CD27 Human, HEK

What is human CD27 and what roles does it play in the immune system?

CD27 is a type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) superfamily that plays important roles in T-cell activation and immune regulation. It exists in both membrane-bound form on T cells, B cells, and NK cells, and as a soluble form (sCD27) in serum and other biological fluids. CD27 interacts with its ligand CD70 to promote T-cell activation, proliferation, and the generation of cytolytic T-cells . The CD27-CD70 interaction has been implicated in regulating cellular immune responses to cancer, with soluble CD27 functioning as an immune modulator that enhances human T-cell activation both in vitro and in vivo . This pathway represents an important target for immunotherapeutic strategies in cancer treatment and potentially other diseases.

How is soluble CD27 (sCD27) produced in the human body?

Soluble CD27 (sCD27) is a 32-kD protein identical to the extracellular domain of membrane-bound CD27. It can be released after lymphocyte activation through two distinct mechanisms: differential splicing of the receptor protein or shedding from the cell surface by metalloproteinases (MMPs) . The production of sCD27 upon T-cell activation has been demonstrated using anti-CD3 or a combination of anti-CD3 and anti-CD2 monoclonal antibodies to stimulate human peripheral blood mononuclear cells (PBMCs) in vitro . Studies show that sCD27 is preferentially derived from activated CD4+ T cells, and its production increases linearly with T-cell stimulation . The regulation of sCD27 production involves the CD27-CD70 interaction itself, as blocking CD70 significantly inhibits sCD27 production, demonstrating that shedding of CD27 from T cells is enhanced by ligation with its ligand CD70 .

How can CD27 be used to distinguish human NK cell subsets?

CD27 serves as a valuable marker for distinguishing functionally distinct subsets of human natural killer (NK) cells. Human NK cells have traditionally been divided into two phenotypically and functionally distinct subsets based on their expression levels of CD56, but research has shown that CD27 can provide additional discriminatory power . The majority of peripheral blood human NK cells are CD27lo/CD56dim NK cells, while the minor CD27hi NK cell population corresponds to the CD56bright phenotype . These subsets differ in their receptor expression patterns and typical NK cell functions such as cytotoxicity and cytokine production. The dual use of CD27 and CD56 as maturation/subset markers provides more refined characterization of human NK cells . This distinction is particularly valuable because CD27 has also been identified as a marker for mouse NK cell subsets, allowing more accurate projections of NK cell behavior between murine models and human pathologies .

What is the relationship between CD27 and its ligand CD70?

CD70 (also known as CD27 ligand or CD27L) is a type II transmembrane glycoprotein belonging to the TNF superfamily (TNFSF) and has been designated TNFSF7 . It functions as the exclusive ligand for CD27 and plays a critical role in T-cell activation . When CD70 binds to CD27, it induces the proliferation of costimulated T-cells and enhances the generation of cytolytic T-cells . This interaction is crucial for effective immune responses against pathogens and tumors. CD70 expression is tightly regulated and primarily found on activated lymphocytes, dendritic cells, and some malignant cells . The CD27-CD70 interaction represents a co-stimulatory pathway that, when engaged, provides additional signals beyond the primary T-cell receptor stimulation . Interestingly, activated T cells express both CD27 and CD70, creating potential for autocrine and paracrine signaling within the activated T-cell population .

How does soluble CD27 (sCD27) functionally influence T-cell activation?

Soluble CD27 (sCD27) is not merely a byproduct of T-cell activation but actively participates in enhancing T-cell responses. Research demonstrates that sCD27 is a functional protein directly involved in T-cell activation, with effects comparable to those of IL-2 in some contexts . When recombinant human sCD27 is added to stimulated peripheral blood mononuclear cells, it significantly upregulates activation markers including CD25, CD70, and 4-1BB (CD137) on CD8+ T cells and CD40L on CD4+ T cells . The addition of sCD27 also enhances T-cell proliferation in a dose-dependent manner, particularly when T cells receive suboptimal stimulation through the TCR/CD3 complex . This effect may mimic physiological situations where tumor cells provide insufficient antigenic stimulation . At the molecular level, sCD27 associates with immunological synapse-related proteins including myosin IIA, HMGB1, and the TCR Vβ chain, with confocal microscopy confirming co-localization of sCD27 and myosin IIA in activated T cells . These findings suggest that sCD27 may contribute to immunological synapse formation or maintenance during T-cell activation.

What experimental approaches can be used to study CD27-expressing lymphoma and leukemia?

