Serine protease inhibitor 6 plays a critical role in protecting murine granzyme B-producing regulatory T cells.

Regulatory T cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. Although Tregs express granzyme-B (GrB) to suppress effector T cells via direct killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. To our knowledge, we show for the first time that both induced Tregs and natural Tregs (nTregs) increase their intracellular expression of GrB and its endogenous inhibitor, serine protease inhibitor 6 (Spi6) upon activation. Subcellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6(-/-) Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6(-/-) nTregs and impaired suppression of alloreactive T cells in vitro. Spi6(-/-) Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6(-/-) nTregs were less effective than wild type nTregs in suppressing graft-versus-host disease because of their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared with wild type recipients. To our knowledge, our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.

Approaches to design non-covalent inhibitors for human granzyme B (hGrB).

A structure-based design campaign for non-covalent small molecule inhibitors of human granzyme B was carried out by means of a virtual screening strategy employing three constraints and probe site-mapping with FTMAP to identify ligand "hot spots". In addition, new scaffolds of diverse structures were subsequently explored with ROCS shape-based superposition methods, following by Glide SP docking, induced fit docking and analysis of QikProp molecular properties. Novel classes of moderately active small molecule blockers (≥25 µM IC50 values) from commercially available libraries were identified, and three novel scaffolds have been synthesized by multi-step procedures. Furthermore, we provide an example of a comprehensive structure-based drug discovery approach to non-covalent inhibitors that relies on the X-ray structure of a covalently bound ligand and suggest that the design path may be compromised by alternative and unknown binding poses.

A deficiency in nucleoside salvage impairs murine lymphocyte development, homeostasis, and survival.

The homeostasis of the immune system is tightly controlled by both cell-extrinsic and -intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function. In this article, we describe a deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway, which affects peripheral T cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosourea-induced mutation in the last α helix of DCK, a functionally null protein has been generated in the mouse and affects the composition of the hematopoietic system. Both B and T lymphocyte development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44(high)CD62L(low) memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notably the result of a cell-extrinsic-driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.

Mesenchymal stem cells express serine protease inhibitor to evade the host immune response.

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow-derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6(-/-)) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6(-/-) MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell-mediated death in vitro. In addition, SPI6(-/-) MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

The novel role of SERPINB9 in cytotoxic protection of human mesenchymal stem cells.

Clinical trials using allogeneic mesenchymal stem cells (MSCs) are ongoing for the purpose of providing therapeutic benefit for a variety of human disorders. Pertinent to their clinical use are the accessibility to sufficient quantities of these cells allowing for repetitive administration, as well as a better understanding of the specific mechanisms by which allogeneic MSCs evade host immune responses that in turn influence their life span following administration. In this report, we sought to characterize and compare human peripheral blood MSCs (hPB-MSCs) with bone marrow-derived MSCs. hPB-MSCs met the established criteria to characterize this cellular lineage, including capacity for self-renewal, differentiation into tissues of mesodermal origin, and expression of phenotypic surface markers. In addition, hPB-MSCs suppressed alloreactive proliferation as well as the production of proinflammatory cytokines. Examination of the mechanisms by which allogeneic MSCs evade the host immune response, which is crucial for their therapeutic use, demonstrated that constitutive expression of serine protease inhibitor 9 (PI-9) on hPB-MSCs and bone marrow-derived MSCs is a major defense mechanism against granzyme B-mediated destruction by NK cells. Similarly, MSCs treated with small interfering RNA for PI-9 increased MSC cellular death, whereas expression of transgenic PI-9 following retroviral transduction protected MSCs. These data significantly advance our understanding of the immunomodulatory role for hPB-MSCs as well as the mechanisms by which they evade host immune responses. These findings contribute to the development of MSC-based therapies for diseases.

RETRACTED: T cell metabolism. The protein LEM promotes CD8⁺ T cell immunity through effects on mitochondrial respiration.

Protective CD8(+) T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named lymphocyte expansion molecule (LEM) that promoted antigen-dependent CD8(+) T cell proliferation, effector function, and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results. Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration, resulting in the production of pro-proliferative mitochondrial reactive oxygen species (mROS). LEM provides a link between immune activation and the expansion of protective CD8(+) T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified cytotoxic CD8(+) T cells.

A simple laboratory practical to illustrate RNA mediated gene interference using drosophila cell culture.

RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode in vitro transcription promoters fused to sequences from Drosophila genes of the Rho/Rac GTPase family. These DNA templates are then used to synthesize double stranded RNAs (dsRNA), which are subsequently used to transfect Drosophila Kc embryonic cells. The resulting RNAi produces simple cellular phenotypes that are observed following fluorescent histochemical staining. These phenotypes are ultimately related to gene ontology data that the students generate through a bioinformatic analysis of the sequences transcribed into dsRNA. Taken together, this laboratory exercise provides "hands on" experience of RNAi in a class setting and provides a framework for the in-depth discussion of how this technique can be applied to studies of gene function.

A novel pyrazolo[1,5-a]pyrimidine is a potent inhibitor of cyclin-dependent protein kinases 1, 2, and 9, which demonstrates antitumor effects in human tumor xenografts following oral administration.

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC50= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G2/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI50= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity.

Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDK-activating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC(50) of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 micromol/L, with CDK2 being inhibited 35-fold less potently (IC(50) 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent.