Scientific Resources

Persistent viral infections are a major health concern worldwide. During persistent infection,overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection,and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously,we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However,the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8(+) and CD4(+) memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers,illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen. View Publication

Cytoplasmic APOBEC3G has been reported to block wild-type human immunodeficiency virus type 1 (HIV-1) infection in some primary cells. It is not known whether cytoplasmic APOBEC3G has residual activity in activated T cells,even though virion-packaged APOBEC3G does restrict HIV-1 in activated T cells. Because we found that APOBEC3G expression is greater in activated CD4(+) T-helper type 1 (Th1) lymphocytes than in T-helper type 2 (Th2) lymphocytes,we hypothesized that residual target cell restriction of incoming Vif-positive virions that lack APOBEC3G,if present,would be greater in Th1 than Th2 lymphocytes. Infection of activated Th1 cells with APOBEC3-negative virions did result in decreased amounts of early and late reverse transcription products and integrated virus relative to infection of activated Th2 cells. Two-long terminal repeat (2-LTR) circles,which are formed in the nucleus when reverse transcripts do not integrate,were increased after APOBEC3-negative virus infection of activated Th1 cells relative to infection of activated Th2 cells. In contrast,2-LTR circle forms were decreased after infection of APOBEC3G-negative cells with APOBEC3G-containing virions relative to APOBEC3G-negative virions and with Th1 cell-produced virions relative to Th2 cell-produced virions. Increasing APOBEC3G in Th2 cells and decreasing APOBEC3G in Th1 cells modulated the target cell phenotypes,indicating causation by APOBEC3G. The comparison between activated Th1 and Th2 cells indicates that cytoplasmic APOBEC3G in activated Th1 cells partially restricts reverse transcription and integration of incoming Vif-positive,APOBEC3G-negative HIV-1. The differing effects of cytoplasmic and virion-packaged APOBEC3G on 2-LTR circle formation indicate a difference in their antiviral mechanisms. View Publication

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. View Publication

The control of tyrosine phosphorylation depends on the fine balance between kinase and phosphatase activities. Protein tyrosine phosphatase 1B (PTP-1B) and T cell protein tyrosine phosphatase (TC-PTP) are 2 closely related phosphatases known to control cytokine signaling. We studied the functional redundancy of PTP-1B and TC-PTP by deleting 1 or both copies of these genes by interbreeding TC-PTP and PTP-1B parental lines. Our results indicate that the double mutant (tcptp(-/-)ptp1b(-/-)) is lethal at day E9.5-10.5 of embryonic development with constitutive phosphorylation of Stat1. Mice heterozygous for TC-PTP on a PTP-1B-deficient background (tcptp(+/-)ptp1b(-/-)) developed signs of inflammation. Macrophages from these animals were highly sensitive to IFN-gamma,as demonstrated by increased Stat1 phosphorylation and nitric oxide production. In addition,splenic T cells demonstrated increased IFN-gamma secretion capacity. Mice with deletions of single copies of TC-PTP and PTP-1B (tcptp(+/-)ptp1b(+/-)) exhibited normal development,confirming that these genes are not interchangeable. Together,these data indicate a nonredundant role for PTP-1B and TC-PTP in the regulation of IFN signaling. View Publication

Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine,we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly,neoadjuvant vaccination was far superior and provided approximately 100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. View Publication

OBJECTIVE: T lymphocytes play an important role in systemic sclerosis (SSc),a connective tissue disease characterized by inflammation,fibrosis,and vascular damage. While their precise role and antigen specificity are unclear,T cell-derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc. METHODS: To identify relationships between a specific cytokine,T cell subset,and the disease course,we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining,we analyzed the intrinsic ability of CD4+ and CD8+ T cell subsets to produce cytokines following in vitro activation. RESULTS: High levels of the profibrotic type 2 cytokine interleukin-13 (IL-13) were produced following activation of peripheral blood effector CD8+ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast,CD4+ T cells showed a lower and more variable level of IL-13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients. CONCLUSION: Dysregulated IL-13 production by effector CD8+ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target. View Publication

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76),hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly,cell death is triggered in macrophages cultured with GMME1,while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation,inhibition of p44/42,AKT and STAT3 phosphorylation,and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17,MOG-specific Ab titers,and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2. View Publication

Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3(+) regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However,it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here,we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2,an isoform of Aldh1a,but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2,but not the other known RA-producing enzymes. Employing a flow cytometric method,we detected ALDH activities in 10-30% of PP-DCs and MLN-DCs. They were CD11c(high)CD4(-/low)CD8alpha(intermediate)CD11b(-/low) F4/80(low/intermediate)CD45RB(low)CD86(high)MHC class II(high)B220(-)CD103(+). Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs,however,additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2(+) DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria,whereas IL-4 receptor-mediated signals were dispensable. GM-CSF(+)CD11c(-)F4/80(+) cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA. View Publication

Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore,bone marrow samples from 100 children with acute lymphoblastic leukemia (ALL) were cultured in the cytokines TNF-alpha,GM-CSF,c-kit ligand,and fetal liver tyrosine kinase 3 ligand,with or without IL-3 and -4 and after administration of HdI valproic acid (VAL),suberoylanilide hydroxamic acid (SAHA),isobutyramid,or trichostatin A. Among the tested samples,25 were positive for the chromosomal translocation t(12;21),encoding the fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 (TEL/AML1). SAHA increased CD83 expression of TEL/AML1-positive blasts in conditions without ILs,and SAHA and VAL increased the number of CD86(+)80(-) cells in the presence of ILs. VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive,leukemic DC; VAL and SAHA reduced those of TEL/AML1-negative DC. Cytotoxic T cells sensitized with leukemic DC produced more IFN-gamma and TNF-alpha upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of nondifferentiated blasts,which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. Therefore,the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended. View Publication

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis,type 1 diabetes mellitus,and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5),containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin,is a potent and selective blocker of these channels. However,a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability,ShK-192,contains a nonhydrolyzable phosphotyrosine surrogate,a methionine isostere,and a C-terminal amide. ShK-192 shows the same overall fold as ShK,and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg,approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24,48,and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells. View Publication

The CD31(+) subset of human naive CD4(+) T cells is thought to contain the population of cells that have recently emigrated from the thymus,while their CD31(-) counterparts have been proposed to originate from CD31(+) cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4(+) T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31(+) naive CD4(+) T cells from adult peripheral blood compared with the CD31(-) subset. IL-7-driven proliferation did not result in loss of CD31 expression,suggesting that CD31(+) naive CD4(+) T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore,IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together,our data suggest that during adulthood CD31(+) naive CD4(+) T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication