Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment

Abstract

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide–MHCII complexes.

Increased Effector Memory Insulin-Specific CD4+ T Cells Correlate With Insulin Autoantibodies in Patients With Recent-Onset Type 1 Diabetes

“…Potential therapeutic approaches could include deletion of InsB-tetramer + –specific T cells using toxin-coupled tetramers, as previously reported for CD8 + T cells in mouse models of T1D ( 51 ), or modulation of the InsB-tetramer + T-cell specific response using monoclonal antibodies targeting InsB peptide in the context of MHCII. This approach has recently been shown to delay diabetes in NOD mice ( 10 , 52 ). Finally, antigen-specific tolerance is a third possibility to modulate the InsB-tetramer + T cells using peptide-pulsed nanoparticles or antigen-coupled cells, as previously described ( 53 , 54 ).…”

Adjuvant selection regulates gut migration and phenotypic diversity of antigen-specific CD4+ T cells following parenteral immunization

“…One day later, when it is known that antigen has drained fully into lymph nodes 30 , CLNs were harvested and single cell preparations were stained with the newly described W6 antibody that preferentially detects 2W1S:I-A b antigen presenting complexes. This allowed us to quantify and phenotype cells specifically presenting the 2W1S peptide 31 . We selected markers to account for the major DC subsets located in the dermis: CD11c + CD11b + ; CD11c + CD11b−; CD11c + CD103 + , and Langerhans cells (EpCam + DEC-205 + ) 32 – 34 .…”

“…This is the first direct evidence specifically demonstrating adjuvant choice guiding differential antigen presentation by distinctive DC subsets. This observation was achieved through the use of a recently described anti-peptide MHC II antibody, which can precisely allow visualization of specific peptide:MHCII complexes 31 . We also show epidermal Langerhans cells are preferentially engaged in the presence of dmLT compared to CpG.…”

Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

“…To identify four clones that produced anti-iglb12 idiotypes, we used multiplexed screening approaches to evaluate thousands of hybridomas. In future studies the screening burden can be further reduced by enrichment or sorting of B cells expressing BCRs with desirable binding properties before and/or after hybridoma fusion (Taylor et al, 2012; Spanier et al, 2016).…”