KLRC2 Human

What is KLRC2 and what is its genomic location in humans?

KLRC2 (NKG2C) is located at 12p13 in the NK gene complex . It encodes an activating receptor primarily expressed on NK cells that binds to HLA-E, transmitting an activating signal that enhances NK cell effector functions. Unlike its inhibitory counterpart NKG2A (KLRC1), NKG2C provides stimulatory signals that are crucial for immune surveillance against viral infections, particularly human cytomegalovirus (HCMV) .

How do KLRC2/NKG2C+ NK cells differ functionally from conventional NK cells?

KLRC2/NKG2C+ NK cells exhibit memory-like properties not typically associated with conventional NK cells. These cells can specifically recognize HCMV strains encoding variable UL40 peptides and can further expand and differentiate in response to pro-inflammatory factors . They show enhanced antibody-dependent cellular cytotoxicity (ADCC) responses and preferentially expand following HCMV infection. While conventional NK cells rely more on natural cytotoxicity receptors (NCRs) for activation, KLRC2+ NK cells demonstrate enhanced CD16-mediated responses despite often having reduced levels of NCRs like NKp46 and NKp30 .

What is the relationship between KLRC2 expression and adaptive NK cells?

While KLRC2/NKG2C expression is strongly associated with adaptive NK cells, it is not an absolute requirement. Adaptive NK cells have been characterized as NK cells lacking FcRγ, tyrosine kinase SYK, EAT-2, and PLZF expression . These cells show epigenetic remodeling similar to memory T cells and display enhanced antibody-dependent functions. Importantly, adaptive NK cells can develop in NKG2C-negative individuals and in those with NKG2C gene deletions, indicating that NKG2C is not the sole driver of the adaptive NK cell phenotype . Research has found that the characteristic molecular footprint of adaptive NK cells exists in NKG2C−/− donors .

How can researchers accurately distinguish between KLRC1 (NKG2A) and KLRC2 (NKG2C) expression?

Traditional antibody-based detection faces significant challenges as anti-NKG2A antibodies often cross-react with NKG2C protein . The most reliable method involves RNA-based flow cytometry (such as PrimeFlow technology) that labels specific gene transcripts of KLRC1 and KLRC2, taking advantage of nucleotide differences between these transcripts . This approach allows simultaneous detection of surface proteins and gene transcripts with single-cell resolution. When designing experiments to differentiate these receptors, researchers should include appropriate controls to validate the specificity of detection methods and consider using combinatorial approaches of antibody staining and RNA labeling .

What is the optimal flow cytometry panel design for studying KLRC2+ NK cells?

An effective panel should include:

• NK cell markers: CD56, CD3 (to exclude T cells)

• KLRC2 detection: anti-NKG2C antibody (clone 134591 or similar) and/or RNA probes for KLRC2

• Adaptive NK cell markers: FcRγ (absent), CD57 (often present)

• Functional markers: CD16, NKG2D, KIRs

• Inhibitory receptors: NKG2A/KLRC1 to identify pure KLRC2+ populations

• Activation/differentiation markers: CD2, CD57, CCR5

When analyzing human peripheral blood mononuclear cells (PBMCs), conjugate the anti-KLRC2 antibody to a bright fluorochrome (such as PE) for optimal detection sensitivity, and use a dump channel to exclude non-NK cells . For RNA-based detection, follow appropriate fixation and permeabilization protocols to preserve cell surface staining while enabling intracellular probe hybridization .

What technical challenges should researchers anticipate when isolating and culturing KLRC2+ NK cells?

KLRC2+ NK cells typically represent a minority population in unexposed individuals but may expand significantly following HCMV infection . Challenges include:

• Low frequency in HCMV-negative donors

• Cross-reactivity issues with antibodies when attempting to sort pure populations

• Sensitivity to cryopreservation, which may affect functional assays

• Requirement for specific cytokines (IL-15) to maintain viability and phenotype in culture

• Heterogeneity within the KLRC2+ population (KLRC1+KLRC2+ vs. KLRC1-KLRC2+)

For optimal results, use freshly isolated cells when possible, employ RNA-based sorting techniques for high purity, and supplement cultures with IL-15 to maintain the survival and phenotypic stability of these cells .

How does HCMV infection specifically drive the expansion of KLRC2+ NK cells?

HCMV infection leads to a dramatic 12-fold increase in KLRC2+ NK cells compared to only a modest 2-fold increase in KLRC1+ NK cells . This expansion involves several mechanisms:

• HLA-E upregulation on infected cells provides ligands for NKG2C

• Pro-inflammatory cytokines (IL-12, IL-15) create a conducive environment for expansion

• Specific recognition of HCMV peptides presented by HLA-E by NKG2C+ NK cells

• Potential involvement of licensing through inhibitory KIRs specific for self-HLA class I molecules

There is a significant positive correlation between increasing KLRC2+ NK cells and rhCMV-binding IgG levels, supporting the specific relationship between KLRC2+ NK cell expansion and CMV infection . The expansion appears to involve clonal or oligoclonal populations, suggesting antigen-specific recognition similar to adaptive immune responses .

What is the phenotypic signature of KLRC2+ NK cells in HCMV-exposed versus unexposed individuals?

In HCMV-unexposed individuals, dual KLRC1+KLRC2+ NK cells predominate, while HCMV-exposed individuals show a shift toward KLRC1-KLRC2+ cells . This suggests downregulation of the inhibitory NKG2A receptor following HCMV exposure. The complete phenotypic signatures include:

HCMV-unexposed:

• Higher proportion of KLRC1+KLRC2+ NK cells

• Lower absolute frequencies of both KLRC1+ and KLRC2+ NK cells

• Less differentiated phenotype with higher expression of NKp30 and NKp46

• Less prominent expression of CD57 and self-KIRs

HCMV-exposed:

• Predominance of KLRC1-KLRC2+ NK cells

• Higher absolute frequencies of KLRC2+ NK cells

• More differentiated phenotype with lower NCR expression

• Higher expression of CD16, enhancing ADCC capacity

• Epigenetic remodeling of the IFNG locus, particularly the CNS1 region

Besides HCMV, which other viral infections are associated with alterations in KLRC2+ NK cell populations?

While HCMV provides the strongest association, elevated NKG2C expression on human NK cells has also been observed in response to HIV infection . The SIV (Simian Immunodeficiency Virus) model also shows expansion of KLRC2+ NK cells, although this may be associated with concurrent CMV infection rather than a direct effect of SIV . Other viral infections that may influence KLRC2+ NK cell populations include hantavirus infections, where inflammatory cytokines and HLA-E can be upregulated similar to HCMV infection . The precise mechanisms driving KLRC2+ NK cell responses in these different viral contexts remain an area of active investigation.

What epigenetic changes characterize KLRC2+ adaptive NK cells?

KLRC2+ NK cells in HCMV-seropositive individuals undergo significant epigenetic remodeling, particularly of the conserved non-coding sequence 1 (CNS1) of the IFNG gene . This remodeling results in:

• An open configuration of CNS1, similar to memory CD8+ T or Th1 cells

• Enhanced transcriptional accessibility necessary for increased IFNG transcription

• Specific association with expanded NKG2Chi self-MHC specific KIRs+ NK cells

• Stable epigenetic imprinting that persists long-term

This epigenetic remodeling occurs exclusively in NKG2C+ cells from HCMV+ individuals, not in NKG2C- cells from HCMV+ individuals or NKG2C+ cells from HCMV- individuals . This suggests that both receptor expression and viral exposure are necessary for these epigenetic changes, which likely contribute to the enhanced recall responses of these memory-like NK cells.

How do KLRC2+ NK cells exhibit memory-like properties despite being innate immune cells?

KLRC2+ NK cells challenge the traditional paradigm of innate immunity by demonstrating several memory-like properties:

• Enhanced response to secondary challenges with the same stimulus

• Clonal or oligoclonal expansion following specific antigen exposure

• Long-term persistence following initial expansion

• Functional reprogramming with specialized effector capabilities

• Stable epigenetic modifications similar to memory T cells

Despite these memory-like features, current evidence does not indicate that adaptive NK cells can recognize various antigens and mount enhanced responses to all of them, which distinguishes them from true adaptive immune memory cells . Their memory appears to be more pathogen-specific or driven by specific receptor-ligand interactions, particularly involving CD16 and ADCC mechanisms .

What is the contribution of cytokines to KLRC2+ NK cell expansion and function?

Cytokines play crucial roles in KLRC2+ NK cell biology:

• IL-12, IL-15, and HLA-E are critical for the expansion of NKG2C+ NK cells

• These factors can be upregulated during HCMV, hantavirus, or HIV infection

• IL-12Rβ1 deficiency impairs the generation of NK cells with adaptive features

• IL-15 appears essential for maintenance of KLRC2+ NK cells

Interestingly, despite the role of IL-12 in initial expansion, adaptive NK cells show low IL-12 receptor expression and marginal response to IL-12 plus IL-18 stimulation in vitro, suggesting a complex relationship between cytokine signaling and the adaptive NK phenotype . This apparent contradiction requires further investigation to fully understand the temporal dynamics of cytokine responsiveness in KLRC2+ NK cells.

How can KLRC2+ NK cells be leveraged for immunotherapy applications?

KLRC2+ NK cells offer several promising features for immunotherapeutic development:

• Enhanced ADCC capacity, particularly valuable for antibody-based therapies

• Potential for expansion and persistence after adoptive transfer

• Memory-like properties that might enable more durable responses

• Reduced inhibitory receptor expression that may enhance anti-tumor activity

Potential approaches include:

• Expansion and adoptive transfer of autologous KLRC2+ NK cells

• Combination with therapeutic antibodies to exploit enhanced ADCC

• Genetic modification to express KLRC2 in conventional NK cells

• Cytokine-based approaches to selectively expand KLRC2+ populations in vivo

Each approach would require careful optimization of expansion protocols, cytokine support, and assessment of persistence and function following therapeutic application .

What is the significance of KLRC2 gene deletions in human populations?

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How do KLRC2+ NK cells interact with other immune cells in coordinating antiviral responses?

While NK cells traditionally function as immune sentinels that eradicate target cells and release various cytokines and chemokines, KLRC2+ adaptive NK cells may have specialized roles in immune coordination . They likely interact with dendritic cells, T cells, and B cells through cytokine production and direct cellular contacts. Understanding these interactions is crucial for developing comprehensive immunotherapeutic strategies that leverage the unique properties of KLRC2+ NK cells while engaging the broader immune system for optimal clinical outcomes.