CD4+ T Cell Help Confers a Cytotoxic T Cell Effector Program Including Coinhibitory Receptor Downregulation and Increased Tissue Invasiveness.

CD4+ T cells optimize the cytotoxic T cell (CTL) response in magnitude and quality, by unknown molecular mechanisms. We here present the transcriptomic changes in CTLs resulting from CD4+ T cell help after anti-cancer vaccination or virus infection. The gene expression signatures revealed that CD4+ T cell help during priming optimized CTLs in expression of cytotoxic effector molecules and many other functions that ensured efficacy of CTLs throughout their life cycle. Key features included downregulation of PD-1 and other coinhibitory receptors that impede CTL activity, and increased motility and migration capacities. "Helped" CTLs acquired chemokine receptors that helped them reach their tumor target tissue and metalloprotease activity that enabled them to invade into tumor tissue. A very large part of the "help" program was instilled in CD8+ T cells via CD27 costimulation. The help program thus enhances specific CTL effector functions in response to vaccination or a virus infection.

High functional CD70 expression on α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia.

Antigen-loaded dendritic cells (DCs) used as anticancer vaccine holds promise for therapy, but needs to be optimized. The most frequently described DC vaccine is being matured with a cocktail containing prostaglandin E2 (PGE2 DC). However, even though PGE2 DCs express both costimulatory and migratory receptors, their IL-12p70-prodcution is low, leading to an insufficient Th1 immune response. As an alternative, α-type-1 polarized DCs (αDC1s) have shown a superior production of IL-12p70 and subsequent activation of effector cells. From chronic lymphocytic leukaemia (CLL) patients, αDC1s can be generated to induce a functional Th1-immune response. Yet, another costimulatory receptor, CD70, appears to be essential for optimal DC function by promotion of T cell survival and function. So far, PGE2 is suggested as one of the most important factors for the induction of CD70 expression on DCs. Therefore, we wanted to investigate whether αDC1s have the ability to express functional CD70. We found that CD70 expression on αDC1s could be upregulated in the same manner as PGE2 DCs. In an allogeneic mixed leucocyte reaction, we found that antibody-blocking of CD70 on αDC1s from controls reduced effector cell proliferation although this could not be found when using CLL αDC1s. Nevertheless, CD70-blocking of αDC1s from both controls and patients with CLL had a negative influence on the production of both IL-12p70 and the Th1 cytokine IFN-γ, while the production of the Th2 cytokine IL-5 was enhanced. Together, this study further suggests that αDC1s should be considered as a suitable candidate for clinical antitumour vaccine strategies in patients with CLL.

Genome-wide pathway analysis in attention-deficit/hyperactivity disorder.

This study aimed to (1) to identify candidate single-nucleotide polymorphisms (SNPs) and mechanisms of attention-deficit/hyperactivity disorder (ADHD) and (2) to generate SNP-to-gene-to-pathway hypotheses. An ADHD genome-wide association study (GWAS) dataset that included 428,074 SNPs in 924 trios (2,758 individuals) of European descent was used in this study. The Identify candidate Causal SNPs and Pathways (ICSNPathway) analysis was applied to the GWAS dataset. ICSNPathway analysis identified 11 candidate SNPs, 6 genes, and 6 pathways, which provided 6 hypothetical biological mechanisms. The strongest hypothetical biological mechanism was that rs2532502 alters the role of CD27 in the context of the pathways of positive regulation of nucleocytoplasmic transport [nominal p < 0.001; false discovery rate (FDR) = 0.028]. The second strongest mechanism was the rs1820204, rs1052571, rs1052576 → CASP9 → mitochondrial pathway (nominal p < 0.001; FDR = 0.032). The third mechanism was the rs1801516 → ATM → CD25 pathway (nominal p < 0.001; FDR = 0.034). By applying the ICSNPathway analysis to the ADHD GWAS data, 11 candidate SNPs, 6 genes that included CD27, CASP9, ATM, CD12orf65, OXER1, and ACRY, and 6 pathways were identified that may contribute to ADHD susceptibility.

Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility.

Ath1 is a quantitative trait locus on mouse chromosome 1 that renders C57BL/6 mice susceptible and C3H/He mice resistant to diet-induced atherosclerosis. The quantitative trait locus region encompasses 11 known genes, including Tnfsf4 (also called Ox40l or Cd134l), which encodes OX40 ligand. Here we report that mice with targeted mutations of Tnfsf4 had significantly (P

TNF and TNF receptor superfamily members in HIV infection: new cellular targets for therapy?

Tumor necrosis factor (TNF) and TNF receptors (TNFR) superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients.

The price of the CD27-CD70 costimulatory axis: you can't have it all.

T cells require costimulatory signals for optimal proliferation, differentiation, and survival and thus to induce protective immune responses. Recent data, however, show that during chronic lymphocyte choriomeningitis virus (LCMV) infection, triggering of the costimulatory receptor CD27 by its ligand CD70 impedes neutralizing antibody production and leads to viral persistence. Thus, while being crucial for the induction of some adaptive effector pathways, costimulation may block the development of others. Pathogens may exploit this Achilles' heal to achieve persistence.

Opposing effects of CD70 costimulation during acute and chronic lymphocytic choriomeningitis virus infection of mice.

T cell costimulation is important for T cell activation. The CD27/CD70 pathway contributes to effector and memory T cell development and is involved in T cell and B cell activation. CD27/CD70 is known for having opposing roles during different models of antigenic challenges. During primary T cell responses to influenza virus infection or during tumor challenges, CD27/CD70 costimulation has a positive role on T cell responses. However, during some chronic infections, constitutive triggering of this signaling pathway has a negative role on T cell responses. It is currently unclear what specific characteristic of an antigen determines the outcome of CD27/CD70 costimulation. We investigated the effect of a transient CD70 blockade during an acute or a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Blockade of this pathway during acute LCMV infection (Armstrong strain) resulted in delayed T cell responses and decreased CD127 (interleukin-7 receptor α [IL-7Rα] chain) conversion. Upregulation of CD127 is an important event in T cell differentiation that heralds the passage of an effector T cell to a long-lived memory T cell. In contrast to the reduced CD8 T cell responses after CD70 blockade during acute infection, CD70 blockade during chronic LCMV infection resulted in increased CD8 T cell responses. Our data show the dual roles of this costimulatory pathway in acute versus persistent antigen challenge. Our findings suggest that antigen persistence may determine the effect of CD27/CD70 signaling on CD8 T cell responses. Tailored triggering or blockade of this costimulatory pathway may be important in vaccination regimens against acute or chronic pathogens.

The Pim kinase pathway contributes to survival signaling in primed CD8+ T cells upon CD27 costimulation.

Stimulation of the costimulatory receptor CD27 by its ligand CD70 has proved important for the generation of primary and memory CD8(+) T cell responses in various models of antigenic challenge. CD27/CD70-mediated costimulation promotes the survival of primed T cells and thereby increases the size of effector and memory populations. In this paper, we reveal molecular mechanisms underlying the prosurvival effect of CD27. CD27 signaling upregulated expression of the antiapoptotic Bcl-2 family member Bcl-x(L). However, genetic reconstitution of Cd27(-/-) CD8(+) T cells with Bcl-x(L) alone or in combination with the related protein Mcl-1 did not compensate for CD27 deficiency in the response to influenza virus infection. This suggested that CD27 supports generation of the CD8(+) effector T cell pool not only by counteracting apoptosis via Bcl-2 family members. Genome-wide mRNA expression profiling indicated that CD27 directs expression of the Pim1 gene. Pim-1 is a serine/threonine kinase that sustains survival of rapidly proliferating cells by antiapoptotic and prometabolic effects that are independent of the mammalian target of rapamycin (mTOR) pathway. In TCR-primed CD8(+) T cells, CD27 could increment Pim-1 protein expression and promote cell survival throughout clonal expansion independent of the mTOR and IL-2R pathways. In addition, introduction of the Pim1 gene in Cd27(-/-) CD8(+) T cells partially corrected their defect in clonal expansion and formation of an effector pool. We conclude that CD27 may contribute to the survival of primed CD8(+) T cells by the upregulation of antiapoptotic Bcl-2 family members but also calls the Pim-1 kinase survival pathway into action.

Inhibition of terminal differentiation of B cells mediated by CD27 and CD40 involves signaling through JNK.

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.

Cutting edge: adaptive versus innate receptor signals selectively control the pool sizes of murine IFN-γ- or IL-17-producing γδ T cells upon infection.

γδ T lymphocytes are commonly viewed as embracing properties of both adaptive and innate immunity. Contributing to this is their responsiveness to pathogen products, either with or without the involvement of the TCR and its coreceptors. This study clarifies this paradoxical behavior by showing that these two modes of responsiveness are the properties of two discrete sets of murine lymphoid γδ T cells. Thus, MyD88 deficiency severely impaired the response to malaria infection of CD27((-)), IL-17A-producing γδ T cells, but not of IFN-γ-producing γδ cells. Instead, the latter compartment was severely contracted by ablating CD27, which synergizes with TCRγδ in the induction of antiapoptotic mediators and cell cycle-promoting genes in CD27((+)), IFN-γ-secreting γδ T cells. Hence, innate versus adaptive receptors differentially control the peripheral pool sizes of discrete proinflammatory γδ T cell subsets during immune responses to infection.

The CD70/CD27 pathway is critical for stimulation of an effective cytotoxic T cell response against B cell precursor acute lymphoblastic leukemia.

For effective immunotherapy, maintaining the frequency and cytotoxic potential of effector cells is critical. In this context costimulation via the CD70/CD27 pathway has been proven essential. CD70 has been reported to be expressed to varying degrees on malignant B cells. However, in B cell precursor acute lymphoblastic leukemia, the most common childhood malignancy, the role of CD70 in stimulation of antileukemic T cell responses has so far not been delineated. Herein we demonstrate that in B cell precursor acute lymphoblastic leukemia expression of CD70 is low but can be induced upon blast activation via CD40. Both CD70 and CD80/CD86 up-regulated on CD40-stimulated blasts contribute to primary stimulation of T cell proliferation and cytokine production in an additive manner. These two signals also cooperate in the prevention of T cell anergy. In contrast to blockade of CD70 during the effector phase, inhibition of CD70-mediated costimulation during generation of antileukemic T cells prevents effector cell proliferation and reduces their cytotoxic capacity. Modulation of the CD70/CD27 pathway may thus represent a novel therapeutic approach for augmenting magnitude and quality of the antileukemic response in B cell precursor acute lymphoblastic leukemia.

Blocking CD27-CD70 costimulatory pathway suppresses experimental colitis.

The pathogenesis of human inflammatory bowel disease (IBD) and most experimental models of IBD is dependent on the activation and expansion of CD4(+) T cells via interaction with mucosal APCs. The costimulatory receptor CD70 is transiently expressed on the surface of conventional dendritic cells, but is constitutively expressed by a unique APC population in the intestinal lamina propria. We used two experimental IBD models to evaluate whether interfering the interaction between CD70 and its T cell ligand CD27 would affect the development of colitis. Adoptive transfer of naive CD27-deficient CD45RB(high) CD4(+) T cells into Rag-1(-/-) mice resulted in significantly less disease than when wild-type CD45RB(high)CD4(+) T cells were used. Moreover, a monoclonal anti-CD70 Ab prevented the disease caused by the transfer of wild-type CD45RB(high) CD4(+) T cells into Rag-1(-/-) mice and the same Ab also ameliorated an established disease. The colitis associated proinflammatory cytokines IL-6, TNF-alpha and IFN-gamma were significantly reduced after anti-CD70 Ab treatment, suggesting an overall reduction in inflammation due to blockade of pathogenic T cell expansion. Anti-CD70 Ab treatment also suppressed trinitrobenzene sulfonic acid-induced colitis in SJL/J mice. Because anti-CD70 Ab treatment suppressed multiple proinflammatory cytokines, this may be a more potent therapeutic approach for IBD than blockade of individual cytokines.

CD27 expression promotes long-term survival of functional effector-memory CD8+ cytotoxic T lymphocytes in HIV-infected patients.

Human immunodeficiency virus (HIV)-specific CD8(+) T cells persist in high frequencies in HIV-infected patients despite impaired CD4(+) T helper response to the virus, but, unlike other differentiated effector cytotoxic T lymphocytes, most continue to express the tumor necrosis factor receptor family member CD27. Because the ligand for CD27 (CD70) is also overexpressed in HIV-infected hosts, we examined the nature of expression and potential functional consequences of CD27 expression on HIV-specific CD8(+) T cells. Analysis of CD27(+) and CD27(-) T cells derived from the same HIV-specific clone revealed that retention of CD27 did not interfere with acquisition of effector functions, and that after T cell receptor stimulation, CD27(+) cells that concurrently were triggered via CD27 exhibited more resistance to apoptosis, interleukin 2 production, and proliferation than CD27(-) T cells. After transfer back into an HIV-infected patient, autologous HIV-specific CD27(-) T cells rapidly disappeared, but CD27(+) T cells derived from the same clone persisted at high frequency. Our findings suggest that the CD27-CD70 interaction in HIV infection may provide CD27(+) CD8(+) T cells with a survival advantage and compensate for limiting or absent CD4(+) T help to maintain the CD8 response.

A critical role for OX40 in T cell-mediated immunopathology during lung viral infection.

Respiratory infections are the third leading cause of death worldwide. Illness is caused by pathogen replication and disruption of airway homeostasis by excessive expansion of cell numbers. One strategy to prevent lung immune-mediated damage involves reducing the cellular burden. To date, antiinflammatory strategies have affected both antigen-specific and naive immune repertoires. Here we report a novel form of immune intervention that specifically targets recently activated T cells alone. OX40 (CD134) is absent on naive T cells but up-regulated 1-2 d after antigen activation. OX40-immunoglobulin fusion proteins block the interaction of OX40 with its ligand on antigen-presenting cells and eliminate weight loss and cachexia without preventing virus clearance. Reduced proliferation and enhanced apoptosis of lung cells accompanied the improved clinical phenotype. Manipulation of this late costimulatory pathway has clear therapeutic potential for the treatment of dysregulated lung immune responses.

CD27 cooperates with the pre-T cell receptor in the regulation of murine T cell development.

CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti-CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3-mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal.

The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response.

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T-dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.

CD27-CD70 interactions in the pathogenesis of Waldenstrom macroglobulinemia.

Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by an IgM monoclonal gammopathy and bone marrow (BM) infiltration with lymphoplasmacytic cells (LPCs). Excess mast cells (MCs) are commonly present in WM, and provide growth and survival signals to LPCs through several TNF family ligands (CD40L, a proliferation-inducing ligand [APRIL], and B-lymphocyte stimulator factor [BLYS]). As part of these studies, we demonstrated that WM LPCs secrete soluble CD27 (sCD27), which is elevated in patients with WM (P < .001 vs healthy donors), and serves as a faithful marker of disease. Importantly, sCD27 stimulated expression of CD40L on 10 of 10 BM MC samples and APRIL on 4 of 10 BM MC samples obtained from patients with WM as well as on LAD2 MCs. Moreover, the SGN-70 humanized monoclonal antibody, which binds to CD70 (the receptor-ligand partner of CD27), abrogated sCD27 mediated up-regulation of CD40L and APRIL on WM MCs. Last, treatment of severe combined immunodeficiency-human (SCID-hu) mice with established WM using the SGN-70 antibody blocked disease progression in 12 of 12 mice, whereas disease progressed in all 5 untreated mice. The results of these studies demonstrate a functional role for sCD27 in WM pathogenesis, along with its utility as a surrogate marker of disease and a target in the treatment of WM.

CD27(+) peripheral blood B-cells are a useful biodosimetric marker in vitro.

The CD8(+) natural killer (NK) subpopulation has recently been identified as a fast and reliable biodosimetric indicator within human peripheral blood mononuclear cells (PBMC) in vitro. In irradiated and subsequently cultivated PBMC, a decrease of the relative number of intact CD3(-)CD8(+) lymphocytes 16 and 48 h after treatment has allowed for estimating the received dose in the range of 0 - 10 Gy and lethal/sublethal dose discrimination, respectively. Here we show that suitable biodosimeters can also be found in the peripheral blood B-cell compartment. Multiparameter flow cytometric analysis of irradiated and subsequently cultivated human PBMC revealed that both the CD27(+) and CD21(-) B-cell subpopulations can be used as biodosimeters and the CD19(+)CD27(+) lymphocytes have proved useful for retrospective determination of the received dose in the range of 0 - 6 Gy. In addition, several CD19(+) lymphocyte subsets characterized by co expression of CD21, CD27 and CD38 have been shown to bear biodosimetric potential, too. However, when important parameters like the original size within the CD19(+) compartment, its radiation-induced changes and data variation had been taken into account, the CD27(+) subpopulation proved superior to the other B-cell subpopulations and subsets. It appears that, in the dose range of 0 - 6 Gy, the relative decrease of CD27(+) B lymphocytes provides more sensitive and reliable data than that of CD8(+) NK-cells due mainly to lower data variation. In contrast to CD27(+) B cells, the proportions of CD27(+) subpopulations of T-cells were not affected by irradiation. We have also proposed a simple experimental protocol based on full blood cultivation and three-color CD27/CD3/CD19 immuno-phenotyping as a time-saving and inexpensive approach for practical biodosimetric evaluations on simple, three-to-four color flow cytometers.

The role of CD27-CD70-mediated T cell co-stimulation in vasculogenesis, arteriogenesis and angiogenesis.

BACKGROUND: T cells have a distinctive role in neovascularization, which consists of arteriogenesis and angiogenesis under pathological conditions and vasculogenesis under physiological conditions. However, the role of co-stimulation in T cell activation in neovascularization has yet to be established. The aim of this study was to investigate the role T cell co-stimulation and inhibition in angiogenesis, arteriogenesis and vasculogenesis. METHODS AND RESULTS: Hind limb ischemia was induced by double ligation of the left femoral artery in mice and blood flow recovery was measured with Laser Doppler Perfusion Imaging in control, CD70-/-, CD80/86-/-, CD70/80/86-/- and CTLA4+/- mice. Blood flow recovery was significantly impaired in mice lacking CD70 compared to control mice, but was similar in CD80/86-/-, CTLA4+/- and control mice. Mice lacking CD70 showed impaired vasculogenesis, since the number of pre-existing collaterals was reduced as observed in the pia mater compared to control mice. In vitro an impaired capability of vascular smooth muscle cells (VSMC) to activate T cells was observed in VSMC lacking CD70. Furthermore, CD70-/-, CD80/86-/- and CD70/80/86-/- mice showed reduced angiogenesis in the soleus muscle 10 days after ligation. Arteriogenesis was also decreased in CD70-/- compared to control mice 10 and 28 days after surgery. CONCLUSIONS: The present study is the first to describe an important role for T cell activation via co-stimulation in angiogenesis, arteriogenesis and vasculogenesis, where the CD27-CD70 T cell co-stimulation pathway appears to be the most important co-stimulation pathway in pre-existing collateral formation and post-ischemic blood flow recovery, by arteriogenesis and angiogenesis.

Tumor necrosis factor receptor superfamily members and their ligands.

The tumor necrosis factor (TNF) receptor family comprises a number of type I integral membrane glycoproteins which exhibit sequence homology in their cysteine-rich extracellular domains. Recently, ligands for many of these receptors have been identified. These molecules all display sequence identity with TNF and lymphotoxin beta, prototype ligands for this receptor family, and have the conformation of type II transmembrane molecules. While certain biological activities are common to many members of this TNF-like family, other activities appear to be shared only by some ligands, or are unique. The identification of the TNF superfamily of ligands has provided the opportunity to compare and contrast the diverse biological effects mediated through the interaction of these related molecules with their receptors.