An endogenous suppressor of HIV-1 transcription
Human immunodeficiency virus-1 infection of brain mononuclear phagocytes can result in HIV-1-associated dementia. The search for transcription factors that regulate HIV-1 expression in the nervous system is at an early stage. Carlson et al. (p. 381 ) used mRNA differential display to identify a gene product, OTK18, that was up-regulated 7 days after HIV-1 infection of human monocyte-derived macrophages (MDM). An OTK18 fusion protein suppressed the promoting activity of a luciferase reporter in a trans manner. Deletion analyses localized the suppressive activity to a region near the N terminus that contained two Kruppel-associated box domains. In addition, an N-terminal region comprised of 13 zinc finger motifs contained a secondary suppressive domain. Immunoblotting of OTK18-transfected cell extracts revealed OTK18 proteins of three sizes, suggesting intracellular processing. OTK18-transfected MDM down-regulated several cytokine and chemokine mRNAs. These cells also had lower levels of HIV-1 reverse transcriptase activity and a significant increase in OTK18 mRNA expression by 5 days after infection compared with controls (HIV-1 infection only or with cells transfected with OTK18 deletion mutant plus HIV-1 infection). A trans-reporting system demonstrated that OTK18 suppressed the activity of the viral tat protein on the HIV-1 long terminal repeat promoter. The data demonstrate that HIV-1 infection of MDM induces synthesis of a transcriptional suppressor (OTK18) and suggest that OTK18 might have therapeutic potential.
Naive T cell homeostasis
Control of peripheral naive T cell homeostasis is mediated through contact with self-MHC/peptide complexes and IL-7. It is not known whether TCR affinity plays a role in homeostatic proliferation. Kieper et al. (p. 40 ) found that proliferation in irradiated hosts of T cell lines transgenic (Tg) for TCRs of different affinities for self-MHC/peptide ligands varied inversely with the density of TCR and directly with the level of CD5 expression. Coinjection of a low dose of bystander wild-type CD8+ T cells or of low affinity Tg TCR CD8+ T cells before injection of a mixture of the TCR Tg and wild-type cells suppressed proliferation of the autologous injected cells. Suppression of the highest affinity cells occurred only with a higher dose of high affinity TCR Tg bystander cells. Proliferation of the high affinity cells 4 wk after injection of the mixture of cells into unirradiated hosts was lowest in the autologous host and highest in the wild-type host and in the host of lower affinity. These levels were increased in hosts bred to an IL-7 Tg background. Most injected cells of lower affinity and wild-type did not proliferate in any host, and recoveries from the heterologous hosts were lower than seen for the highest affinity cells. The authors conclude that the survival advantage of high affinity over low affinity cells is a result of increased competitiveness for specific self-MHC/peptide ligands and an enhanced responsiveness to IL-7.
Lymphopenia-induced CD8+ T cell proliferation
Naive CD8+ T cells can be activated by self-MHC/peptide complexes in mice rendered lymphopenic by irradiation or gene deletion. Newborn mice provide a natural lymphopenic environment to study the acquisition of a memory phenotype as a result of lymphopenia-induced proliferation (LIP). Schüler et al. (p. 15 ) reconstituted neonatal C57BL6 (B6) mice with purified, CFSE-labeled CD8+ T cells from naive adult congenic mice. A total of 67%, 64%, and 41% of cells recovered from spleens of recipients injected 1 day, 4 days, and 7 days, respectively, after birth had proliferated (CFSElow) and had a memory (CD44high) phenotype. Nearly all cells recovered from similarly treated recombination-activating gene-deficient mice were also CFSElowCD44high, whereas most cells from injected B6 adult mice were CFSEhighCD44low. The majority of CD8+ T cells expressing a fluorescent protein maintained their CD44high phenotype at 8 wk after transfer. CFSE-labeled CD44lowCD8+ T cells transferred into B6 neonates proliferated and became CD44high. Pretreatment of B6 neonates with a mixture of mAbs against IL-7 and the IL-7R α-chain prevented LIP above background values seen in adult B6 recipients. Both injected and endogenous CD8+ T cells recovered 2 wk later from spleens of neonates injected on day 1 produced IFN-γ. The authors conclude that LIP-induced generation of memory CD8+ T cells in the neonate occurs before reconstitution of the naive T cell repertoire.
Inactivating bacterial virulence factors
During the inflammatory response, neutrophils release serine proteinases (NSPs) that act on a variety of substrates. Lopez-Boado et al. (p. 509 ) were interested in determining whether flagellin, the structural component of flagella in many bacterial species, was a substrate. The authors found that two NSPs, neutrophil elastase (NE) and cathepsin G (CG), degraded purified flagellin from Pseudomonas aeruginosa; degradation was prevented by preincubation of NE and CG with a serine protease inhibitor. Incubation of intact bacteria with NE and CG or incubation of purified flagellin with neutrophils from wild-type mice, but not with neutrophils from CG-deficient or NE/CG-deficient mice, resulted in flagellin degradation. The NSPs also degraded flagellin from Salmonella typhimurium. The cleavage sites for both NSPs were within the N-terminal domain of the protein. Intact flagellin, but not flagellin cleaved by NE or CG, induced expression of host defense genes in epithelial cell lines. Both flagellin and free active NE were detected by immunoblotting in cell-free bronchoalveolar lavage fluid from mice infected with P. aeruginosa. The data show that NSPs can modulate the host inflammatory response by degrading the bacterial virulence factor flagellin.
Clearing established HSV infection
Herpes simplex virus is complex in that it infects skin and travels to neural ganglia where the initial lytic infection becomes latent. Re-emergence of the virus occurs via retro-axonal flow to secondary skin sites. It is not clear at which stage HSV-specific CD8+ T cells clear the infection. van Lint et al. (p. 392 ) used a mouse zosteriform model of flank skin infection to study infection stages in a TCR transgenic animal whose CD8+ T cells recognized the HSV glycoprotein B (gB). Resting gB-specific CD8+ T cells, but not control transgenic T cells, transferred into the transgenic mice were found in the ganglia 5 days postinfection (p.i.) when secondary lesions began to appear. Recombination-activating gene-deficient mice injected with in vitro-activated gB T cells 4–5 days p.i. completely cleared virus from ganglia within 5 days after transfer, whereas all control mice died by 13 days p.i. Adoptive transfer of gB-peptide activated gB-specific T cells between 1 day before infection and 24 h p.i. lowered virus titers in the primary site of skin infection and in the neural ganglia by 100-fold; virus spread was not inhibited by transfer after 24 h p.i. Activated control cells had no impact on virus titers at either site. The authors suggest that HSV-specific CD8+ T cells normally clear replicating virus from established lytic infections in skin and nerves.
Antileukemic allogeneic NK cells
Allogeneic NK cells are used clinically to treat hematologic malignancies. Efforts are made to attain the “perfect mismatch” toachieve maximum NK cell-mediated lysis of the HLA-mismatched recipient leukemic cells. Leung et al. (p. 644 ) looked at the degree of mismatch between inhibitory killer-cell Ig-like receptors (KIR) on the donor’s NK cells and in the recipient’s HLA repertoire in stem cell transplants. This receptor-ligand model was most successful in predicting relapse of primary hematologic malignancies in 36 pediatric patients compared with a KIR ligand-ligand model and a NK cell cytotoxicity model. Recipient NK cells in 22 patients receiving purified CD34+ cells from an HLA-haploidentical donor assumed a donor specific KIR expression pattern within 3 mo of transplantation. NK cell-mediated lysis in vitro was highest against leukemia cells expressing the lowest numbers of transfected KIR HLA ligands. In vivo experiments in nonobese diabetic/SCID mice transplanted with the human NK cells and human leukemia cells showed that leukemia incidence was lowest in animals expressing the lowest numbers of transfected KIR HLA ligands. Prestimulation with both IL-12 and IL-18, or IL-12 alone, of leukemia cells expressing the highest number of KIR HLA ligands (the cells most resistant to NK-mediated lysis) rendered the cells sensitive to NK killing. The authors conclude that the best donor for NK transplantation would have the largest number of mismatch pairs but would have at least one KIR that recognizes an HLA on the recipient’s tumor cells.
IL-12 and Th1 differentiation
Development of Th1 responses is dependent on IL-12 activation of STAT4. The possibility that IFN-α may be an alternative route to Th1 differentiation of naive T cells has not been rigorously studied. Athie-Morales et al. (p. 61 ) used an APC-free human in vitro Th1/Th2 differentiation model to determine the direct actions of IL-12 and IFN-α. IFN-α only doubled the proportion of IFN-γ-producing cells, whereas IL-12 caused a nearly 5-fold increase compared with T cells activated without polarizing factors. However, only the IL-12-induced STAT4 activation was sustained (up to 48 h) and the constant presence of IL-12 was required. Cells pretreated with IFN-α were refractory to further IFN-α stimulation but could be stimulated by IL-12. Cell surface expression of one subunit of each cytokine receptor was down-regulated by 4 h after exposure to its ligand. IL-12 receptor subunit expression recovered by 24 h in the presence of IL-12; in contrast, IFN-α receptor subunit expression did not recover in the presence of IFN-α. IL-2 strongly increased the IL-12/STAT4 response through up-regulated expression of the IL-12 receptor subunit. The ability of IL-12 to activate T cells was lost in the presence of IL-2 Abs. Prolonged exposure (72 h) to IL-12 in the presence of IL-2 promoted optimal Th1 cell differentiation. IL-2 had no effect on the IFN-α/STAT4 response. The data show the requirement for cytokine signals of long duration, as occurs with IL-12 but not with IFN-α, in Th1 development.
Summaries written by Dorothy L. Buchhagen, Ph.D.