CD47 Human

What is the molecular structure and basic function of human CD47?

CD47, also known as integrin-associated protein (IAP), is a ubiquitous 50 kDa multipass transmembrane protein with a single V-type Ig-like domain at its N-terminus. It belongs to the immunoglobulin superfamily and is encoded by the CD47 gene in humans. Structurally, CD47 contains five transmembrane domains with an extracellular N-terminal domain and a short C-terminal cytoplasmic tail. The protein functions primarily as a cellular marker that inhibits phagocytosis by binding to signal-regulatory protein alpha (SIRPα) on macrophages, essentially serving as a "don't eat me" signal that prevents the engulfment of viable cells .
Alternative splicing generates several isoforms with truncated cytoplasmic domains, allowing for tissue-specific functions. Within the N-terminal extracellular domain, human CD47 shares approximately 63% amino acid sequence identity with mouse and rat CD47, highlighting evolutionary conservation of this important immune regulatory protein .

What are the primary binding partners of CD47 and their functional significance?

CD47 interacts with multiple binding partners that mediate distinct cellular functions:

How does CD47 contribute to cancer progression and immune evasion?

CD47 is expressed on the surface of all human solid tumor cells, serving as a critical immune evasion mechanism. Cancer cells upregulate CD47 to prevent phagocytosis by macrophages, allowing tumors to evade immune surveillance and clearance. This mechanism operates through the CD47-SIRPα axis, where CD47 on cancer cells engages SIRPα on phagocytic cells to transmit inhibitory signals that prevent engulfment .
The ubiquitous expression of CD47 across diverse cancer types indicates its fundamental role in cancer cell survival. Research has demonstrated that CD47 upregulation correlates with poorer clinical outcomes in various malignancies. Interestingly, cancer cells often simultaneously upregulate both CD47 (the "don't eat me" signal) and calreticulin (an "eat me" signal), but the inhibitory CD47 signal dominates in the absence of therapeutic intervention .
Methodologically, researchers typically assess CD47 expression in cancer tissues using immunohistochemistry, flow cytometry, or Western blotting. Flow cytometric analysis is particularly valuable for quantifying CD47 surface expression levels, with increased mean fluorescence intensity (MFI) serving as a key measurement parameter when comparing malignant versus normal tissues .

What is the role of CD47 in neurodevelopmental disorders associated with macrocephaly?

Recent research has revealed an unexpected role for CD47 in neurodevelopmental disorders, particularly in 16p11.2 deletion syndrome that presents with macrocephaly. Studies on patient-derived induced pluripotent stem cells (iPSCs) show that CD47 is significantly overexpressed in neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs) from individuals with 16p11.2 deletion syndrome who exhibit brain enlargement .
The overexpression of CD47 in these neural cells disrupts normal phagocytic clearance mechanisms, contributing to brain overgrowth. Specifically, 16p11.2 deletion NPCs and OPCs show:

• Significantly upregulated CD47 expression at both mRNA and protein levels

• Increased cell surface expression of calreticulin (a prophagocytic "eat me" signal)

• Reduced rates of phagocytosis by macrophages and microglia

• Normalization of phagocytosis rates when treated with CD47-blocking antibodies
  These findings establish a novel connection between CD47 and neurodevelopmental disorders, suggesting that dysregulation of cellular clearance mechanisms may contribute to the pathophysiology of conditions associated with brain overgrowth. Importantly, CD47 expression and related phagocytosis rates were similar across control and 16p11.2 deletion groups at the pluripotent stage before differentiation, highlighting the developmental specificity of this mechanism .

What are the optimal techniques for detecting and quantifying CD47 expression in different tissue types?

Multiple complementary techniques can be employed to detect and quantify CD47 expression, each with specific advantages depending on the research question:

How can researchers effectively block CD47 function in experimental models?

Blocking CD47 function is a critical approach in both basic research and therapeutic development. Several methodological approaches have proven effective:

• Blocking antibodies: Anti-CD47 antibodies such as clone B6.H12 can effectively block the CD47-SIRPα interaction. When using blocking antibodies, it's essential to:

  • Titrate antibody concentrations (typically 5-20 μg/mL) to determine optimal blocking without non-specific effects

  • Include appropriate isotype controls

  • Confirm blocking efficacy using functional assays such as phagocytosis assays

• Genetic approaches:

  • CRISPR/Cas9-mediated knockdown or knockout

  • shRNA or siRNA targeting CD47 mRNA

  • Dominant-negative CD47 constructs

• Small molecule inhibitors:

  • Peptide mimetics of SIRPα that compete for CD47 binding

  • Small molecules that disrupt the CD47-SIRPα interface
    In phagocytosis assays, researchers typically co-culture target cells (e.g., NPCs, cancer cells) with macrophages or microglia at defined ratios (often 1:1 or 2:1) and quantify engulfment through fluorescent labeling and microscopy or flow cytometry. Treatment with CD47-blocking antibodies has been shown to restore phagocytosis of 16p11.2 deletion NPCs and OPCs to control levels, particularly in cells with increased surface expression of calreticulin .

What is the relationship between CD47 expression and cellular senescence?

Recent research has uncovered a significant relationship between CD47 expression and cellular senescence. Quantitative PCR analyses demonstrate that CD47 mRNA levels increase during replicative senescence and aging. This upregulation appears to be part of the senescence-associated secretory phenotype (SASP) and may contribute to age-related tissue dysfunction .
The increased expression of CD47 in senescent cells has several potential implications:

• It may protect senescent cells from immune clearance, contributing to their accumulation in aging tissues

• It could affect tissue homeostasis by altering the phagocytic removal of damaged cells

• It might represent a potential therapeutic target for senolytic approaches
  Methodologically, researchers studying this relationship typically induce senescence through various means (replicative exhaustion, oncogene activation, irradiation, or chemical induction), followed by assessment of CD47 expression via qPCR, Western blotting, and flow cytometry. The relationship between senescence markers (p16, p21, SA-β-gal) and CD47 expression provides valuable insights into the role of this protein in aging processes .

How does the CD47-SIRPα axis interact with other phagocytic signals in determining cell fate?

The regulation of phagocytosis involves a complex interplay between pro-phagocytic "eat me" signals and anti-phagocytic "don't eat me" signals, with CD47-SIRPα representing a dominant inhibitory pathway. This complex system operates as follows:

• Calreticulin (CRT): A major pro-phagocytic signal that translocates to the cell surface in response to cellular stress or damage. CRT binds to LDL-receptor-related protein (LRP) on phagocytes to promote engulfment.

• Phosphatidylserine (PS): Exposed on the outer leaflet of the plasma membrane during apoptosis, PS is recognized by various receptors on phagocytes, including TIM-4, BAI1, and stabilin-2.

• CD47-SIRPα interaction: Provides a dominant inhibitory signal that can override pro-phagocytic signals.
  Research in 16p11.2 deletion syndrome has revealed important insights into this balance. 16p11.2 deletion NPCs and OPCs simultaneously upregulate both CD47 and cell surface calreticulin, but the high levels of CD47 override the pro-phagocytic CRT signal. Importantly, treatment with CD47-blocking antibodies restored phagocytosis, particularly in cells with high CRT expression, demonstrating the dominance of the CD47 signal in this context .
  Methodologically, researchers investigating this axis often use flow cytometry to simultaneously measure surface expression of CD47, calreticulin, and other markers such as asialoglycan binding sites (detected with PHA-L). The balance between these signals determines cellular fate, with therapeutic manipulation of this balance showing promise for treating conditions ranging from cancer to neurodevelopmental disorders .

What are the current approaches for targeting CD47 in cancer immunotherapy?

The recognition of CD47 as a nearly universal tumor immune evasion mechanism has spurred significant therapeutic development. Current approaches include:

What potential exists for targeting CD47 in neurodevelopmental disorders?

The discovery of CD47 overexpression in 16p11.2 deletion syndrome with macrocephaly opens new therapeutic possibilities for neurodevelopmental disorders. Research has demonstrated that blocking CD47 with antibodies restores normal phagocytic clearance of neural progenitor cells and oligodendrocyte progenitor cells derived from individuals with 16p11.2 deletion syndrome .
This suggests several potential therapeutic approaches:

• Targeted CD47 blockade: Specific delivery of CD47-blocking agents to the central nervous system during critical developmental windows

• Modulation of downstream pathways: Targeting components of the CD47-SIRPα signaling cascade to normalize phagocytic function

• Combinatorial approaches: Simultaneously enhancing pro-phagocytic signals while blocking CD47
  Significant research challenges remain, including the delivery of therapeutics across the blood-brain barrier, identifying optimal developmental windows for intervention, and ensuring specificity to abnormal neural cells. Future research directions should include comprehensive investigation of CD47 expression patterns across various neurodevelopmental disorders associated with brain overgrowth, development of CNS-penetrant CD47-targeting agents, and detailed characterization of the effects of CD47 modulation on neural circuit development .

How should researchers optimize flow cytometry protocols for accurate CD47 quantification?

Flow cytometric analysis of CD47 requires careful optimization to ensure accurate quantification, particularly given its ubiquitous expression and the importance of detecting subtle expression differences in pathological states:

• Sample preparation:

  • Fresh isolation is preferred when possible

  • Careful enzymatic dissociation to preserve surface epitopes

  • Appropriate fixation (if needed) using paraformaldehyde at concentrations that maintain epitope integrity

• Antibody selection and titration:

  • Use of validated anti-CD47 antibodies (e.g., clone 472603 for human CD47)

  • Proper titration to determine optimal antibody concentration

  • Inclusion of appropriate isotype controls to establish baseline fluorescence

• Panel design:

  • Include markers for cell identification and viability

  • Consider simultaneous detection of related markers (SIRPα, calreticulin)

  • Careful compensation when using multiple fluorophores

• Analysis considerations:

  • Report CD47 expression as mean fluorescence intensity (MFI)

  • Use consistent gating strategies between experiments

  • Consider the percentage of CD47+ cells and the intensity distribution
    Human peripheral blood lymphocytes can serve as positive controls for CD47 expression, as demonstrated in multiple studies using techniques such as flow cytometry with APC-conjugated monoclonal antibodies against CD47 .

What are the key considerations for designing valid phagocytosis assays to study CD47 function?

Phagocytosis assays are central to functional studies of CD47. Critical methodological considerations include:

• Target cell preparation:

  • Fluorescent labeling (e.g., CFSE, CellTracker dyes) for tracking

  • Assessment of baseline CD47 and calreticulin expression

  • Standardization of cell numbers and viability

• Phagocyte selection and preparation:

  • Source of phagocytes (primary macrophages, microglia, cell lines)

  • Consistent activation state (M0, M1, M2 polarization)

  • Fluorescent labeling distinct from target cells

• Co-culture conditions:

  • Optimized target:phagocyte ratio (typically 1:1 to 5:1)

  • Appropriate incubation time (2-24 hours depending on system)

  • Culture medium selection to support both cell types

• Analysis methods:

  • Flow cytometry (double-positive events indicating engulfment)

  • Confocal microscopy with Z-stack confirmation of internalization

  • Live cell imaging for kinetic studies

• Controls and interventions:

  • CD47 blocking antibodies (e.g., B6.H12 clone)

  • SIRPα blocking antibodies

  • Cytochalasin D to inhibit all phagocytosis as negative control
    Phagocytosis is typically quantified as the percentage of phagocytes containing engulfed target cells. When studying 16p11.2 deletion NPCs and OPCs, researchers have demonstrated that treatment with CD47-blocking antibodies restores phagocytosis rates to control levels, particularly in cells with increased cell surface expression of calreticulin .