CCX5 Antibody

What is the CX5 monoclonal antibody and what epitope does it recognize?

The CX5 monoclonal antibody is a functional grade reagent that specifically recognizes mouse NKG2D (CD314), a lectin-like molecule expressed on both human and mouse natural killer cells. This antibody binds to NKG2D, which serves as an activating receptor on NK cells and plays a crucial role in immune surveillance. Mouse NKG2D interacts with retinoic acid-inducible ligands RAE-1α, -β, -γ, -δ, -ε, and the minor histocompatibility molecule H60 .

NKG2D signaling occurs through association with the adapter molecules DAP12 and DAP10, which facilitate downstream signal transduction. The CX5 antibody is particularly valuable in research settings because it can block the binding of NKG2D to its natural ligands, thereby enabling functional studies of NKG2D-dependent immune responses both in vitro and in vivo .

In which mouse cell populations is NKG2D expressed?

NKG2D expression follows a specific pattern across lymphoid populations in mice. According to flow cytometric analyses, NKG2D is expressed by:

• All spleen and liver NK cells

• NK1.1+ thymocytes

• In vitro activated lymphokine-activated killer (LAK) cells

• A subset of splenic NKT cells

Expression patterns have been confirmed in C57BL/6 and NWNA mouse strains, while notably absent or significantly reduced in Balb/c and DBA/2 mice, highlighting important strain-specific differences that researchers must consider when designing experiments . This differential expression across mouse strains suggests genetic regulation of NKG2D that may impact experimental outcomes when using CX5 antibody in different mouse models.

What are the recommended parameters for flow cytometry using CX5 antibody?

When using CX5 antibody for flow cytometric analysis, researchers should adhere to the following methodological guidelines:

• Optimal concentration: ≤0.5 μg per test, where a test is defined as the amount of antibody required to stain a cell sample in a final volume of 100 μL

• Cell number: Should be determined empirically but typically ranges from 10^5 to 10^8 cells per test

• Secondary antibody requirement: When using the CX5 antibody for staining, it is recommended to use biotin anti-rat as the secondary antibody

• Staining controls: Include isotype controls and unstained samples to establish background fluorescence levels

• Cross-reactivity: No cross-reactivity to Ly-49A, C, D or G2 has been observed, supporting the specificity of the CX5 antibody

These parameters ensure optimal detection of NKG2D-expressing cells while minimizing background and non-specific binding.

How can the CX5 antibody be used to investigate NK cell-mediated cytotoxicity?

The CX5 antibody serves as an essential tool for studying NKG2D-dependent NK cell cytotoxicity through several methodological approaches:

• Blocking studies: The CX5 antibody has been validated for blocking the interaction between NKG2D and its ligands (RAE-1 and H60), allowing researchers to assess the specific contribution of NKG2D to NK cell activation and cytotoxicity .

• Inhibition assays: When added to in vitro cultures, CX5 can inhibit NKG2D-dependent NK cell-mediated cytotoxicity against tumor cells bearing NKG2D ligands. This application enables researchers to quantify the proportion of target cell lysis that is specifically attributable to NKG2D engagement .

• In vivo blockade: CX5 antibody has been used in vivo to block NKG2D-dependent tumor cell clearance, providing insights into the physiological relevance of this receptor in anti-tumor immunity .

• Mechanistic studies: By combining CX5 blockade with other receptor-specific antibodies, researchers can delineate the relative contributions and potential synergism between different NK cell activating receptors.

The functional grade preparation of CX5 antibody is particularly suitable for these applications as it lacks potential contaminants that might interfere with cellular functions in biological assays.

What is the relationship between CXCR5+CD8+ T cells and antibody responses?

Recent research has uncovered a novel population of CD8+ T cells that express CXCR5 and play significant roles in shaping antibody responses. These cells exhibit several key characteristics and functions:

• Development context: CXCR5+CD8+ T cells arise in response to protein immunization and peripheral viral infection, even in the absence of follicular infection. Approximately 60% of endogenous CD44hiCD8+ T cells express CXCR5 following OVA/alum immunization, and about 45% express this receptor after x31-OVA viral infection .

