slowed proliferation, mutant B had an intermediate effect, and mutants C, D, or E did not slow proliferation, each in three independent experiments. The increased activities of mutants A or B in the latter setting may reflect the fact that Ba/F3 cells accumulate three times faster than myeloid progenitors and so perhaps are more sensitive to subtle effects. Sin3A, a co-repressor shown to interact with CBFβ-SMMHC, retained the ability to bind mutants A–E. Analysis of mutants N1 and F–H for mSin3A binding, nuclear localization, and their effects on normal progenitor proliferation is in progress. Together, these findings indicate that a surface near the C-terminus of the CBFβ-SMMHC rod domain, encompassing much of the "extended ACD", is required for multimerization and inhibition of AML1. Helices N1 and H demarcate the boundaries of this surface, with helix H been the very last helix of the rod domain. Further characterization of the molecular interactions which allow this surface to mediate SMMHC multimerization may enable the rationale design of drugs for the therapy of AML associated with inv(16).

CBFβ-SMMHC, encoded by the inv(16) or t(16;16) translocations in approximately 8% of acute myeloid leukemia (AML) cases, is a fusion protein containing amino acids 1-165 of the 182 residue core binding factor β (CBFβ) and the rod domain of smooth muscle myosin heavy chain (SMMHC). The CBFβ domain of CBFβ-SMMHC retains the ability to interact with AML1/RUNX1. The SMMHC domain both mediates multimerization and interacts directly with corepressors, including mSin3A. CBFβ-SMMHC inhibits the expression of AML1-regulated genes, by sequestering AML1 in multimeric complexes and by directly repressing AML1-regulated genes. CBFβ-SMMHC was previously found to slow G1 to S cell cycle progression in hematopoietic cell lines, reflecting repression of AML1-regulated genes required for cell cycle, including cyclin D3. This effect was overcome be exogenous c-Myc or cdk4. In this study, murine marrow or human CD34+ cells were transduced with retroviral or lentiviral vectors, respectively, expressing CBFβ-SMMHC or two mutant variants. CBFβ-SMMHC reduced murine or human myeloid cell proliferation 3- to 4-fold in liquid culture, during a period when control murine cells accumulated 5-fold and human cells 20-fold. CBFβ-SMMHC decreased the formation of myeloid, but not erythroid, colonies 2- to 4-fold, and myeloid colonies expressing CBFβ-SMMHC were markedly reduced in size. Lack of effect on erythroid colonies reflects their lack of expression of AML1. The mutant variant CBFβ-SMMHC(Δ2-11) does not bind AML1 due to a deletion near its N-terminus, and CBFβ-SMMHC(ΔACD) does not multimerize or efficiently bind corepressors due to a 28 residue deletion near its C-terminus. Neither of these mutants, which were expressed at levels similar to wild-type, slowed proliferation or reduced myeloid colonies. CBFβ-SMMHC increased the G1/S ratio in wild-type murine and human progenitors. Proliferation was still slowed in p15(−/−) murine marrow cells transduced with CBFβ-SMMHC, suggesting that additional mutations, such as activation of growth factor receptors and consequent c-myc induction, are required in primary AMLs to allow enhanced proliferation. AML1-ER(T), which contains full-length AML1 and accelerates G1 to S progression in cell lines when activated by 4HT, had an effect opposite to CBFβ-SMMHC, stimulating proliferation of murine or human myeloid progenitors 2-fold. In summary, CBFβ-SMMHC inhibits the proliferation of myeloid progenitors dependent upon inhibition of AML1 and integrity of its Assembly Competence Domain. Targeting the CBFβ-SMMHC ACD or its CBFβ domain may uncover novel therapeutics useful for AML cases expressing this oncoprotein. Furthermore, these findings support a model we have proposed previously which states that mutations which accelerate G1 are required during leukemogenesis by CBFβ-SMMHC and other CBF oncoproteins. Finally, our results lend support to the conclusion that AML1 participates in the regulation of normal myeloid stem-progenitor cell proliferation. Exogenous AML1 may therefore be useful for expansion of hematopoietic stem-progenitor cells.