Cerebrospinal fluid soluble CD27 is associated with CD8+ T cells, B cells and biomarkers of B cell activity in relapsing-remitting multiple sclerosis.

Cerebrospinal fluid (CSF) soluble CD27 (sCD27) is a sensitive biomarker of intrathecal inflammation. Although generally considered a biomarker of T cell activation, CSF sCD27 has been shown to correlate with biomarkers of B cell activity in multiple sclerosis. We analyzed CSF from 40 patients with relapsing-remitting multiple sclerosis (RRMS) and nine symptomatic controls using flow cytometry and multiplex electrochemiluminescence immunoassays. CSF sCD27 levels were increased in RRMS and correlated with IgG index, soluble B cell maturation antigen, cell count, B cell frequency and CD8+ T cell frequency. We provide new data indicating that CSF sCD27 is associated with CD8+ T cells and B cells in RRMS.

Conditioning treatment with CD27 Ab enhances expansion and antitumor activity of adoptively transferred T cells in mice.

Cyclophosphamide plus fludarabine (C/F) are currently used to improve the expansion and effectiveness of adoptive cell therapy (ACT). However, these chemotherapeutics cause pan-leukopenia and adverse events, suggesting that safer and more effective conditioning treatments are needed to improve ACT outcomes. Previously, we reported that varlilumab, a CD27-targeting antibody, mediates Treg -preferential T cell depletion, CD8-T cell dominant costimulation, and systemic immune activation in hCD27 transgenic mice and cancer patients. We reasoned that the activities induced by varlilumab may provide an effective conditioning regimen for ACT. Varlilumab pretreatment of hCD27 +/+mCD27 - /- mice resulted in prominent proliferation of transferred T cells isolated from wild-type mice. These studies uncovered a critical role for CD27 signaling for the expansion of transferred T cells, as transfer of T cells from CD27 deficient mice or treatment with a CD70 blocking antibody greatly reduced their proliferation. In this model, varlilumab depletes endogenous hCD27+/+ T cells and blocks their subsequent access to CD70, allowing for more CD70 costimulation available to the mCD27 +/+ transferred T cells. CD27-targeted depletion led to a greater expansion of transferred T cells compared to C/F conditioning and resulted in longer median survival and more cures than C/F conditioning in the E.G7 tumor model receiving OT-I cell therapy. We propose that translation of this work could be achieved through engineering of T cells for ACT to abrogate varlilumab binding but preserve CD70 ligation. Thus, varlilumab could be an option to chemotherapy as a conditioning regimen for ACT.

Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex.

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.

Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters ("cord factor") form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.

Cerebrospinal fluid sCD27 levels indicate active T cell-mediated inflammation in premanifest Huntington's disease.

INTRODUCTION: Huntington's disease (HD) is a neurodegenerative disorder, but evidence also suggests neuroinflammation in the pathogenesis. The immune mechanisms involved and the timing of their activation need further clarification. METHODS: A clinically well-characterized HD cohort and gene negative controls were enrolled. YKL-40 reflecting innate immunity and sCD27, a marker of adaptive immunity, were measured across disease stages. Comparisons were made with markers of neurodegeneration: neurofilament light (NFL), total-tau (T-tau), and phospho-tau (P-tau). RESULTS: 52 cross-sectional cerebrospinal fluid samples and 23 follow-up samples were analyzed. sCD27 was elevated in manifest HD and premanifest gene expansion carriers, whereas controls mostly had undetectable levels. YKL-40 showed a trend toward increase in manifest HD. sCD27 correlated with YKL-40 which in turn was closely associated to all included markers of neurodegeneration. YKL-40, NFL, and both forms of tau could all independently predict HD symptoms, but only NFL levels differed between groups after age-adjustment. CONCLUSION: Increased sCD27 in premanifest HD is a sign of T cell-mediated neuroinflammation. This finding is novel since other reports almost exclusively have found early involvement of innate immunity. Validation of sCD27 in a larger HD cohort is needed. The role of adaptive immunity in HD needs further clarification, as it may hasten disease progression.

Crystal structure of CD27 in complex with a neutralizing noncompeting antibody.

CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70. To provide insight into the mechanism of action of the mAb, the crystal structure of the CD27 extracellular domain in complex with the Fab fragment of mAb 2177 was determined at 1.8 Šresolution. CD27 exhibits the assembly of cysteine-rich domains characteristic of the TNF receptor superfamily. The structure reveals a unique binding site of mAb 2177 at the edge of the receptor molecule, which allows the mAb to sterically block the cell-bound form of CD70 from reaching CD27 while leaving the ligand epitope clear. This mode of action suggests a potential dual use of mAb 2177 either as an antagonist or as an agonist.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity.

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate "on-target" toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and "on-target off-tumor" toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs.

CD27 expression discriminates porcine T helper cells with functionally distinct properties.

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α- T helper cells but divides CD8α+ T helper cells into a CD27+ and a CD27- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α-CD27+ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α+CD27+ and CD8α+CD27- T helper cells were found in blood, spleen and liver. Both CD8α+CD27+ and CD8α+CD27- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α-CD27+, CD8α+CD27+ and CD8α+CD27- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α+CD27- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α+CD27- T helper cells were mostly CCR7- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α+CD27+ T helper cells, which also had a proliferation rate similar to naïve CD8α-CD27+ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α+CD27+ T helper cells displayed a phenotype and functional properties of central memory cells.

