Scientific Resources

Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies,but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally,strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently,using a xenograft model,we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study,we show that MYXV binds to resting,primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-?,interleukin-2 (IL-2),and soluble IL-2R?,but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM,we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells,thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM,ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens. View Publication

BACKGROUND Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples,making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However,it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology,immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells,according to proposed guidelines published by the European Myeloma Network (EMN) in 2012. METHOD Bone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification,13q14 deletion,17p deletion,t(4;14),and t(14;16)] in CD138(+) cells purified by magnetic cell sorting. RESULTS This test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14) were found in two cases. CONCLUSION This technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition,magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies. View Publication

Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells,polymorphonuclear (PMN) cells,and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126) showed that,of 84 patients with 100% donor DNA in PMN,16 (19%) had evidence of clinical relapse and textgreater10% recipient DNA in the plasma. Additional 16 patients of the 84 (19%) showed textgreater10% recipient DNA in plasma,but without evidence of relapse. Eight patients had mixed chimerism in granulocytes,lymphocytes,and plasma,but three of these patients had textgreater10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes,but cfDNA showed various levels of chimerism. Of these patients 14 (41%) showed laboratory or clinical evidence of relapse and all had textgreater10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse. View Publication

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients,eliminate virus infections,then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs),they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs,but there was little apparent expansion of these cells in patients. In that study,VSTs were gene-modified on day 19 of culture and we hypothesized that by this time,sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism,we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV),Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates,so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively,and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2,TNF-α,IFN-γ,MIP-1α,MIP-1β and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype,they should expand and persist in vivo,simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR). View Publication

Following haematopoietic stem cell transplantation,monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood,bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood,a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid,inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit,Accumol,Calgary,AB,Canada) (P = 0.59). Of the 303 samples tested by flow cytometry,290 (95.7%) exceeded 90% purity,and 215 (70.95%) were over 99% pure. There were some outlying samples,showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols,allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature,assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results. View Publication