Recombinant Human Chemokine-like factor (CKLF)

What is Recombinant Human Chemokine-like factor (CKLF)?

Chemokine-like factor 1 (CKLF1) is a protein that exhibits chemotactic effects on leukocytes. Its amino acid sequence shares significant similarity with TARC/CCL17 and MDC/CCL22, which are established cognate ligands for the CCR4 receptor . Recombinant Human CKLF refers to artificially produced CKLF protein using recombinant DNA technology for research applications. CKLF belongs to the CMTM (CKLF-like MARVEL transmembrane domain-containing) family of proteins, which have diverse roles in immune regulation and cancer biology .

What receptors does CKLF bind to?

CKLF1 functions as a ligand for the C-C chemokine receptor type 4 (CCR4). This has been conclusively demonstrated through multiple experimental approaches:

• CKLF1 induces migration of CCR4-transfected HEK293 cells in a dose-dependent manner

• The chemotactic effects of CKLF1 for CCR4-transfected cells can be desensitized by TARC/CCL17 pretreatment (100 nM at 37°C for 30 min)

• CKLF1 induces calcium flux in CCR4-transfected cells and fully desensitizes subsequent responses to TARC/CCL17

• CKLF1 causes significant receptor internalization in pCCR4-EGFP transfected cells

CKLF1 does not appear to bind CCR6, as CCR6-transfected HEK293 cells failed to migrate when treated with CKLF1 .

In which cell types is CKLF predominantly expressed?

CKLF shows distinct expression patterns across human tissues:

This tissue-specific expression pattern has been confirmed through comprehensive analyses including the Genotype-Tissue Expression (GTEx) portal and BioGPS database containing profiles from 745 samples representing diverse primary human cells .

What experimental methods are used to assess CKLF functionality in immune cell migration?

Several robust methodological approaches are employed to evaluate CKLF's functional effects:

• Transwell migration assays: These measure CKLF1's chemotactic effects on leukocytes or receptor-transfected cell lines. Studies typically use classic bell-shaped migration response curves with maximal migration observed at approximately 20 nM concentration .

• Receptor transfection studies: Cells (e.g., HEK293) are transiently transfected with receptor-expressing plasmids (pcDI-CCR4, pcDI-CCR6) via electroporation, with expression confirmed after 48 hours .

• Desensitization experiments: Pretreatment with known receptor ligands (e.g., TARC/CCL17 for CCR4) to desensitize receptors before CKLF stimulation, confirming shared receptor mechanisms .

• G-protein inhibition studies: Treatment with pertussis toxin (PTX, 100 ng/ml, 16h pretreatment) to assess dependency on Gi/o pathways .

• Calcium flux assays: Measuring intracellular calcium mobilization following CKLF treatment to evaluate receptor activation and signaling .

• Receptor internalization assays: Using fluorescently tagged receptors (pCCR4-EGFP) to visualize and quantify receptor trafficking after CKLF exposure .

How can researchers effectively measure CKLF expression in clinical samples?

Several approaches can be employed for measuring CKLF expression:

RNA-based methods:

• Microarray analysis: Used in large cohort studies including the CRCSC study, which employed three distinct probesets corresponding to CKLF .

• RNA-seq: Provides comprehensive gene expression data with greater dynamic range than microarrays, as used in the GTEx portal .

• RT-qPCR: For targeted, sensitive quantification of CKLF mRNA levels.

Cell-specific expression analysis:

• Fluorescence Activated Cell Sorting (FACS): Can isolate specific cell populations from dissociated fresh primary tumors for subsequent expression analysis, as demonstrated in colorectal cancer studies .

• Single-cell RNA-seq: Provides expression data at the individual cell level, particularly useful in heterogeneous samples like tumors.

When analyzing CKLF expression in clinical samples, researchers should consider cellular heterogeneity, as CKLF expression is predominantly associated with specific immune cell populations rather than being uniformly distributed across all tissue components .

How is CKLF1 functionally related to CCR4 receptor binding and signaling?

CKLF1's interaction with CCR4 involves several functional mechanisms:

• G-protein coupling: CKLF1-induced chemotaxis is completely abolished by pertussis toxin (PTX) treatment, confirming that CKLF1 signals through CCR4's coupling to PTX-sensitive Gi/o family proteins .

• Calcium signaling dynamics: CKLF1 induces calcium flux in CCR4-transfected cells and fully desensitizes subsequent responses to TARC/CCL17. Interestingly, TARC/CCL17 can only partly desensitize the response to CKLF1, suggesting potentially different binding characteristics or receptor activation mechanisms .

• Cross-desensitization patterns: The pretreatment with 100 nM TARC/CCL17 desensitizes CCR4 to subsequent stimulation with CKLF1, confirming a shared receptor mechanism but potentially different binding dynamics .

These findings collectively confirm CKLF1 as a functional ligand for CCR4 while suggesting unique characteristics in its receptor interaction compared to canonical CCR4 ligands.

What role does CKLF play in cancer progression and immune surveillance?

CKLF demonstrates context-dependent roles in cancer that vary by tumor type:

Colorectal Cancer (CRC):

• High CKLF expression is associated with favorable prognosis specifically in the CMS1 (Consensus Molecular Subtype 1) subtype of colorectal cancer .

• In CMS1 patients, high CKLF expression correlates with a 79% reduced risk of recurrence compared to low expression (HR 0.18, 95% CI 0.04-0.89) .

• This risk reduction remains statistically significant after adjusting for confounding factors including age, stage, tumor location, and adjuvant treatment .

• CKLF expression in CRC tumors is primarily derived from tumor-infiltrating leukocytes rather than tumor cells themselves .

Hepatocellular Carcinoma (HCC):

These contrasting findings highlight the context-dependent nature of CKLF's role in cancer biology, potentially related to differences in the tumor microenvironment, immune landscape, or molecular subtypes between cancer types.

How do CKLF expression patterns differ between healthy and disease states?

The available research provides insights into CKLF expression differences in certain conditions:

Colorectal Cancer:

• CKLF expression is significantly higher in tumor-infiltrating leukocytes compared to other cell types within the tumor microenvironment .

• Expression levels vary within CRC subtypes, with particular relevance in the CMS1 (Consensus Molecular Subtype 1) subtype .

• The prognostic value of CKLF appears restricted to tumors within the highest-expressing tertile, indicating potential threshold effects .

Hepatocellular Carcinoma:

• Paired data analysis of 50 cases showed significantly increased mRNA expression of CKLF in HCC compared to adjacent normal tissues .

• High CKLF expression correlates with worse survival outcomes in HCC, unlike in colorectal cancer .

These findings suggest that CKLF's expression and biological significance must be interpreted in the specific context of each disease and its unique pathological features.

What is the relationship between CKLF and other members of the CMTM family in cancer?

The CMTM family consists of CKLF and CMTM1-8, which show varied relationships to cancer progression:

Expression patterns in Hepatocellular Carcinoma:

• Paired data analysis (50 cases) showed significantly increased mRNA expression of CKLF, CMTM1, CMTM3, CMTM4, CMTM7, and CMTM8 in HCC compared to adjacent normal tissues .

Prognostic significance in HCC:

This reveals a complex relationship between different CMTM family members and cancer outcomes, with some members (including CKLF) appearing to promote cancer progression while others demonstrate tumor-suppressive effects.