MHC CLASS II, I-E Antibody

What is the specificity profile of the commonly used MHC Class II antibody clone M5/114.15.2?

The M5/114.15.2 monoclonal antibody recognizes polymorphic determinants shared by mouse I-Ab/d/q and I-Ed/k MHC class II alloantigens. This antibody specifically detects epitopes on APCs from mice carrying the H-2b, H-2d, H-2q, H-2p, H-2r, and H-2u haplotypes, but not from mice carrying the H-2s or H-2f haplotypes . The antibody binds to both I-A and I-E subregion-encoded glycoproteins (I-Ab, I-Ad, I-Aq, I-Ed, I-Ek, but not I-Af, I-Ak, or I-As) and is commonly used to identify antigen-presenting cells including B cells, monocytes, macrophages, dendritic cells, and activated T lymphocytes .

How does cross-linking MHC Class II molecules with antibodies affect antigen presentation function?

Cross-linking MHC Class II molecules with antibodies serves as a surrogate for T-cell engagement and promotes several functional changes in antigen-presenting cells:

• It induces clustering of MHC-II present in lipid raft membrane microdomains

• This clustering leads to MHC-II endocytosis and degradation in lysosomes

• The degradation affects both immunologically relevant and irrelevant MHC-II molecules

• Functionally, antibody cross-linking (using F(ab')2 fragments) significantly inhibits the ability of dendritic cells to stimulate antigen-specific T cells

• Mock cross-linking (without F(ab')2) does not affect T cell proliferation, confirming specificity

This mechanism serves as an important immune regulation pathway, attenuating additional activation of naïve CD4 T cells after initial T cell engagement .

How do different pathogens modulate MHC Class II expression, and what experimental approaches can detect these changes?

Pathogens have evolved various mechanisms to modulate MHC Class II expression as immune evasion strategies:

Experimental approaches to study these changes include:

• Flow cytometry quantification of surface and intracellular MHC-II expression

• Western blotting for total protein levels

• qRT-PCR for gene expression analysis

• Cell-specific knockout models to analyze resistance mechanisms

• Confocal microscopy to visualize trafficking patterns

How can I effectively design experiments to study MHC Class II engagement with T cells?

When studying MHC Class II-T cell interactions, consider these experimental designs:

• Antibody cross-linking studies:

  • Use both anti-I-A and anti-I-E antibodies separately to determine isotype-specific effects

  • Compare cross-linking (F(ab')2) with non-cross-linking conditions

  • Measure downstream effects on T cell proliferation using tritiated thymidine assays

• Alloreactive T cell assays:

  • Use purified CD4 T lymphocytes (1×10^5 to 1.25×10^4 cells/well)

  • Stimulate with T cell-depleted splenocytes (3.1×10^4 to 50×10^4 cells/well)

  • Include blocking antibodies against I-A, I-E, or both

  • Quantify IL-2 producing cells by ELISPOT

• Antigen-specific T cell activation:

  • Pretreat antigen-pulsed DCs with anti-MHC-II antibodies

  • Test their ability to stimulate antigen-specific T cells

  • Compare the effects of antibody binding vs. cross-linking

  • Include time-course analysis to track MHC-II downregulation kinetics

What controls should be included when investigating MHC Class II down-regulation mechanisms?

Essential controls for MHC Class II down-regulation studies include:

How can I determine if altered MHC Class II expression stems from defects in the antigen-processing pathway versus direct regulation?

This requires a systematic approach examining multiple components of the MHC-II pathway:

• Assess CLIP-MHC-II complexes: High levels indicate peptide-loading defects. Use anti-CLIP/MHC-II antibodies in flow cytometry to measure CLIP occupancy on MHC-II molecules .

• Evaluate HLA-DM and HLA-DO levels: These molecules facilitate CLIP release and peptide loading.

  • Measure protein levels through flow cytometry

  • Analyze gene expression through qRT-PCR

  • In human samples, DM-deficient cells show increased MHCII-CLIP despite normal total MHC-II levels

• Examine transcriptional regulation:

  • Analyze MHC Class II, DM, and DO mRNA levels

  • Assess CIITA (MHC-II transactivator) expression and function

  • Perform ChIP assays to measure promoter activity

• Traffic tracking experiments:

  • Use fluorescent antibodies to track MHC-II internalization

  • Monitor co-localization with endosomal/lysosomal markers

  • Compare surface vs. intracellular expression ratios

A case study from Type 1 diabetes research showed that despite similar MHC-II levels between patients and controls, patients had significantly higher MHCII-CLIP levels in all APC subsets, with B cells showing a 3.4-fold increase, indicating a peptide-loading defect rather than altered expression .

What mechanisms explain the isotypic shifts in MHC Class II restriction observed in certain experimental systems?

Research has revealed intriguing isotype-dependent mechanisms in MHC Class II antigen presentation:

• DM-dependent peptide loading differences:

  • CD4 T cell responses in wild-type mice are primarily restricted to I-A molecules

  • DM-deficient mice show a shift toward I-E-restricted responses

  • This suggests differential DM dependency between I-A and I-E

• Peptide repertoire differences:

  • I-A and I-E molecules have distinct peptide binding preferences

  • In the absence of DM-mediated editing, a different repertoire of peptides binds to I-E

  • This can be tested by blocking I-A vs. I-E in T cell stimulation assays

• Differential regulation mechanisms:

  • I-A and I-E molecules may be differentially affected by certain stimuli

  • For example, T. gondii infection specifically reduces I-A/I-E+ cells without affecting H-2D expression

  • The specificity for I-A/I-E vs. H-2D suggests targeted regulation rather than global MHC effects

• T cell development influences:

  • Thymic selection may generate different repertoires of I-A vs. I-E restricted T cells

  • This creates inherent biases in the peripheral T cell pool

  • Can be assessed using alloreactive T cell assays with isotype-specific blocking

How do aberrant self-antigens complexed with MHC Class II molecules contribute to autoimmunity?

