CD74 Human, Sf9

What is CD74 and what are its main functions?

CD74, also known as Invariant chain (Ii) or p33, is a type 2 transmembrane glycoprotein with multiple biological functions. Its primary role is as a chaperone for MHC class II molecules on antigen-presenting cells, where it undergoes progressive proteolysis during class II trafficking and antigenic peptide loading . Beyond this canonical function, CD74 serves as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine involved in various inflammatory conditions, cardiovascular diseases, and cancer . When expressed on the cell surface, CD74 can assemble into trimers which associate with class II molecules in nonameric complexes . Additionally, CD74 associates with CD44 and mediates inflammatory leukocyte responses, protection from tissue fibrosis, B cell proliferation and survival signaling, and the upregulation of angiogenic factors .

What cell types express CD74 and how is this expression regulated?

CD74 is primarily expressed in antigen-presenting cells including B cells, monocytes/macrophages, and dendritic cells, consistent with its classical role in MHC II-mediated antigen presentation . Recent evidence shows that CD74 is also expressed in T cells, specifically CD4+ T cells, which constitutively express CD74 intracellularly . Upon T-cell activation, CD74 expression is significantly and rapidly upregulated, post-translationally modified by chondroitin sulfate, and translocated to the cell surface to function as a MIF receptor . CD74 is also upregulated on non-immune cells at sites of inflammation, including amyloid beta plaques and atherosclerotic plaques . In pathological conditions like severe COVID-19, CD74 surface expression is significantly upregulated on both CD4+ and CD8+ T cells compared to patients with mild disease .

How does CD74 interact with MIF receptors to induce T-cell migration?

CD74 functions as part of a complex MIF receptor network that includes classical chemokine receptors CXCR2, CXCR4, and ACKR3/CXCR7. In activated CD4+ T cells, CD74 interacts specifically with CXCR4 to form heterocomplexes visualized through proximity ligation assay (PLA) . These CD74/CXCR4 complexes are significantly diminished after MIF treatment, suggesting a MIF-mediated internalization process . Functional studies using 3D-matrix-based live cell imaging and receptor pathway-specific inhibitors have demonstrated a causal involvement of both CD74 and CXCR4 in MIF-induced CD4+ T-cell migration . Mechanistically, MIF-mediated signaling via CD74 depends on receptor complex formation with CD44, CXCR2, CXCR4, and ACKR3/CXCR7, which induces downstream signaling pathways including PI3K/Akt, AMPK, NF-κB, calcium signaling, and ERK pathways . These pathways collectively drive immune cell recruitment and activate various cellular responses including proliferation and metabolism .

What is the significance of CD74 isoforms and post-translational modifications?

CD74 exists in multiple isoforms ranging from 31 kDa to 41 kDa, resulting from alternative splicing and the use of a second initiation start site . These isoforms differ by the presence or absence of a 16 amino acid N-terminal extension and/or a 64 amino acid internal section of the extracellular domain . Post-translational modifications, particularly glycosylation and chondroitin sulfate addition, significantly affect CD74's function and localization. In activated CD4+ T cells, CD74 is post-translationally modified by chondroitin sulfate, which appears to be important for its surface expression and function as a MIF receptor . Additionally, MIF binding to CD74 induces proteolytic cleavage of the CD74 intracellular domain, which promotes B cell differentiation . These various modifications and isoforms likely contribute to the diverse functions of CD74 across different cell types and biological contexts.

How is CD74 involved in disease pathophysiology, particularly in COVID-19?

CD74 has emerged as a significant marker in various pathological conditions. In COVID-19, CD74 surface expression is significantly upregulated on CD4+ and CD8+ T cells in patients with severe disease (WHO grade ≥ 5) compared to patients with only mild disease (WHO grade 1–3) . This upregulation is accompanied by increased CD74 expression on classical monocytes . Given that MIF has been identified as a prognostic marker to predict disease severity and patient outcomes in COVID-19, and that impaired T cell responses during SARS-CoV-2 infections often display sustained T-cell activation, the CD74-MIF axis appears to play a crucial role in COVID-19 pathophysiology . Beyond COVID-19, CD74 is upregulated at sites of inflammation, including amyloid beta plaques involved in neurodegenerative diseases and atherosclerotic plaques in cardiovascular disease . In cancer biology, CD74 contributes to B cell proliferative and survival signaling and the upregulation of angiogenic factors .

What are the optimal methods for CD74 detection and quantification?

Researchers employ multiple complementary techniques to detect and quantify CD74 expression:

Flow Cytometry:
For surface and intracellular CD74 detection in different cell populations. T cells can be isolated by positive depletion from the enriched PBMC fraction using CD3+ microbeads, and CD74 expression can be determined in CD3+-selected cells that are further characterized by CD4, CD8, and HLA-DR surface expression . Quantification is typically performed using flow cytometry software such as FlowJo .

Western Blot:
For total protein detection and verification of siRNA knockdown efficiency. Western blotting is particularly useful for monitoring CD74 expression over time after transient knockdown, where the lowest level of CD74 is typically detected 72 hours post-transfection (5-10% of wild-type expression remaining) and downregulation remains stable for up to 144 hours post-transfection .

Immunohistochemistry:
For tissue localization studies and visualization of CD74 distribution in different cellular compartments .

RNA-sequencing and Proteomic Analysis:
For comprehensive analysis of CD74 expression and regulation at both transcriptomic and proteomic levels .

How can researchers effectively manipulate CD74 expression for functional studies?

Transient knockdown of CD74 using siRNA provides an effective method for studying CD74 function:

• siRNA Selection: Test various siRNA constructs to identify the most efficient single siRNA (>90% downregulation) for experimental use .

• Transfection Protocol:

  • Plate cells 24 hours prior to transfection

  • Change medium 1 hour before starting transfection

  • Transfect siRNA CD74 (50 nM) into target cells using Lipofectamine 2000 according to manufacturer's instructions

  • Use appropriate negative control siRNA (e.g., MISSION® siRNA Universal Negative Control)

• Validation of Knockdown: Monitor knockdown efficiency and duration by western blotting, with optimal knockdown typically achieved 72 hours post-transfection .

This approach allows researchers to effectively reduce CD74 expression to study its role in various cellular processes, including MIF binding, signal transduction, and cell migration.

How can protein-protein interactions involving CD74 be detected and analyzed?

Proximity Ligation Assay (PLA) is a powerful technique for visualizing protein-protein interactions involving CD74:

• Protocol Implementation: Use commercially available kits (e.g., Duolink In Situ) following manufacturer's instructions to detect direct interaction between CD74 and binding partners .

• Imaging and Analysis:

  • Capture images using confocal laser scanning microscopy (e.g., LSM710 with Plan-Apochromat 63×/1.40 oil objective)

  • Take multiple z-stack sections spanning entire cells

  • Create maximum projections of cross-sections for quantification

  • Count red puncta (PLA signals) per cell using ImageJ software

  • Normalize to cell number to obtain mean PLA puncta/cell values

  • Maintain constant microscope settings for all images to enable direct comparison

  • Evaluate significance using ANOVA with a significance level of 0.05

This method has successfully visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing toward a MIF-mediated internalization process .

What functional assays can be used to study CD74-mediated cellular responses?

Several functional assays can effectively measure CD74-mediated responses:

Cell Adhesion Assays:
When 5 x 10^4 cells per well are added to Recombinant Human CD74 coated plates, cell adhesion is enhanced in a dose-dependent manner with an ED50 of 0.6-3 μg/mL . This assay allows for quantitative assessment of CD74's role in cellular adhesion.

3D-Matrix-Based Live Cell Imaging:
This technique, combined with receptor pathway-specific inhibitors, has been used to determine the causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration . The assay allows for real-time visualization and quantification of cell movement in response to MIF stimulation under various inhibitory conditions.

Surface Plasmon Resonance (SPR):
SPR has been utilized to study interactions between CD74 and various binding partners, including redox-dependent plasticity of oxMIF that facilitates its interaction with CD74 and therapeutic antibodies . This technique provides quantitative binding kinetics and affinity measurements.