CIITA Antibody

What is CIITA and why is it important in immunological research?

CIITA functions as the master regulator of Major Histocompatibility Complex Class II (MHC-II) gene expression, which is essential for the presentation of antigens to CD4+ T cells. This process is vital for the activation of adaptive immunity, as CD4+ T cells recognize and respond to pathogens. CIITA's function is particularly important in immune system development, as defects in CIITA can lead to severe immunodeficiencies, such as Bare Lymphocyte Syndrome, characterized by the absence of MHC class II molecules on the cell surface . Expression of CIITA is induced by interferon-gamma through JAK1 and Stat1 signaling pathways, highlighting its role in immune responses to infections .

What experimental applications are suitable for CIITA antibodies?

CIITA antibodies can be used in multiple experimental applications including:

• Western blotting (WB) - For detecting CIITA protein expression levels

• Immunoprecipitation (IP) - For isolating CIITA and associated protein complexes

• Immunofluorescence (IF) - For visualizing cellular localization of CIITA

• Immunohistochemistry (IHC) - For examining tissue distribution

When selecting antibodies for these applications, researchers should consider the specific reactivity (human, mouse, rat) and the availability of different conjugated forms (agarose, horseradish peroxidase, phycoerythrin, fluorescein isothiocyanate, and Alexa Fluor® conjugates) depending on the experimental design .

How should researchers validate CIITA antibody specificity for their experimental system?

Validation of CIITA antibody specificity should include:

• Positive controls - Using cell lines known to express CIITA (e.g., B cell lines like Raji)

• Negative controls - Using CIITA-deficient cell lines (e.g., RJ2.2.5, a CIITA-deficient mutant of Raji)

• Comparison of results across multiple detection methods

• Blocking experiments with the immunogenic peptide

• Analysis of band size in Western blots (CIITA is reported to be approximately 123.5 kilodaltons)

How can ChIP-chip approaches be optimized for genome-wide identification of CIITA target genes?

For researchers aiming to identify novel CIITA target genes using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip), multiple experimental designs should be considered:

• Comparison between wild-type and CIITA-deficient cells (e.g., Raji vs. RJ2.2.5)

• Comparison between cell states with differential CIITA expression (e.g., immature vs. mature dendritic cells)

• Comparison between CIITA-ChIP samples and input genomic DNA

• Use of multiple biological replicates to ensure reproducibility

When designing primers for validation via quantitative ChIP experiments, they should be targeted within the regions at which peaks were observed in the ChIP-chip experiments. This approach has previously identified nine new CIITA target genes, seven of which function in processes related to antigen presentation .

How should researchers address contradictory findings regarding CIITA's role in Th1/Th2 balance?

Contradictory findings exist regarding CIITA's role in T helper cell differentiation. While studies in CIITA-deficient mice suggested that CIITA suppresses Th2 cytokine production by CD4+ T cells, results from CIITA-transgenic mice showed that overexpression of CIITA in T cells led to enhanced IL-4 secretion and impaired Th1 polarization .

To address these contradictions, researchers should:

• Compare both in vitro and in vivo approaches to study Th differentiation

• Examine both gain-of-function (transgenic) and loss-of-function (knockout) models

• Utilize disease models that are dependent on either Th1 or Th2 responses (e.g., oxazolone-induced colitis as a Th2-mediated model)

• Measure multiple cytokines simultaneously (IL-4, IFN-γ)

• Consider contextual factors such as tissue environment and activation state

What are the recommended approaches for studying CIITA's interactions with the transcriptional machinery?

CIITA influences transcription through multiple protein-protein interactions. To study these interactions:

• DNA-dependent coimmunoprecipitation assay: This approach can demonstrate that CIITA recruitment depends on multiple, synergistic protein-protein interactions with DNA-bound factors constituting the MHC-II enhanceosome .

• Pull-down assay using immobilized promoter templates: This allows examination of protein complex formation on promoter DNA .

• In vitro phosphorylation assays: These can be used to study CIITA's effect on RNA Polymerase II phosphorylation, particularly by examining the phosphorylation status of Ser5 residues of the RNA Pol II C-terminal domain (CTD) .

How can researchers accurately measure changes in CIITA-dependent MHC-II expression?

Measuring CIITA-dependent MHC-II expression can be approached through several complementary methods:

• Flow cytometry: For quantifying surface MHC-II expression at the protein level

• RT-qPCR: For measuring MHC-II and CIITA mRNA levels

• Luciferase reporter assays: Using MHC-II promoter constructs to assess transcriptional activity

• ChIP assays: To measure CIITA occupancy at MHC-II promoters and correlate with expression levels

Data should be normalized appropriately using housekeeping genes or proteins, and statistical analysis should account for biological variability .

What technical considerations should be addressed when investigating CIITA's physical association with MHC-II promoters?

When investigating CIITA's physical association with promoters:

• Cross-linking conditions must be optimized for chromatin immunoprecipitation (ChIP) assays

• Primer design should target the S-Y enhancers of MHC-II genes where CIITA binding has been observed

• ChIP-chip experiments should employ multiple control strategies, including comparison with CIITA-deficient cells

• Test/control signal ratios typically range from 6 to over 30 for true CIITA binding sites

• The sensitivity of ChIP-chip approaches should be evaluated (false-negative rates around 5% have been reported for well-established target genes)

How can researchers differentiate between direct and indirect effects of CIITA on gene expression?

Differentiating between direct and indirect CIITA effects requires:

• Temporal analysis of target gene expression after CIITA induction

• ChIP experiments to confirm physical binding of CIITA to promoters

• Mutational analysis of promoter elements to identify CIITA-responsive regions

• Sequential ChIP (re-ChIP) to determine if CIITA is part of specific transcriptional complexes

• RNA-seq combined with ChIP-seq to correlate binding with expression changes

How can CIITA antibodies be used to investigate the role of CIITA in immune evasion by cancer cells?

CIITA plays a significant role in cancer immune evasion, particularly in primary mediastinal large B-cell lymphoma (PMLBCL) where genomic alterations in CIITA are frequent. Researchers investigating this connection should:

• Use CIITA antibodies to compare expression levels between normal and malignant cells

• Employ ChIP techniques to examine CIITA binding to MHC-II promoters in tumor samples

• Analyze the correlation between CIITA mutations/expression and MHC-II levels

• Investigate CIITA interaction with other pathways implicated in immune escape (JAK-STAT and NFκB)

• Consider using CIITA gene expression profiles as a pattern for identifying diffuse large B-cell lymphomas with PMLBCL characteristics

What are the best experimental approaches for studying CIITA's interaction with kinases and its effect on transcriptional regulation?

To investigate CIITA's interaction with kinases:

• Coimmunoprecipitation assays: Can demonstrate interaction between CIITA and kinases such as CDK7 and CDK9

• In vitro kinase assays: Using recombinant CIITA protein and immunoprecipitated Pol II holoenzyme complex to study phosphorylation

• Immunodepletion experiments: To examine the relative contribution of different kinases (CDK7 vs. CDK9)

• Western blot analysis: Using antibodies against phosphorylated Ser2 or Ser5 CTD to determine specific phosphorylation sites

• Bacterially expressed CTD: As a phosphorylation substrate for kinases like CDK7

How can transgenic and knockout mouse models be effectively used to study CIITA function in vivo?

Mouse models have provided valuable but sometimes contradictory insights into CIITA function. When using these models, researchers should:

• Consider models with tissue-specific CIITA expression or deletion (e.g., Dlck-CIITA Tg mice with CIITA expression restricted to peripheral T cells)

• Evaluate multiple disease models (e.g., EAE for Th1 responses, oxazolone-induced colitis for Th2 responses)

• Perform ex vivo analysis of isolated cell populations

• Compare results from both CIITA-deficient and CIITA-overexpressing models

• Analyze multiple immune parameters (cytokine production, cell surface molecule expression)

• Consider the potential for compensatory mechanisms in genetic models

What are common challenges in CIITA antibody-based experiments and how can they be overcome?

Common challenges include:

• Background staining: Optimize blocking conditions, antibody dilutions, and consider using IgG controls

• Low signal: Enhance detection with signal amplification methods or using more sensitive conjugates

• Non-specific bands in Western blots: Optimize lysate preparation, blocking conditions, and antibody concentration

• Variability in ChIP experiments: Standardize chromatin preparation, fragment size, and antibody amounts

• Cross-reactivity: Validate specificity using CIITA-deficient cells or knockdown approaches

How should researchers address experimental variability when studying CIITA-dependent gene regulation?

To address variability:

• Include appropriate positive and negative controls in each experiment

• Use multiple biological and technical replicates

• Standardize experimental conditions (cell density, passage number, treatment durations)

• Consider the influence of cell type and activation state on CIITA expression

• Employ multiple complementary techniques to confirm findings

• Normalize data to appropriate reference genes or proteins