• Phenotypic profile: These cells display a follicular-homing phenotype similar to CD4+ T follicular helper (Tfh) cells, with diminished cytotoxic potential compared to conventional CD8+ T cells .

• Functional impact: CXCR5+CD8+ T cells promote IgG2c class switching in responding B cells, demonstrating their ability to shape antibody responses in vivo .

• Antibody suppression: A subset of alloprimed CXCR5+CXCR3-CD8+ T cells mediates in vitro cytotoxicity against alloprimed "self" B cells and suppresses alloantibody production when transferred into transplant recipients .

Understanding the interplay between these cells and antibody responses could provide new insights into immune regulation and potential therapeutic strategies for transplantation and autoimmune disorders.

How does CXCR5 expression influence CD8+ T cell function in germinal centers?

CXCR5 expression on CD8+ T cells is critically important for their localization to germinal centers and subsequent functions in regulating humoral immunity:

• Follicular migration: CXCR5 facilitates CD8+ T cell migration to B cell follicles by responding to CXCL13, the CXCR5 ligand produced by follicular dendritic cells. Adoptive transfer studies with CXCR5 knockout CD8+ T cells demonstrate that this receptor is essential for antibody-suppressor function .

• Cellular interactions: Within germinal centers, CXCR5+CD8+ T cells interact with B cells and can mediate cytotoxicity against activated IgG+ B cells in an MHC I-restricted manner. Flow-sorted CXCR5+CXCR3-CD8+ T cells, but not CXCR3+CXCR5-CD8+ T cells, significantly downregulate alloantibody production .

• Impact on germinal center dynamics: CXCR5+CD8+ T cells influence germinal center reactions by reducing the number of germinal center B cells and cytokine-expressing CD4+ Tfh cells. This suggests that these cells have a regulatory role in limiting excessive antibody responses .

• Cytokine production: The majority (>70%) of alloprimed CD44+CXCR5+CD8+ T cells express IFN-γ, which appears to be critical for their antibody-suppressive function. This defines a functional phenotype of CXCR5+IFN-γ+CD8+ T cells that regulate humoral immunity .

These findings establish CXCR5 as a key determinant of CD8+ T cell function in germinal centers, specifically in regulating antibody responses through direct interactions with germinal center B cells.

What are the optimal methods for identifying and isolating CXCR5+CD8+ T cells?

Researchers studying CXCR5+CD8+ T cells should consider the following methodological approaches for accurate identification and isolation:

• Flow cytometric identification: CXCR5+CD8+ T cells can be identified by staining for CD8, CD44 (to identify activated cells), and CXCR5. Additional markers such as CXCR3 can help distinguish different functional subsets. According to experimental data, researchers should look for CD44hiCD8+ T cells expressing CXCR5, as naïve CD44loCD8+ T cells display minimal CXCR5 positivity .

• Timing considerations: In response to OVA/alum immunization, optimal detection of CXCR5+CD8+ T cells occurs around day 7 post-immunization in the spleen. For influenza virus infection models, assessment around day 8 post-infection in the mediastinal lymph node (mLN) yields approximately 45% CXCR5-expressing CD44hiCD8+ T cells .

• Cell sorting strategy: When isolating CXCR5+CD8+ T cells for functional studies, researchers should implement a sorting strategy that distinguishes CXCR5+CXCR3- from CXCR3+CXCR5- subsets, as these populations exhibit distinct functional properties. The CXCR5+CXCR3- subset specifically demonstrates antibody-suppressor function .

• Functional validation: Following isolation, validation of CXCR5+CD8+ T cell function can be performed through in vitro cytotoxicity assays against activated B cells or through adoptive transfer experiments to assess impact on antibody responses in vivo .

These methodological considerations ensure accurate identification and isolation of functional CXCR5+CD8+ T cell populations for downstream applications.

How can researchers differentiate between cytotoxic and helper functions of CXCR5+CD8+ T cells?

Distinguishing between the cytotoxic and helper functions of CXCR5+CD8+ T cells requires specific experimental approaches:

• Cytotoxicity assays: To assess the cytotoxic potential of CXCR5+CD8+ T cells against B cells, researchers can co-culture flow-sorted CXCR5+CXCR3-CD8+ T cells with alloprimed "self" IgG1+ B cells and measure B cell death. In experimental settings, these cells demonstrate significant cytotoxicity (12.7±1.8%) against alloprimed self B cells but not against allogeneic B cells .