In vitro HIV-1 LTR integration into T-cell activation gene CD27 segment and the decoy effect of modified-sequence DNA.

Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors.

Porcine CD27: identification, expression and functional aspects in lymphocyte subsets in swine.

Up to now for Swine Workshop Cluster 2 (SWC2) the orthologous human CD molecule was unknown. By use of the SWC2-specific mAb b30c7 and a retroviral cDNA expression library derived from stimulated porcine peripheral blood mononuclear cells we could identify SWC2 as porcine CD27. Phenotypic analyses of lymphocytes isolated from blood and lymphatic organs revealed that mature T cells in thymus and T cells in the periphery with a naïve phenotype were CD27(+). However, within CD8α(+) T helper and CD8α(+) γδ T cells also CD27(-) cells were present, indicating a down-regulation after antigen contact in vivo. B cells lacked CD27 expression, whereas NK cells expressed intermediate levels. Furthermore, plate-bound mAb b30c7 showed a costimulatory capacity on CD3-activated T cells for proliferation, IFN-γ and TNF-α production. Hence, our data indicate an important role of porcine CD27 for T-cell differentiation and activation as described for humans and mice.

Monitoring the T cell response to genital tract infection.

To date, it has not been possible to study antigen-specific T cell responses during primary infection of the genital tract. The low frequency of pathogen-specific T cells in a naïve mouse makes it difficult to monitor the initial events after antigen encounter. We developed a system to examine the response of pathogen-specific T cells in the genital mucosa after intrauterine infection. We identified the protective CD4(+) T cell antigen Cta1 from Chlamydia trachomatis and generated T cell receptor (TCR) transgenic (tg) mice with specificity for this protein. By transferring TCR tg T cells into naïve animals, we determined that Chlamydia-specific T cells were activated and proliferated in the lymph nodes draining the genital tract after primary intrauterine infection. Activated T cells migrated into the genital mucosa and secreted IFN-gamma. The development of Chlamydia-specific TCR tg mice provides an approach for dissecting how pathogen-specific T cells function in the genital tract.

NF-kappaB activation in CD27 signaling: involvement of TNF receptor-associated factors in its signaling and identification of functional region of CD27.

CD27 belongs to TNF receptor family, and it is unique in this family for its disulfide-linked homodimerization of 55-kDa monomers. In the present study we demonstrate that overexpression of CD27 in 293 cells induces a low level of NF-kappaB activation, and the ligation of the receptor by its corresponding ligand (CD70) augments this signal dramatically. Either TNF receptor-associated factor-2 (TRAF2) or TRAF3 binds to the CD27 molecule from the coimmunoprecipitation experiment. This NF-kappaB activation signal is inhibited by dominant negative TRAF2 or intact TRAF3, indicating that TRAF2 and TRAF3 works as a mediator and an inhibitor, respectively. The activated NF-kappaB complex contains at least two components, p50 and p65, but not p52. All these phenomena have also been observed in the TNF receptor type II, CD30 and CD40 signaling system, indicating that this receptor family uses the common or similar molecules for this signal. Finally, we identified the 13-amino acid alignment in the cytoplasmic region of the CD27 molecule (residues 238-250 amino acids), which is critical for the NF-kappaB activation signal and also for its association with TRAFs. This amino acid alignment contains the EEEG sequence, which is essential for interaction of CD30 or CD40 with TRAFs (TRAF1 and TRAF2, but not TRAF3), and also contains the PIQED sequence, which is similar to PXQXT that is known to be necessary for interaction of TNF receptor II and CD30 with TRAFs (TRAF1, 2, and 3).

CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein.

Members of the tumor necrosis factor receptor (TNFR) superfamily are important for cell growth and survival. In addition to providing costimulatory signals for cell proliferation, ligation of both TNFR1 and Fas can result in programmed cell death or apoptosis. The underlying mechanism requires an intact 80-aa stretch present in the cytoplasmic tails of both TNFR1 and Fas, termed the death domain (DD). Here we show that CD27, a member of the TNFR family, expressed on discrete subpopulations of T and B cells and known to provide costimulatory signals for T and B cell proliferation and B cell Ig production, can also induce apoptosis. Co-crosslinking of surface Ig receptors along with ligation of CD27 augments CD27-mediated apoptosis. Unlike TNFR1 and Fas, the cytoplasmic tail of CD27 is relatively short and lacks the DD. Using the yeast two-hybrid system, we have cloned a novel protein (Siva) that binds to the CD27 cytoplasmic tail. It has a DD homology region, a box-B-like ring finger, and a zinc finger-like domain. Overexpression of Siva in various cell lines induces apoptosis, suggesting an important role for Siva in the CD27-transduced apoptotic pathway.

Gene structure and chromosomal localization of the mouse homologue of rat OX40 protein.

The OX40 protein is expressed only on activated rat CD4+ T blasts and is a member of a superfamily of cell surface molecules which includes CD40, CD30, CD95 (Fas), CD27, 4-1BB antigens and the receptors for tumor necrosis factor (TNF) and nerve growth factor (NGF). The proteins of this group are related to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes.