CD58 Human

What is CD58 and what is its primary function in the human immune system?

CD58, also known as Lymphocyte Function-Associated Antigen 3 (LFA-3), is a heavily glycosylated surface glycoprotein of 40-70 kDa that is extensively expressed on both hematopoietic and nonhematopoietic cells. It functions primarily as a costimulatory molecule that interacts with CD2 primarily expressed on T cells and NK cells .

The primary functions of CD58 in the human immune system include:

• Facilitating cell-cell adhesion, which is crucial for leukocyte-mediated chemotaxis, phagocytosis, cytotoxicity, and induction of lymphocyte differentiation and proliferation

• Providing an effective second signal for T cell activation, thereby optimizing the proliferative response mediated through TCR/CD3 signaling

• Participating in the formation of the immunological synapse (IS) that induces activation and proliferation of T/NK cells

• Triggering distinct intracellular signaling cascades in both T/NK cells and target cells

To methodologically study CD58 function, researchers commonly employ blocking antibodies, recombinant soluble CD58 proteins, and genetic manipulation approaches to modulate CD58 expression in target cells. Flow cytometry, confocal microscopy, and functional assays measuring T/NK cell activation can effectively quantify these interactions.

What experimental approaches can be used to study the CD58-CD2 interaction in human immunological synapses?

The CD58-CD2 interaction can be studied through multiple complementary approaches:

• Rosette formation assays: The classic "E-rosette test" where human T lymphocytes are mixed with sheep red blood cells (SRBC) to form rosettes. This formation depends on the binding of CD2 in T lymphocytes with a structure functionally homologous to CD58 on SRBC . Anti-CD58 and anti-CD2 mAbs can inhibit rosette formation, providing a functional readout.

• Structural analysis techniques:

  • Site-directed mutagenesis coupled with NMR structural studies have defined the CD58 binding site on CD2 as a charged surface area covering approximately 770 Ų on the AGFCC'C" face of the CD2 beta barrel

  • X-ray crystallography to determine the 3D structure of the CD58-CD2 complex

  • Molecular dynamics simulations to study the dynamics of this interaction

• Live cell imaging:

  • Fluorescence microscopy with labeled CD58 and CD2 proteins to visualize immunological synapse formation in real-time

  • FRET (Förster Resonance Energy Transfer) to measure the proximity of these molecules during cell-cell interactions

• Functional readouts:

  • Calcium flux assays following CD58-CD2 engagement

  • Cytokine production analysis

  • Proliferation assays to measure T cell activation strength

• Binding kinetics measurement:

  • Surface plasmon resonance to determine on/off rates and affinity constants

  • Bio-layer interferometry to characterize binding dynamics

These methodologies provide complementary information about different aspects of the CD58-CD2 interaction and its functional consequences in the immunological synapse.

How do the different isoforms of CD58 influence its biological function?

CD58 exists in two main isoforms with distinct biological functions:

Contrary to common understanding of GPI-anchors, in CD58 the GPI-anchored form primarily enhances adhesion while the transmembrane form is more critical for signal transduction . This structural distribution is significant for CD58's dual roles in both cellular adhesion and transmembrane signaling.

Methodologically, researchers can distinguish between these isoforms using:

• Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment which selectively cleaves GPI-anchored proteins

• Isoform-specific antibodies

• Expression of recombinant constructs containing only one isoform

• Domain-swapping experiments to determine which regions confer specific functions

Understanding the distribution and regulation of these isoforms provides important insights into CD58 function in different tissues and disease states.

What methods provide the most accurate quantification of CD58 expression in human tissues and cells?

Multiple complementary approaches ensure accurate quantification of CD58 expression:

• Flow cytometry:

  • Gold standard for quantifying surface CD58 expression

  • Enables analysis of expression levels on specific cell populations within heterogeneous samples

  • Can determine both percentage of positive cells and mean fluorescence intensity

  • Antibody clones should be carefully validated for specificity

• Quantitative PCR (qPCR):

  • Measures CD58 mRNA expression with high sensitivity

  • Important for studying transcriptional regulation

  • Should include assessment of multiple housekeeping genes for normalization

  • Can distinguish between transmembrane and GPI-anchored isoforms with proper primer design

• RNA sequencing:

  • Provides comprehensive gene expression analysis

  • Single-cell RNA-seq enables analysis of expression heterogeneity

  • Detects alternative splicing events affecting different CD58 isoforms

  • Requires computational analysis expertise for accurate interpretation

• Western blotting:

  • Detects CD58 protein in cell/tissue lysates

  • Provides information about protein size and post-translational modifications

  • Can distinguish between different isoforms based on molecular weight

  • Requires careful optimization of lysis conditions to solubilize membrane proteins

• Immunohistochemistry/Immunofluorescence:

  • Visualizes CD58 expression in tissue context

  • Provides spatial information about expression patterns

  • Can be combined with other markers for colocalization studies

  • Requires quantitative image analysis for accurate measurement

• ELISA:

  • Primarily for detecting soluble CD58 in biological fluids

  • High sensitivity for quantifying sCD58 levels

  • Requires careful standardization with recombinant proteins

Each method has distinct advantages and limitations, and combining multiple approaches provides the most complete assessment of CD58 expression in experimental and clinical samples.

How is CD58 expression regulated by cytokines in different cell types?

The regulation of CD58 expression by cytokines demonstrates notable cell type specificity:

This differential regulation has important implications for immune responses in different tissues and disease states. The mechanisms underlying this cell type-specific regulation likely involve differences in transcription factor expression, promoter accessibility, and signaling pathway activation.

Methodologically, researchers investigating cytokine-mediated regulation should:

• Perform time-course experiments to capture both early and late responses

• Test physiologically relevant cytokine concentrations

• Assess both surface protein expression and mRNA levels

• Consider the effects of cytokine combinations rather than individual cytokines

• Evaluate potential post-transcriptional regulatory mechanisms

Understanding the context-dependent regulation of CD58 expression can inform therapeutic strategies targeting this pathway in inflammatory and malignant conditions.

How do CD58 genetic alterations contribute to immune evasion in diffuse large B-cell lymphoma (DLBCL)?

CD58 genetic alterations play a significant role in immune evasion in diffuse large B-cell lymphoma (DLBCL) through multiple mechanisms:

• Prevalence and clinical impact:

  • Mutation rate: 9.1% of DLBCL patients

  • Copy number loss: 44.7% of DLBCL patients

  • These alterations significantly correlate with reduced response to R-CHOP therapy and inferior survival outcomes

• Molecular mechanisms of immune evasion:

  • Impaired T-cell activation: Loss of CD58 reduces costimulatory signaling through CD2 on T cells

  • JAK2/STAT1 pathway dysregulation: CD58 normally inhibits this pathway via the LYN/CD22/SHP1 axis

  • Increased PDL1 expression: CD58 deficiency leads to elevated PDL1 levels, promoting T cell exhaustion

  • Elevated IDO expression: Similar mechanism increases IDO, creating an immunosuppressive microenvironment

  • Enhanced CD8+ T-cell exhaustion: Single-cell RNA-seq demonstrates CD58 expression in tumor cells negatively correlates with T-cell exhaustion markers

• Therapeutic resistance mechanisms:

  • CD58-deficient DLBCL shows resistance to chimeric antigen receptor (CAR) T-cell therapy

  • This resistance can be overcome by combining CD58-CD2 costimulatory activation with anti-PDL1 blockade or IDO inhibition

These findings demonstrate that CD58 functions beyond simple costimulation, playing a critical role in regulating immune checkpoint pathways. The high frequency of CD58 alterations in DLBCL suggests this represents a major mechanism of immune evasion in this malignancy.

Methodologically, researchers investigating CD58 in lymphoma should employ:

• Multi-omics approaches (genomic, transcriptomic, proteomic)

• Single-cell analyses to understand heterogeneity

• Functional validation in patient-derived models

• Combination therapy testing to overcome resistance mechanisms

What signaling pathways are activated downstream of CD58-CD2 interactions, and how might they be therapeutically targeted?

The CD58-CD2 interaction activates distinct signaling pathways with therapeutic implications:

• Signaling in T cells (CD2-mediated):

  • Activation of LCK (lymphocyte-specific protein tyrosine kinase)

  • Phosphorylation of CD3ζ chains and ZAP-70 recruitment

  • Activation of MAPK, PLCγ1 (calcium mobilization), and PI3K/AKT pathways

  • Enhanced formation and stabilization of the immunological synapse

• Signaling in target cells (CD58-mediated):

  • CD58 activates the LYN/CD22/SHP1 axis

  • This axis inhibits the JAK2/STAT1 pathway

  • Inhibition of this pathway limits PDL1 and IDO expression

  • In CD58-deficient cells, JAK2/STAT1 becomes hyperactive

• Therapeutic targeting strategies:

  Approach	Mechanism	Potential Applications
  CD2 agonists	Direct activation of CD2 signaling	Enhancing T cell responses against tumors
  Recombinant CD58	Engaging CD2 on T/NK cells	Overcoming CD58 deficiency in tumors
  JAK2/STAT1 inhibitors	Blocking downstream effects of CD58 loss	CD58-deficient malignancies
  Combined checkpoint blockade	Targeting PDL1 upregulation from CD58 loss	Enhancing immunotherapy efficacy
  IDO inhibitors	Counteracting IDO upregulation	Reversing immunosuppression
  Bispecific engagers	Linking CD2 activation to tumor targeting	Directing T cell responses

• Synergistic approaches:

  • Direct activation of CD58-CD2 costimulation combined with anti-PDL1 blockade or IDO inhibitors has shown efficacy in sensitizing CD58-deficient DLBCL to CAR T-cell therapy

  • This approach addresses both the loss of costimulation and the secondary immune evasion mechanisms

For researchers studying these pathways, methodological approaches should include:

• Phospho-flow cytometry to quantify pathway activation at the single-cell level

• Proximity ligation assays to detect protein-protein interactions

• CRISPR screens to identify additional pathway components

• Pharmacological inhibitor studies with careful dose-response analyses

• In vivo models to validate therapeutic combinations

What is the specific cytokine profile induced by CD58 co-stimulation of T cells, and how does it differ from other co-stimulatory pathways?

CD58 co-stimulation induces a distinctive cytokine profile in human T cells that differs significantly from other co-stimulatory pathways:

• Cytokine production pattern:

  Cytokine	Response to CD58 Co-stimulation	Comparison to CD80 Co-stimulation
  IL-10	Potently induced (protein and mRNA)	Similar high induction
  IFN-γ	Significantly increased	Similar high induction
  TGF-β	Increased (mRNA level)	Similar induction
  IL-2	Low to absent	Significantly higher with CD80
  IL-4	Low to absent	Variable, often higher with CD80
  IL-5	Low to absent	Variable, often higher with CD80
  IL-13	Low to absent	Variable, often higher with CD80
  TNF-α	Variable effects	Generally higher with CD80

• Unique immunoregulatory profile:

  • The IL-10/IFN-γ combination represents a distinctive signature

  • This pattern combines aspects of both Th1 and regulatory responses

  • May serve to promote specific immune functions while limiting excessive inflammation

• Experimental approaches to study this phenomenon:

  • Co-culture systems using CD58-transfected P815 cells with anti-CD3 as primary stimulus

  • Measurement of cytokine production by ELISA, cytometric bead arrays, and intracellular staining

  • Evaluation of mRNA expression by qPCR or RNA-seq

  • Comparison with other co-stimulatory molecules like CD80 to identify unique effects

• Functional implications:

  • The high IL-10 production may provide regulatory feedback to limit immune activation

  • Combined IL-10 and IFN-γ production may be particularly important in chronic immune responses

  • This profile may help explain the therapeutic potential of targeting the CD58-CD2 axis

This unique cytokine induction profile distinguishes CD58 co-stimulation from other pathways and suggests it may have specialized immunoregulatory functions beyond simple T cell activation.

What molecular mechanisms connect CD58 deficiency to increased PDL1 and IDO expression in cancer cells?

CD58 deficiency leads to increased PDL1 and IDO expression through a specific molecular pathway:

• The LYN/CD22/SHP1/JAK2/STAT1 axis:

  In normal CD58-expressing cells:

  • CD58 activates the LYN kinase

  • LYN phosphorylates CD22

  • Phosphorylated CD22 recruits SHP1 (SH2 domain-containing phosphatase 1)

  • SHP1 dephosphorylates and inhibits JAK2

  • Inhibited JAK2 cannot activate STAT1

  • Reduced STAT1 activation limits PDL1 and IDO transcription

  In CD58-deficient cells:

  • Reduced activation of the LYN/CD22/SHP1 axis

  • Disinhibition of JAK2/STAT1 signaling

  • Increased STAT1 phosphorylation and nuclear translocation

  • Enhanced transcription of STAT1 target genes, including PDL1 and IDO

  • Elevated PDL1 and IDO protein expression

• Functional consequences:

  • Increased PDL1 expression promotes T cell exhaustion through PD-1 engagement

  • Elevated IDO expression leads to tryptophan depletion and kynurenine production

  • The immunosuppressive microenvironment created by these changes impairs anti-tumor immunity

  • This mechanism contributes to resistance to various immunotherapies, including CAR T-cell therapy

• Experimental validation approaches:

  • Phospho-specific western blotting to assess pathway activation

  • Chromatin immunoprecipitation to examine STAT1 binding to target gene promoters

  • Reporter assays to monitor transcriptional activity

  • Pharmacological pathway manipulation (JAK inhibitors, SHP1 modulators)

  • CRISPR-based genetic validation

  • Reconstitution experiments with wild-type CD58

This mechanistic understanding connects CD58 alterations to broader immune evasion strategies through immune checkpoint upregulation, explaining why CD58 loss provides such a significant selective advantage to cancer cells.

What are the most effective experimental models for studying CD58 function given the absence of a murine homolog?

The absence of a CD58 homolog in mice presents a significant challenge for studying CD58 function. Researchers have developed several alternative experimental approaches:

• Zebrafish model system:

  • Functional CD58 and CD2 homologs have been identified in zebrafish

  • This model can compensate for the limitation of murine models

  • Zebrafish provide an attractive system for studying both innate and adaptive immunity

  • Advantages include optical transparency, genetic tractability, and rapid development

• Human cell-based systems:

  • Primary human lymphocytes and cell lines expressing CD58 and CD2

  • CRISPR/Cas9-engineered cell lines with CD58 modifications

  • 3D organoid models incorporating CD58-expressing cells

  • Co-culture systems using defined cell populations

• Humanized mouse models:

  • Immunodeficient mice engrafted with human immune system components

  • Allow for in vivo study of human CD58-CD2 interactions

  • Particularly valuable for evaluating therapeutic approaches

  • Enable assessment of spatial and temporal aspects of immune responses

• Non-human primate models:

  • Express CD58 homologs with functional similarity to human CD58

  • Provide physiologically relevant system for studying CD58 function

  • Essential for preclinical evaluation of therapeutic approaches

  • Allow for longitudinal studies not possible with other models

• In silico approaches:

  • Computational modeling of CD58-CD2 interactions

  • Systems biology analysis of CD58's role in immune networks

  • Virtual screening to identify modulators of CD58 function

Each model system has specific advantages and limitations. For rigorous experimental design, researchers should:

• Validate the functional equivalence of CD58 homologs in non-human systems

• Develop species-specific reagents (antibodies, recombinant proteins)

• Perform cross-species comparisons to identify conserved mechanisms

• Combine multiple model systems for comprehensive understanding

How does soluble CD58 (sCD58) modulate immune responses in the tumor microenvironment?

Soluble CD58 (sCD58) plays a complex, context-dependent role in modulating immune responses within the tumor microenvironment:

• Immunomodulatory effects:

  • At high concentrations, sCD58 can bind to CD2 on T cells, potentially blocking interactions with membrane-bound CD58

  • sCD58 can restrain rosette formation of human T cells with erythrocytes

  • It can profoundly dampen mixed lymphocyte reactions

  • sCD58 alleviates the cytotoxicity of human NK clones (CD2+ CD3-)

  • Paradoxically, sCD58 and mitotic CD2R mAb can act synergistically in triggering T cell activation

• Sources in the tumor microenvironment:

  • Direct secretion by tumor cells

  • Higher production by cells with GPI-anchoring defects

  • Proteolytic cleavage of membrane-bound CD58

  • Release by tumor-associated immune cells

• Role in immune evasion:

  • Release of substantial sCD58 from melanoma cells results in accumulation within tumor tissue

  • High concentrations can inhibit cellular immune responses and reduce immunotherapeutic sensitivity

  • sCD58 can curb the lysis of neoplastic cells through competitively suppressing binding to CD2

• Measurement approaches:

  • ELISA is the gold standard for quantifying sCD58 in biological fluids

  • Western blotting can confirm the molecular weight and integrity

  • Functional assays measuring the impact on T/NK cell activation

• Potential therapeutic implications:

  • Elevated sCD58 levels may predict immunotherapy resistance

  • Neutralizing antibodies against sCD58 could enhance anti-tumor immunity

  • The ratio of membrane-bound to soluble CD58 might serve as a biomarker

This dual immunomodulatory function of sCD58 highlights the complexity of CD58 biology in the tumor microenvironment and suggests that both membrane-bound and soluble forms must be considered when targeting this pathway therapeutically.

Current Knowledge Gaps and Future Research Directions

Despite significant advances in understanding CD58 biology, several important knowledge gaps remain that represent promising areas for future research:

• Structural biology: While we have information about the CD58 binding site on CD2, the complete structure of CD58 and the CD58-CD2 complex remains to be fully characterized. Advanced techniques like cryo-EM could provide critical insights into the conformational changes that occur during receptor engagement.

• Signaling complexity: The precise signaling pathways activated in CD58-expressing cells following CD2 binding require further elucidation. Phosphoproteomic approaches could reveal additional components and regulatory mechanisms.

• Tissue-specific functions: CD58 is expressed on diverse cell types, but its function may vary in different tissue contexts. Single-cell approaches in various human tissues could uncover specialized roles.

• Evolutionary aspects: The absence of CD58 in mice but presence in zebrafish raises questions about evolutionary conservation and divergence. Comparative studies across species could provide insights into the core functions of this pathway.

• Therapeutic targeting: While CD58 alterations clearly influence immunotherapy responses, direct targeting of this pathway remains underdeveloped. Structure-based drug design and novel biologics could exploit this pathway for cancer immunotherapy.

• Regulation of soluble CD58: The mechanisms controlling sCD58 production and its physiological roles in healthy individuals need further investigation to understand its dysregulation in disease states.