• Antibody suppression assays: The helper/regulatory function of CXCR5+CD8+ T cells can be assessed through adoptive transfer experiments. Transfer of 2×10^6 flow-sorted alloprimed CXCR5+CXCR3-CD8+ T cells into CD8 KO hepatocyte recipients results in decreased alloantibody production (titer=90±40 versus titer=1,300±500 in controls) and enhanced graft survival (MST=32 days versus MST=14 days) .

• Germinal center analysis: Examining germinal center dynamics after CXCR5+CD8+ T cell transfer can reveal their impact on B cell responses. Researchers should quantify germinal center B cells (GL7+Fas+B220+) and CD4+ Tfh cells (CXCR5+PD-1+CD4+) using flow cytometry .

• Cytokine profiling: Intracellular cytokine staining for IFN-γ and other relevant cytokines helps characterize the functional phenotype of CXCR5+CD8+ T cells. The majority of alloprimed CD44+CXCR5+CD8+ T cells (>70%) express IFN-γ, which is critical for their antibody-suppressive function .

These complementary approaches allow researchers to comprehensively characterize both the cytotoxic and helper/regulatory functions of CXCR5+CD8+ T cells in humoral immune responses.

How do strain-specific differences affect CX5 antibody applications?

Strain-specific variations in NKG2D expression significantly impact experimental approaches using the CX5 antibody:

• Expression variations: NKG2D expression has been confirmed in C57BL/6 and NWNA mouse strains, while Balb/c and DBA/2 mice show minimal expression. This strain-dependent variation must be considered when designing experiments, as it directly affects the utility of CX5 antibody in different mouse models .

• Experimental controls: When using CX5 antibody across different mouse strains, researchers should include strain-specific positive and negative controls to account for variable NKG2D expression. Flow cytometric analysis of splenocytes from different strains can establish baseline expression levels before proceeding with experimental interventions .

• Interpretation challenges: Results from CX5 antibody experiments may vary between laboratories using different mouse strains. Researchers should clearly report the mouse strain used and avoid direct comparisons of NKG2D-dependent functions between strains with known differences in receptor expression .

• Alternative approaches: For studies in strains with low NKG2D expression, alternative approaches such as genetic models or targeting other NK cell receptors may be more appropriate than using CX5 antibody blockade .

Understanding these strain-specific differences is essential for accurate experimental design and interpretation when using the CX5 antibody in immunological research.

What are the intersections between NKG2D biology and CXCR5+CD8+ T cell functions?

While the search results don't directly address the relationship between NKG2D (the target of CX5 antibody) and CXCR5+CD8+ T cells, several potential intersections can be hypothesized based on the provided information:

• Functional specialization: Both NKG2D-expressing NK cells and CXCR5+CD8+ T cells represent specialized lymphocyte populations with roles in immune regulation. NKG2D mediates cytotoxicity against stressed cells expressing induced ligands, while CXCR5+CD8+ T cells regulate antibody responses through B cell interactions .

• Cytotoxic mechanisms: NKG2D activates NK cell cytotoxicity against target cells, while CXCR5+CD8+ T cells can mediate cytotoxicity specifically against activated B cells. These parallel cytotoxic functions could potentially involve overlapping molecular mechanisms .

• IFN-γ production: Both NKG2D activation in NK cells and CXCR5+CD8+ T cell function involve IFN-γ production. The majority of alloprimed CD44+CXCR5+CD8+ T cells express IFN-γ, which is critical for their antibody-suppressive function .

• Potential co-expression: While not directly addressed in the search results, it is possible that CXCR5+CD8+ T cells might co-express NKG2D, particularly given that some activated CD8+ T cells are known to upregulate this receptor. This hypothesis would require direct experimental validation through co-staining for both markers.

Further research is needed to directly investigate potential functional and phenotypic overlaps between NKG2D-expressing cells and CXCR5+CD8+ T cells in immune responses.