CD27 is recognized as a cell-surface marker on various B- and T-cell malignancies, making it a potential therapeutic target . Several experimental approaches can be employed to study CD27-expressing lymphomas and leukemias:

First, researchers can utilize monoclonal antibodies specific for CD27, such as the fully human antibody 1F5 (later developed as CDX-1127/varlilumab), which binds with high affinity and specificity to human CD27 and competes with CD70 ligand binding . These antibodies can be characterized through analytical and functional assays in vitro to assess their potential utility against CD27-expressing malignancies .

Animal models provide another valuable approach, as demonstrated by studies using severe combined immunodeficient (SCID) mice inoculated with human CD27-expressing lymphoma cells such as Raji or Daudi tumors . Administration of anti-CD27 antibodies in these models has shown significant enhancement of survival, potentially through direct effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) .

Safety assessment in non-human primates represents an important preclinical step, with studies showing that administration of up to 10 mg/kg of anti-CD27 antibodies can be well-tolerated without evidence of significant toxicity or depletion of circulating lymphocytes . This supports the potential clinical development of CD27-targeting therapeutic approaches.

What methodologies are used to measure sCD27 in clinical and research samples?

Accurate quantification of sCD27 in biological samples is crucial for both research and potential clinical applications. Several methodologies have been developed and validated for this purpose:

Enzyme-Linked Immunosorbent Assay (ELISA) is the most commonly employed method, with commercial kits available from manufacturers like eBioscience (San Diego, CA) and Sanquin (Amsterdam, Netherlands) . These assays provide sensitive and specific quantification of sCD27 in serum, plasma, cell culture supernatants, and other biological fluids.

For experimental studies investigating the production of sCD27 upon T-cell activation, researchers typically stimulate peripheral blood mononuclear cells (PBMCs) with anti-CD3/CD28 beads plus IL-2 or other activation protocols, and then collect the supernatant at various time points (e.g., days 3, 7, and 15) for sCD27 quantification . The linear increase in sCD27 production over time can be measured and correlated with other parameters of T-cell activation .

In some research contexts, sCD27 depletion experiments may be necessary to determine the specific contribution of sCD27 to observed effects. This can be accomplished by coating plates with anti-human CD27 antibody or IgG control, incubating with serum or recombinant sCD27, and then confirming depletion by ELISA before using the depleted samples in functional assays .

How can recombinant human CD27 and CD70 proteins be produced using HEK expression systems?

HEK293 cell lines provide an excellent platform for expressing recombinant human CD27 and CD70 proteins due to their capacity to perform proper post-translational modifications, particularly glycosylation, which is crucial for these proteins' functionality. For recombinant CD70 (CD27 ligand) production, the protein can be expressed from HEK293 cells with human Fc (hFc) at the N-terminus, typically containing amino acids Gln39-Pro193 of the native protein .

For soluble CD27 production, researchers have successfully generated recombinant human sCD27 by transducing HEK293 cells with a construct encoding the extracellular domain (amino acids 1 to 189) of the CD27 protein . This approach yields functional sCD27 that can be purified from the cell culture supernatant and used for various experimental applications, including studies of T-cell activation enhancement .

The purification process typically involves affinity chromatography, often utilizing the Fc tag if present, followed by additional purification steps such as size exclusion chromatography to ensure high purity. Quality control steps include verification of protein identity by mass spectrometry, purity assessment by SDS-PAGE, and functional validation through binding and activity assays .

What controls should be included when studying CD27-CD70 interactions?

When investigating CD27-CD70 interactions, researchers should implement several key controls to ensure experimental validity and interpretability:

For binding interaction studies, both positive and negative controls are essential. Positive controls might include previously validated antibodies known to bind CD27 or CD70, while negative controls should incorporate irrelevant proteins of similar structure or isotype-matched control antibodies . Competition experiments using excess unlabeled ligand can confirm binding specificity.

When studying T-cell activation enhanced by sCD27, critical controls include: stimulation without added sCD27; sCD27-depleted conditions (using anti-CD27 antibody depletion); and dosage controls with varying concentrations of sCD27 . Additionally, experiments blocking CD70 with antibodies can demonstrate the dependency of sCD27 effects on CD70 interaction, as studies have shown significant inhibition of sCD27 production when CD70 is blocked .

For cellular assays, controls should address the timing of sCD27 addition relative to TCR stimulation, as temporal dynamics can significantly impact results. Experiments comparing different T-cell subsets (CD4+ vs. CD8+) help identify population-specific effects, as sCD27 has been shown to upregulate different activation markers on these populations (e.g., CD40L on CD4+ cells; CD25, CD70, and 4-1BB on CD8+ cells) .

How can researchers troubleshoot low expression of recombinant CD27 in HEK cells?

