Gregor S. D. Reid

The Cationic Antimicrobial Peptide LL-37 Modulates Dendritic Cell Differentiation and Dendritic Cell-Induced T Cell Polarization

Dendritic cells (DC) are instrumental in orchestrating an appropriately polarized Th cell response to pathogens. DC exhibit considerable phenotypic and functional plasticity, influenced by lineage, Ag engagement, and the environment in which they develop and mature. In this study, we identify the human cationic peptide LL-37, found in abundance at sites of inflammation, as a potent modifier of DC differentiation, bridging innate and adaptive immune responses. LL-37-derived DC displayed significantly up-regulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated costimulatory molecule expression, enhanced secretion of Th-1 inducing cytokines, and promoted Th1 responses in vitro. LL-37 may be an attractive therapeutic candidate for manipulating T cell polarization by DC.

mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL.

TAP expression provides a general method for improving the recognition of malignant cells in vivo.

A major class of tumors lack expression of the transporters associated with antigen processing (TAP). These proteins are essential for delivery of antigenic peptides into the lumen of the endoplasmic reticulum (ER) and subsequent assembly with nascent major histocompatibility complex (MHC) class I, which results in cell surface presentation of the trimeric complex to cytolytic T lymphocytes. Cytolytic T lymphocytes are major effector cells in immunosurveillance against tumors. Here we have tested the hypothesis that TAP downregulation in tumors allows immunosubversion of this effector mechanism, by establishing a model system to examine the role of TAP in vivo in restoring antigen presentation, immune recognition, and effects on malignancy of the TAP-deficient small-cell lung carcinoma, CMT.64. To test the potential of providing exogenous TAP in cancer therapies, we constructed a vaccinia virus (VV) containing the TAP1 gene and examined whether VV-TAP1 could reduce tumors in mice. The results demonstrate that TAP should be considered for inclusion in cancer therapies, as it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic complement of the tumor or the MHC haplotypes of the host.

Noninvasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a strategy for preclinical modeling

The efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic leukemia (ALL) cell lines and primary samples has facilitated identification of the antileukemia activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and endpoint analysis at high disease burden. In this study, we report that bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This approach provides a noninvasive, longitudinal measure of leukemia burden and localization that enhances the sensitivity of treatment response detection and provides greater insight into the mechanism of action of antileukemia agents. In addition, this study reveals significant cell line- and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Overall, this study demonstrates that the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects at earlier, previously unquantifiable disease burdens and thus provides the means to standardize and expedite the evaluation of anti-ALL activity in preclinical xenograft studies.

The role of the ovary and nutritional signals in the regulation of fat body yolk protein gene expression in Drosophila melanogaster

Abstract The yolk-protein genes of Drosophila are expressed in the ovarian follicle cells and fat bodies of adult females. Adults must relate egg production to environmental conditions, thus the regulation of these genes in the fat body is complex and their expression is modulated by the sex determination hierarchy, the hormones ecdysone and juvenile hormone, and the nutritional status of the fly. In some insects the ovary also affects the level of yolk-protein synthesis in the fat body. In this paper we show that the presence of a vitellogenic ovary has little effect on levels of yolk-protein synthesis in the fat body, though there are fewer yolk-protein transcripts in “ovaryless” Drosophila females. The nutritional effect acts rapidly to stop yolk-protein uptake and then reduces yolk-protein transcript levels. The fat body of starved females develops normally and is unlikely to account for differences in yolk-protein transcript levels. We show here that the nutritional effect on the fat body is not mediated by a developing ovary. Thus, it seems that we must look for a signal from the gut, which acts on both the ovary and the fat body or a signal which activates the release of hormone, but not ecdysone or juvenile hormone, which can rapidly affect these two tissues.

Novel molecular and cellular therapeutic targets in acute lymphoblastic leukemia and lymphoproliferative disease

While the outcome for pediatric patients with lymphoproliferative disorders (LPD) or lymphoid malignancies, such as acute lymphoblastic leukemia (ALL), has improved dramatically, patients often suffer from therapeutic sequelae. Additionally, despite intensified treatment, the prognosis remains dismal for patients with refractory or relapsed disease. Thus, novel biologically targeted treatment approaches are needed. These targets can be identified by understanding how a loss of lymphocyte homeostasis can result in LPD or ALL. Herein, we review potential molecular and cellular therapeutic strategies that (i) target key signaling networks (e.g., PI3K/AKT/mTOR, JAK/STAT, Notch1, and SRC kinase family-containing pathways) which regulate lymphocyte growth, survival, and function; (ii) block the interaction of ALL cells with stromal cells or lymphoid growth factors secreted by the bone marrow microenvironment; or (iii) stimulate innate and adaptive immune responses.

ETV6 (TEL)-AML1 pre-B acute lymphoblastic leukaemia cells are associated with a distinct antigen-presenting phenotype.

Summary. The recently recognized translocation t(12;21)(p13;q22), which results in the ETV6‐AML1 fusion product, is the most common genetic rearrangement found in childhood pre‐B acute lymphoblastic leukaemia (ALL). It has been associated with a more favourable prognosis and a distinct immunophenotype in terms of myeloid and B cell‐associated antigen expression. Using flow cytometry, we investigated whether the unique ETV6‐AML1 phenotype extended to molecules associated with antigen presentation by analysing 50 diagnostic bone marrow samples from paediatric pre‐B ALL patients. Reverse transcription polymerase chain reaction for the ETV6‐AML1 fusion transcript was positive in 14 patients. ETV6‐AML1‐positive samples were characterized by a significantly higher expression of the co‐stimulatory molecule CD40 (P < 0·0001), as well as a significantly higher class II HLA‐DR mean channel fluorescence (P = 0·001). In contrast, CD86 expression was significantly lower on fusion‐positive samples (P = 0·010) while there was no difference in expression of CD80 or major histocompatibility complex class I between ETV6‐AML1‐positive and ‐negative samples. This is the first observation in acute leukaemia that the distinct immunophenotype associated with specific translocations includes the expression of molecules associated with antigen presentation. In the case of ETV6‐AML1 pre‐B ALL, this characteristic immunophenotype may have implications for the immunogenicity of the leukaemic cells.

Differential killing of pre-B acute lymphoblastic leukaemia cells by activated NK cells and the NK-92 ci cell line

The use of NK cells in adoptive therapy for malignant disease is an area of great potential. Currently the only NK cell line in clinical trials is NK‐92, an activated NK cell line with a broad range of cytotoxicity against malignant cells. The activity of NK‐92 against pre‐B acute lymphoblastic leukaemias, however, is highly variable. In this study we compare the cytotoxic mechanisms and signalling pathways utilized by NK‐92 ci and IL‐2 activated NK cells to mediate killing of pre‐B acute lymphoblastic leukaemia cell lines. Deficiencies in TNF family mediated apoptosis, phosphoinositide‐3 kinase dependent and phosphoinositide‐3 kinase independent killing limit the efficiency of NK‐92 ci against pre‐B acute lymphoblastic leukaemia cells. Importantly, treatment of the poorly killed leukaemia cells with TNF‐α augmented both phosphoinositide‐dependent and ‐independent cytolysis.

Long-term protection from syngeneic acute lymphoblastic leukemia by CpG ODN-mediated stimulation of innate and adaptive immune responses.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a major cause of mortality in children with recurrent disease and in adults. Despite observed graft-versus-leukemia effects after stem cell transplantation, successful immune therapies for ALL have proven elusive. We previously reported immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODN) enhance allogeneic T(h)1 responses and reduce leukemic burden of primary human ALL xenografts. To further the development of CpG ODN as a novel ALL therapy, we investigated the antileukemia activity induced by CpG ODN in a transplantable syngeneic pre-B ALL model. CpG ODN induced early killing of leukemia by innate immune effectors both in vitro and in vivo. Mice were treated with CpG ODN starting 7 days after injection with leukemia to mimic a minimal residual disease state and achieved T cell-dependent remissions of more than 6 months. In addition, mice in remission after CpG ODN treatment were protected from leukemia rechallenge, and adoptive transfer of T cells from mice in remission conferred protection against leukemia growth. To our knowledge, this is the first demonstration that CpG ODN induce a durable remission and ongoing immune-mediated protection in ALL, suggesting this treatment may have clinical utility in patients with minimal residual disease.

Expression of the adaptor protein BLNK/SLP-65 in childhood acute lymphoblastic leukemia

Deficient expression of BLNK, an adaptor molecule crucial for normal B-cell development, is associated with increased pro-B/pre-B-cell expansion in mice. It has been proposed that BLNK deficiency is a primary cause of B-lineage acute lymphoblastic leukemia (ALL). We studied BLNK expression in the leukemic cells from 352 patients with childhood ALL (309 B-lineage; 43 T-lineage). By HG_U95Av2 Affymetrix GeneChip analysis, BLNK was expressed in 275 of 284 (96.8%) B-lineage ALL samples but in only one of 43 (2.3%) T-lineage ALL samples. Of 118 B-lineage ALL samples analyzed with the HG_U133A GeneChip, 117 (99.2%) expressed BLNK. All 30 primary B-lineage ALL samples studied by RT-PCR expressed BLNK transcripts; all 19 samples studied by Western blotting or flow cytometry expressed BLNK protein. Levels of BLNK in B-lineage ALL were as high as those of their normal counterparts; they were not related with genetic subgroups or differentiation stage. These results indicate that BLNK deficiency is a rare occurrence in childhood B-lineage ALL and is unlikely to be a common leukemogenic event as previously proposed.