Recent research has revealed critical insights into how MHC Class II-self-antigen complexes contribute to autoimmunity:

• Altered antigenic properties:

  • Self-antigens complexed with MHC Class II molecules can exhibit properties different from normal self-antigens

  • These altered complexes can abrogate self-tolerance mechanisms

  • Self-antigens complexed with MHC class II molecules of autoimmune disease risk alleles are major autoantibody targets in several autoimmune diseases

• Disease-specific associations:

  • In Graves' disease, specific HLA class II alleles (like HLA-DPB1*05:01 in Japanese populations) are strongly associated with disease risk

  • Aberrant HLA class II expression is observed on thyroid follicular cells in patients

  • These factors contribute to the presentation of thyroid self-antigens in an immunogenic manner

• Misfolded self-antigens:

  • Research suggests that misfolded self-antigens transported by MHC class II molecules have antigenic properties that differ from normal self-antigens

  • This misfolding-related presentation may be a key mechanism in breaking self-tolerance

• Experimental evidence:

  • Autoantibody binding to self-antigens complexed with MHC class II molecules correlates with disease risk in rheumatoid arthritis and ANCA-associated vasculitis

  • This indicates these complexes may be causally involved in autoimmune pathogenesis

How can I address inconsistent results when using anti-MHC Class II antibodies across different experimental platforms?

When facing inconsistent results with anti-MHC Class II antibodies, consider these approaches:

• Clone-specific considerations:

  • Verify the exact epitope specificity of your antibody clone

  • M5/114.15.2 doesn't recognize I-Af, I-Ak, or I-As - confirm your test cells express compatible haplotypes

  • Use alternative validated clones if needed

• Protocol optimization by platform:

  Platform	Critical Parameters	Troubleshooting Approach
  Flow cytometry	Cell viability, Fc blocking	Titrate antibody, optimize incubation conditions (4°C, 30 min)
  Western blotting	Denaturation conditions	Consider non-reducing conditions for conformational epitopes
  IHC	Fixation, antigen retrieval	Test multiple fixatives, optimize retrieval methods
  Functional assays	Cross-linking conditions	Compare F(ab')2 vs. whole antibody effects

• Post-collection analysis:

  • For flow cytometry, calculate MFI by subtracting isotype control values

  • Use correct compensation for multicolor panels

  • When analyzing rare populations, collect sufficient events

  • Consider the activation state of cells, as this affects MHC-II expression levels

• Antibody storage and handling:

  • Store at recommended temperature (typically 2-8°C)

  • Avoid repeated freeze-thaw cycles

  • Check for precipitates and filter if necessary

  • Verify antibody functionality using positive control samples

What factors might explain contradictory findings regarding MHC Class II expression changes in disease models?

Several factors can explain contradictory findings about MHC Class II expression in disease models:

• Temporal dynamics: Expression changes follow specific kinetics

  • In T. gondii infection, reduction begins approximately 20 hours post-infection

  • Different sampling timepoints can yield opposing results

• Cell type-specific effects:

  • In Type 1 diabetes, B cells showed a 3.4-fold increase in MHCII-CLIP levels

  • Different APC subsets may show variable regulation patterns

• Pathway component differences:

  • Total MHC-II levels may remain stable while CLIP occupancy changes

  • HLA-DM and HLA-DO levels influence peptide loading but may be independently regulated

• Methodological variations:

  • Surface vs. intracellular staining captures different populations

  • Antibody clones recognize distinct epitopes that may be differentially affected

  • Flow cytometry vs. microscopy vs. Western blot can yield different results

• Disease heterogeneity:

  • In Type 1 diabetes, patients with residual C-peptide showed different gene expression profiles than those without

  • Patient subgroups may have distinct pathophysiological mechanisms

• Stimulation protocols:

  • IFN-γ doses and timing significantly impact MHC-II upregulation

  • Some pathogens specifically impair IFN-γ-induced upregulation while others affect basal expression

How can I verify the specificity and functionality of anti-MHC Class II antibodies before using them in critical experiments?

To ensure antibody specificity and functionality before key experiments:

• Haplotype validation:

  • Verify your antibody's haplotype recognition pattern

  • For M5/114.15.2, test on cells from H-2b/d/q (positive) and H-2s/f (negative) backgrounds

• Functional assays:

  • Test antibody's ability to block antigen presentation in T cell proliferation assays

  • Confirm cross-linking effects on MHC-II down-regulation

  • Verify ability to deplete specific cell populations (if being used for this purpose)

• Antibody quality assessment:

  • Check purity (should be ≥95% by SDS-PAGE)

  • Verify endotoxin levels (<1.0 EU/mg for in vivo use)

  • Examine for aggregates or precipitation

• Control experiments:

  • Always include isotype controls at matched concentrations

  • Use cells known to be negative for your antibody's target as specificity controls

  • Include positive controls from validated sources