Gα13 Mediates a Signal That Is Essential for Proliferation and Survival of Thymocyte Progenitors

G protein signaling via the Gα12 family (Gα12 and Gα13) has not been well studied in T cells. To investigate whether Gα12 and Gα13 are involved in thymopoiesis, we expressed the regulator of G protein signaling domain of p115RhoGEF to inhibit Gα12 and Gα13 during thymopoiesis. Fetal thymus organ cultures seeded with p115ΔDH-expressing progenitor cells showed impaired thymopoiesis with a block at the CD4−CD8−CD44−CD25+ (DN3) stage. Using Gα13 or Gα12 minigenes, we demonstrated that Gα13, but not Gα12, is required for thymopoiesis. T progenitor cells expressing p115ΔDH showed reduced proliferation and increased cell death. T cell receptor stimulation of the fetal thymus organ cultures did not rescue the block. Overexpression of the antiapoptotic gene Bcl2 rescued the defect in DN3 cells and partially rescued T cell development. Therefore, Gα13-mediated signaling is necessary in early thymocyte proliferation and survival

TSLP is involved in expansion of early thymocyte progenitors

© 2007 Jiang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

A genetic approach to inactivating chemokine receptors using a modified viral protein

We have developed a genetic system, called degrakine, that specifically and stably inactivates chemokine receptors (CKR) by redirecting the ability of the HIV-1 protein, Vpu, to degrade CD4 in the endoplasmic reticulum (ER) via the host proteasome machinery. To harness Vpu’s proteolytic targeting capability to degrade new receptors, we fused a chemokine with the C terminal region of Vpu. The fusion protein, or degrakine, accumulates in the ER, trapping and functionally inactivating its target CKR. We have demonstrated that degrakines based on SDF-1 (CXCL12), MDC (CCL22) and RANTES (CCL5) specifically inactivate their respective receptor functions. Using a retroviral vector expressing the SDF-1 degrakine, we have established that CXCR4 is required for the homing of hematopoietic stem/progenitor cells (HSPC) to the bone marrow immediately after transplantation. Thus the degrakine provides an effective genetic tool to dissect receptor functions in a number of biological systems in vitro and in vivo

The CATERPILLER protein Monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor α-, and Mycobacterium tuberculosis-induced pro-inflammatory signals

The CATERPILLER (CLR, also NOD and NLR) proteins share structural similarities with the nucleotide binding domain (NBD)-leucine-rich repeat (LRR) superfamily of plant disease-resistance (R) proteins and are emerging as important immune regulators in animals. CLR proteins contain NBD-LRR motifs and are linked to a limited number of distinct N-terminal domains including transactivation, CARD (caspase activation and recruitment), and pyrin domains (PyD). The CLR gene, Monarch-1/Pypaf7, is expressed by resting primary myeloid/monocytic cells, and its expression in these cells is reduced by Toll-like receptor (TLR) agonists tumor necrosis factor (TNF) α and Mycobacterium tuberculosis. Monarch-1 reduces NFκB activation by TLR-signaling molecules MyD88, IRAK-1 (type I interleukin-1 receptor-associated protein kinase), and TRAF6 (TNF receptor (TNFR)-associated factor) as well as TNFR signaling molecules TRAF2 and RIP1 but not the downstream NFκB subunit p65. This indicates that Monarch-1 is a negative regulator of both TLR and TNFR pathways. Reducing Monarch-1 expression with small interference RNA in myeloid/monocytic cells caused a dramatic increase in NFκB activation and cytokine expression in response to TLR2/TLR4 agonists, TNFα, or M. tuberculosis infection, suggesting that Monarch-1 is a negative regulator of inflammation. Because Monarch-1 is the first CLR protein that interferes with both TLR2 and TLR4 activation, the mechanism of this interference is significant. We find that Monarch-1 associates with IRAK-1 but not MyD88, resulting in the blockage of IRAK-1 hyperphosphorylation. Mutants containing the NBD-LRR or PyD-NBD also blocked IRAK-1 activation. This is the first example of a CLR protein that antagonizes inflammatory responses initiated by TLR agonists via interference with IRAK-1 activation

Soluble Rhesus Lymphocryptovirus gp350 Protects against Infection and Reduces Viral Loads in Animals that Become Infected with Virus after Challenge

Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans

TSLP is involved in expansion of early thymocyte progenitors

Abstract Background Thymic stromal derived lymphopoietin (TSLP) is preferentially and highly expressed in the thymus, but its function in T cell development is not clear. Results We report here that TSLP, independently or in combination with IL-7, enhances thymopoiesis in the murine fetal thymic organ culture (FTOC) model. Furthermore, TSLP preferentially increases the number and proliferation of the (DN1 and DN2) pro-T progenitor cells, and FTOC lobes from TSLP receptor-null mice show a decreased number of these cells. Finally, DN1-DN2 cells expanded with TSLP in vitro are functional T progenitors that are able to differentiate into mature T cells in fetal or adult thymus organs. Conclusion Together, these data suggest that TSLP plays an important role in expansion of thymocyte progenitors and may be of value for expanding T progenitor cells in vitro.</p

DN1 and DN2 cells are preferentially increased by TSLP in FTOC and decreased in TSLPRFTOC

<p><b>Copyright information:</b></p><p>Taken from "TSLP is involved in expansion of early thymocyte progenitors"</p><p>http://www.biomedcentral.com/1471-2172/8/11</p><p>BMC Immunology 2007;8():11-11.</p><p>Published online 18 Jul 2007</p><p>PMCID:PMC1940266.</p><p></p> A and B, FTOCs using WT E15 thymic lobes were performed with different doses of TSLP (10, 30 or 100 ng/ml) for 2 weeks. Thymocytes that were negative for expression of CD3/CD4/CD8 were further analyzed for DN subpopulation based on the expression of CD44 and CD25. Shown are representative data from at least 4 independent experiments. SD is shown as error bars. *: P < 0.05, * *: P < 0.01, compared with medium control. C and D, FTOCs from littermate TSLPRor TSLPRembryos were cultured for 2 weeks and DN cells were analyzed. The data is a summary of 3 independent experiments. Error bars indicate SD, * P < 0.05, * *: P < 0.01, compared with TSLPRcontrol lobes

TSLP and IL-7 both enhances early T progenitor cells and thymopoiesis in mouse fetal thymic organ culture

<p><b>Copyright information:</b></p><p>Taken from "TSLP is involved in expansion of early thymocyte progenitors"</p><p>http://www.biomedcentral.com/1471-2172/8/11</p><p>BMC Immunology 2007;8():11-11.</p><p>Published online 18 Jul 2007</p><p>PMCID:PMC1940266.</p><p></p> E15 fetal thymus organs were cultured with either TSLP (30 ng/ml) and/or IL-7 (100 ng/ml) for 2 weeks. Total thymocytes were harvested and counted (A). Thymocytes that were negative for expression of CD3/CD4/CD8 were further analyzed for DN subpopulation based on the expression of CD44 and CD25 (B). The data is a summary of 2 independent experiments. Standard deviation (SD) is shown as error bars. *: P < 0.05, * *: P < 0.01, compared with medium control

DN1 and DN2 cells from TSLP-treated FTOC have T progenitor activity to generate phenotypically mature thymocytes

<p><b>Copyright information:</b></p><p>Taken from "TSLP is involved in expansion of early thymocyte progenitors"</p><p>http://www.biomedcentral.com/1471-2172/8/11</p><p>BMC Immunology 2007;8():11-11.</p><p>Published online 18 Jul 2007</p><p>PMCID:PMC1940266.</p><p></p> DN1 and DN2 cells from FTOC lobes (Ly5.2) cultured with TSLP (30 ng/ml) or medium control were purified. Sorted DN1 cells (4 × 10/mouse) were injected directly into thymi of irradiated (500 rad) Ly5.1 recipient mice with fresh carrier cells (Ly5.1thymocytes, 1 × 10/mouse). Two weeks after injection, thymocytes from recipient mice were analyzed for % donor cells (A), total number donor cells in the thymus (B), and % and number (C and D) of different donor thymocyte subsets. E and F, Sorted DN2 cells (1 × 10/well) were seeded into 2-dG treated E15 thymic lobes by hanging drop culture, and cultured as FTOC for 2 weeks. Thymocytes were harvested and analyzed as above. The data is representative of two independent experiments. Bars indicate SD, * P < 0.05, compared with medium control