Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34+/CD38─ CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4+ SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26+ LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26+ CML LSC engrafted NOD-SCID-IL-2Rγ−/− (NSG) mice with BCR/ABL1+ cells, whereas CD26─ SC from the same patients produced multilineage BCR/ABL1– engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1+ cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy

Non-Hematopoietic Cells in Lymph Nodes Drive Memory CD8 T Cell Inflation during Murine Cytomegalovirus Infection

During human and murine cytomegalovirus (MCMV) infection an exceptionally large virus-specific CD8 T cell pool is maintained in the periphery lifelong. This anomalous response is only seen for specific subsets of MCMV-specific CD8 T cells which are referred to as 'inflationary T cells'. How memory CD8 T cell inflation is induced and maintained is unclear, though their activated phenotype strongly suggests an involvement of persistent antigen encounter during MCMV latency. To dissect the cellular and molecular requirements for memory CD8 T cell inflation, we have generated a transgenic mouse expressing an MHC class I-restricted T cell receptor specific for an immunodominant inflationary epitope of MCMV. Through a series of adoptive transfer experiments we found that memory inflation was completely dependent on antigen presentation by non-hematopoietic cells, which are also the predominant site of MCMV latency. In particular, non-hematopoietic cells selectively induced robust proliferation of inflationary CD8 T cells in lymph nodes, where a majority of the inflationary CD8 T cells exhibit a central-memory phenotype, but not in peripheral tissues, where terminally differentiated inflationary T cells accumulate. These results indicate that continuous restimulation of central memory CD8 T cells in the lymph nodes by infected non-hematopoietic cells ensures the maintenance of a functional effector CD8 T pool in the periphery, providing protection against viral reactivation events

Functionally Relevant Domains of the Prion Protein Identified In Vivo

The prion consists essentially of PrPSc, a misfolded and aggregated conformer of the cellular protein PrPC. Whereas PrPC deficient mice are clinically healthy, expression of PrPC variants lacking its central domain (PrPΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrPC (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrPΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrPΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrPΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants

Differential effects of TNF and LTalpha in the host defense against M. bovis BCG

Signaling via TNF receptor type 1 (TNFR1) was shown to be crucial in host defense against the intracellular pathogens L. monocytogenes, M. tuberculosis and M. bovis. To investigate the function of TNF and LTalpha in host defense against M. bovis, mice double deficient for TNF and LTalpha (TNF / LTalpha (- / -)), TNF / LTalpha (- / -) mice complemented with a murine LTalpha transgene (TNF(- / -)) and LTalpha (- / -) mice were infected with BCG and the ensuing pathology was investigated. Control mice showed a normal host defense with early clearance of bacteria. The granulomatous reaction in the liver was accompanied by recruitment of activated macrophages characterized by their acid phosphatase positivity and differentiation into epithelioid cells as well as a coordinated expression of proinflammatory transcripts. In contrast, TNF / LTalpha (- / -) mice showed no comparable recruitment of activated macrophages in the liver. Furthermore, these mice showed extensive necrotic pulmonary lesions with massive growth of acid fast bacilli. Reintroduction of LTalpha as a transgene into TNF / LTalpha (- / -) mice prolonged survival but did not restore resistance to BCG. This, at least partially protective role of LTalpha was further supported by data demonstrating that LTalpha -deficient mice as well were susceptible to BCG infection. In contrast to the deleterious effect of TNF / LTalpha deficiency in BCG infection, BCG-infected TNF / LTalpha (- / -) mice were tolerant to LPS-induced shock. These results demonstrate that TNF as well as LTalpha are involved in murine host defense against BCG and that absence of TNF / LTalpha protects BCG-infected mice from LPS mediated shock

Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.-Katter, K., Geurts, A. M., Hoffmann, O., Mates, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bosze, Z., Rulicke, T., Izsvak, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits

Lethal recessive myelin toxicity of prion protein lacking its central domain

PrP(C)-deficient mice expressing prion protein variants with large amino-proximal deletions (termed PrP(ΔF)) suffer from neurodegeneration, which is rescued by full-length PrP(C). We now report that expression of PrP(ΔCD), a PrP variant lacking 40 central residues (94–134), induces a rapidly progressive, lethal phenotype with extensive central and peripheral myelin degeneration. This phenotype was rescued dose-dependently by coexpression of full-length PrP(C) or PrP(C) lacking all octarepeats. Expression of a PrP(C) variant lacking eight residues (114–121) was innocuous in the presence or absence of full-length PrP(C), yet enhanced the toxicity of PrP(ΔCD) and diminished that of PrP(ΔF). Therefore, deletion of the entire central domain generates a strong recessive-negative mutant of PrP(C), whereas removal of residues 114–121 creates a partial agonist with context-dependent action. These findings suggest that myelin integrity is maintained by a constitutively active neurotrophic protein complex involving PrP(C), whose effector domain encompasses residues 94–134