When facing challenges with low expression of recombinant CD27 in HEK cells, researchers can implement several troubleshooting strategies:

First, optimize the expression vector by ensuring appropriate promoter strength, codon optimization for human cell expression, and inclusion of efficient signal peptides. For CD27, which is normally expressed on lymphocytes, the native signal peptide might not function optimally in HEK293 cells; alternative signal peptides known to work well in this system should be considered.

Address protein stability issues by examining post-translational modifications, particularly glycosylation. CD27 contains N-linked glycosylation sites that influence protein folding and stability. Analysis of glycosylation patterns using treatments with glycosidases followed by western blotting can reveal problems in this area. If glycosylation appears problematic, consider using glycosylation-modified HEK293 cell lines.

For membrane-bound CD27 expression, verify surface localization using flow cytometry with multiple anti-CD27 antibodies recognizing different epitopes. Some antibodies may not recognize recombinant CD27 efficiently due to conformational differences. For soluble CD27, analyze the culture supernatant using ELISA or western blot to confirm secretion.

If CD27 appears to be produced but rapidly degraded, consider adjusting culture conditions (temperature reduction to 30-32°C can sometimes improve folding) or co-expressing chaperone proteins. Protease inhibitors in the culture medium may help if proteolytic degradation is identified as an issue.

What factors influence the production of soluble CD27 (sCD27) in experimental settings?

Several factors significantly influence sCD27 production in experimental systems, and understanding these is crucial for consistent and interpretable results:

The mode of T-cell stimulation dramatically affects sCD27 production. Studies have shown that T-cell receptor/CD3 plus CD28 stimulation effectively induces sCD27 production, while PMA-ionomycin stimulation, which bypasses the TCR/CD3 signal, results in minimal sCD27 production despite inducing T-cell activation markers like IFN-γ . This suggests specific signaling pathways are required for optimal sCD27 release.

The CD27-CD70 interaction itself regulates sCD27 production. Experiments using antibodies that block CD70 have demonstrated significant inhibition of sCD27 production, indicating that binding of CD27 to CD70 enhances the shedding of CD27 from T cells . This creates a potential positive feedback loop where initial activation leads to CD70 expression, CD27-CD70 binding, and subsequent sCD27 production.

Time course is another critical factor, as sCD27 production typically increases linearly over time during T-cell activation. In studies where PBMCs were stimulated with anti-CD3/CD28 beads plus IL-2, sCD27 levels continued to rise through day 15, possibly due to accumulation of activated T cells and/or increased shedding per cell .

How can researchers determine if soluble CD27 detected in experiments is functional?

Determining the functionality of soluble CD27 in experimental systems requires multiple complementary approaches:

T-cell activation marker analysis provides a key readout of sCD27 functionality. Flow cytometry should be used to measure the upregulation of activation markers including CD25, CD70, and 4-1BB on CD8+ T cells and CD40L on CD4+ T cells following addition of purified sCD27 to stimulated T cells . Significant enhancement of these markers compared to stimulation without sCD27 suggests functional activity.

Proliferation assays offer another measure of functionality. Researchers should assess T-cell proliferation (using methods such as CFSE dilution or tritiated thymidine incorporation) in response to suboptimal TCR stimulation with and without added sCD27 . Functional sCD27 typically enhances proliferation in a dose-dependent manner.

Specificity confirmation is essential through depletion experiments where sCD27 is specifically removed using anti-CD27 antibodies . If the activating effects disappear when sCD27 is depleted and are restored when recombinant sCD27 is added back, this strongly supports specific sCD27 functionality.

Molecular association studies can provide mechanistic insight into sCD27 functionality. Techniques such as immunoprecipitation followed by mass spectrometry have identified associations between sCD27 and immunological synapse-related proteins including myosin IIA, HMGB1, and the TCR Vβ chain . Confocal microscopy showing co-localization of sCD27 with these proteins in activated T cells further supports functional involvement in T-cell activation processes .

How do sCD27 levels in serum correlate with disease states and immunotherapy outcomes?

Soluble CD27 (sCD27) levels in serum have significant clinical correlations across various disease states and therapeutic interventions. In healthy individuals, sCD27 is detectable in serum, plasma, and urine samples, establishing a baseline "sCD27-pool" that fluctuates in response to immune activation . Interestingly, research has shown that this pool is generally greater in healthy donors compared to cancer patients .

In patients with certain disease conditions, sCD27 levels show characteristic patterns. Increased levels have been documented in systemic lupus erythematosus, viral infections, and lymphoid malignancies . The level of sCD27 in plasma samples has been used as a marker of disease burden in patients with Waldenström's macroglobulinemia and to monitor immune activation during antiretroviral therapy in patients with HIV .

What role does CD27 play in the development of therapeutic antibodies for lymphoma and leukemia?

CD27 has emerged as a promising target for therapeutic antibody development in lymphoma and leukemia treatment strategies. As a cell-surface marker expressed on various B- and T-cell malignancies, CD27 offers an opportunity for targeted therapy approaches .

The development of the fully human monoclonal antibody 1F5 (later advanced to clinical development as CDX-1127/varlilumab) illustrates the potential of CD27-targeting strategies . This antibody binds with high affinity and specificity to human CD27 and competes with CD70 ligand binding . Its mechanism of action includes direct antitumor effects against CD27-expressing malignancies, potentially through antibody-dependent cellular cytotoxicity (ADCC), while also possessing immunomodulatory properties through its ability to activate T cells when combined with T-cell receptor stimulation .

Preclinical studies have demonstrated the efficacy of CD27-targeting antibodies in relevant models. In severe combined immunodeficient (SCID) mice inoculated with human CD27-expressing lymphoma cells such as Raji or Daudi tumors, administration of anti-CD27 antibodies significantly enhanced survival . Importantly, these antibodies did not induce proliferation of primary CD27-expressing tumor cells, addressing a potential safety concern for targeted therapies .

Safety assessment in non-human primates has shown that administration of up to 10 mg/kg of anti-CD27 antibodies can be well-tolerated without evidence of significant toxicity or depletion of circulating lymphocytes . This favorable safety profile, combined with demonstrated efficacy in preclinical models, supports the clinical development of CD27-targeting therapeutic approaches for lymphoma and leukemia.

How can CD27 expression in HEK cells be utilized for developing and screening novel immunotherapeutics?

HEK293 cell systems expressing CD27 provide valuable platforms for developing and screening novel immunotherapeutics targeting the CD27-CD70 pathway. These systems offer several advantages for therapeutic development workflows:

For antibody development, stable HEK293 cell lines expressing full-length human CD27 at consistent levels ensure reproducible screening results for potential therapeutic candidates . These cells can be used in flow cytometry-based binding assays to determine antibody affinity and specificity, with parental HEK293 cells serving as negative controls . Competition assays with the natural ligand CD70 can identify antibodies that block or do not interfere with ligand binding, depending on the desired mechanism of action .

HEK expression systems also facilitate the production of recombinant soluble CD27 for functional studies and potential therapeutic applications. Researchers have successfully generated recombinant human sCD27 by transducing HEK293 cells with constructs encoding the extracellular domain of CD27 . These recombinant proteins can be tested for their ability to enhance T-cell activation and proliferation, potentially opening avenues for sCD27-based immunotherapeutic strategies .

For mechanistic studies, HEK293 cells can be engineered with reporter systems where CD27 signaling is coupled to easily measurable outputs like luciferase expression. This approach allows for high-throughput screening of compounds that might modulate CD27 signaling. Additionally, mutagenesis of CD27 in HEK cells enables mapping of binding epitopes for promising therapeutic candidates and structure-function relationship studies .

What considerations are important when using CD27 as a marker in NK cell research?

When employing CD27 as a marker in natural killer (NK) cell research, several important considerations should guide experimental design and interpretation:

First, CD27 expression patterns differ between species, requiring careful translation between animal models and human studies. While CD27 distinguishes subsets of mature mouse NK cells, its application to human NK cells reveals that the majority of peripheral blood human NK cells are CD27lo/CD56dim NK cells, with the minor CD27hi NK cell population corresponding to CD56bright NK cells . This correlation between CD27 and CD56 expression patterns provides a valuable cross-species reference point, allowing more accurate projections of NK cell behavior between murine models and human pathologies .

Functional differences between CD27-defined NK subsets must be characterized comprehensively. CD27lo and CD27hi NK cells show distinctions in receptor expression patterns and typical NK cell functions such as cytotoxicity and cytokine production . These distinctions should be accounted for when designing experiments or interpreting results involving heterogeneous NK populations.

Technical considerations include antibody selection, as different anti-CD27 clones may have varying specificities and affinities. Multiparameter flow cytometry panels should include both CD27 and CD56 for comprehensive human NK cell phenotyping, along with additional markers to define functional status and activation state .

The dual use of CD27 and CD56 as maturation/subset markers provides more refined characterization than either marker alone . This combined approach allows researchers to track developmental relationships between NK subsets and correlate phenotypic markers with functional capabilities more precisely.

How might advances in protein expression systems improve CD27-based therapeutics?

Advances in protein expression technologies will likely drive significant improvements in CD27-based therapeutics through several emerging approaches:

Site-specific glycoengineering in HEK293 expression systems represents a promising frontier. By controlling the glycosylation profile of recombinant CD27 or anti-CD27 antibodies, researchers can potentially enhance stability, reduce immunogenicity, and optimize pharmacokinetic properties. This could be achieved through CRISPR-Cas9 modification of glycosylation enzymes in HEK293 cells or through chemoenzymatic approaches for glycan remodeling post-expression.

Membrane-mimetic expression platforms may better recapitulate the natural presentation of CD27. Technologies such as nanodiscs, liposomes, or cell-derived membrane vesicles containing CD27 in its native membrane environment could provide more physiologically relevant systems for screening therapeutics targeting membrane-bound CD27.

High-throughput expression and characterization systems will accelerate the development pipeline. Automated platforms for parallel expression of CD27 variants, coupled with integrated analytical technologies, could enable rapid screening of stability-enhancing mutations or identification of optimal expression conditions. This approach could significantly reduce the time from concept to candidate selection.

Computational protein design represents another promising direction. Structure-based computational approaches, leveraging advances in protein structure prediction (such as AlphaFold), could guide the rational design of CD27 variants with enhanced properties for therapeutic applications. This might include engineering CD27 constructs with extended half-life, reduced aggregation propensity, or novel binding properties.

These technological advances will likely contribute to the development of next-generation CD27-based therapeutics with improved efficacy, safety profiles, and manufacturing consistency.

What emerging technologies might enhance our understanding of CD27-CD70 signaling dynamics?

Several cutting-edge technologies are poised to revolutionize our understanding of CD27-CD70 signaling dynamics:

Advanced imaging techniques such as single-molecule localization microscopy can visualize CD27 clustering and organization at the immunological synapse with unprecedented resolution. These approaches could reveal how sCD27 associates with synapse-related proteins like myosin IIA, HMGB1, and the TCR Vβ chain . Additionally, lattice light-sheet microscopy enables long-term imaging of living cells with minimal phototoxicity, potentially tracking the dynamic redistribution of CD27 during T-cell activation.

Proximity-based proteomics methods, including BioID and TurboID, allow mapping of protein interactions in living cells. By expressing CD27 fused to a promiscuous biotin ligase, researchers could identify proteins that come into proximity with CD27 during signaling, potentially revealing novel interaction partners beyond those currently known .

Multi-omics integration approaches combining phosphoproteomics, transcriptomics, and metabolomics data can provide a systems-level view of CD27 signaling cascades. This holistic perspective could identify previously unrecognized signaling nodes and feedback mechanisms governing T-cell activation downstream of CD27 engagement.

Optogenetic and chemogenetic tools offer precise temporal control over CD27-CD70 interactions. By engineering light-sensitive or small molecule-responsive variants of CD27 or CD70, researchers could trigger signaling with spatiotemporal precision, dissecting the kinetics and compartmentalization of downstream signaling events.

CRISPR screens targeting components of the CD27 signaling pathway could systematically map the genetic dependencies of CD27-mediated T-cell activation. This approach might identify novel therapeutic targets for modulating CD27 function in cancer immunotherapy or autoimmune disease contexts.

How might CD27-sCD27 research contribute to next-generation cancer immunotherapies?

Research into CD27 and its soluble form (sCD27) holds substantial promise for advancing cancer immunotherapies through several innovative approaches:

Combination therapies incorporating CD27 agonism represent another promising direction. The findings that soluble CD27 enhances T-cell activation, particularly under conditions of suboptimal TCR stimulation (which may mimic insufficient antigenic stimulation by tumor cells), suggests that CD27-targeted therapies might be particularly effective when combined with treatments that enhance tumor antigen presentation or release tumor antigens .

Biomarker development based on sCD27 dynamics could improve patient selection and monitoring for immunotherapy. The observation that cancer patients have lower baseline sCD27 pools than healthy donors, but show increases following effective immunotherapy, suggests that sCD27 measurements might help identify patients likely to respond to immunotherapy or provide early indication of treatment efficacy .

Engineered sCD27 variants with enhanced stability or activity could serve as novel therapeutics. Structure-guided protein engineering could potentially create "super-agonist" forms of sCD27 with increased half-life or enhanced binding to relevant partners at the immunological synapse, such as myosin IIA, HMGB1, or other identified interaction partners .

These approaches, informed by mechanistic understanding of CD27-sCD27 biology, could contribute to more effective and precisely targeted cancer immunotherapies.