A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents

Reed, Brendan; Chopp, Laura B.; Malo, Courtney S.; Renner, Danielle N.; Keulen, Virginia S. Van; Girtman, Megan A.; Nevala, Wendy; Pavelko, Kevin D.; Gil, Diana; Schrum, Adam G.; Johnson, Aaron J.; Pease, Larry R.

doi:10.1371/journal.pone.0137984

Cited by 6 publications

(8 citation statements)

References 19 publications

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“…Given the laborious nature of using cell samples to validate tetramer performance, we aimed to develop an easy and robust assay that could be used before, or within every experiment. For this, we expanded upon previously developed bead-based assays (226; 132). For this, eComp Ultra plus compensation beads were incubated with monoclonal antibodies specific for SARS-CoV-2 RBD, positive control antibodies specific for APC, or negative control antibodies specific for PE (Fig.…”

Section: Resultsmentioning

confidence: 99%

Validation of ligand tetramers for the detection of antigen-specific lymphocytes

Fitzpatrick¹,

Poljakov²,

Bibby³

et al. 2022

Preprint

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The study of antigen-specific lymphocytes has been a key advancement in immunology over the past few decades. One innovation allowing for this type of research was the development of multimerized probes containing antigens, peptide:MHC complexes, or other ligands that can be used to study antigen-specific lymphocytes by flow cytometry. While these types of studies are common and performed by thousands of laboratories, quality control and assessment of probe quality is often minimal. In fact, many of these types of probes are made in-house and protocols vary between labs. While peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for antigen multimers. To ensure high quality and consistency with antigen probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind antibodies specific for the antigen of interest. Using this assay, we have sensitively assessed the performance of antigen tetramers and find considerable batch-to-batch variability in performance and stability over time. This assay can also reveal common production errors such as miscalculation of antigen concentration. Unexpectedly, probes including the fluorochrome allophycocyanin exhibited more rapid performance decline compared to probes including the fluorochrome R-phycoerythrin when both were stored at 4 degrees C. This performance decline was reduced for most, but not all, batches when antigen tetramers were instead stored at -20 degrees C in 50% glycerol. This work could set the stage for the development of standardized assays for all commonly used antigens to limit lab-to-lab technical variation, and experimental failure due to tetramer underperformance.

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Section: Resultsmentioning

confidence: 99%

Validation of ligand tetramers for the detection of antigen-specific lymphocytes

Fitzpatrick¹,

Poljakov²,

Bibby³

et al. 2022

Preprint

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“…Isolated brain lymphocytes were washed twice with fluorescence‐activated cell sorting (FACS) buffer and incubated with the following peptide‐MHC tetramer conjugated to APC for 40 min: D b : VP2 121–130 (FHAGSLLVFM) followed by a 20‐min incubation with Abs against CD8α (PE‐Cy7; 561097), CD4 (PerCP for 1 wk postinfection analysis and BV650 for 6‐wk postinfection analysis; BioLegend, San Diego, CA, USA), and CD45 (PE for 1 wk postinfection analysis and PerCP for 6 wk postinfection analysis; BD Biosciences, San Jose, CA, USA). The H‐2D b MHC class I tetramer D b : VP2 121–130 was constructed as previously described (32, 33). Cells were then washed twice with FACS buffer and fixed in 4% paraformaldehyde (PFA) and 1× PBS.…”

Section: Methodsmentioning

confidence: 99%

Brain atrophy in picornavirus‐infected FVB mice is dependent on the H‐2D^bclass I molecule

Kelcher

Atanga²,

Gamez³

et al. 2017

FASEB j.

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Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the D class I molecule. FVB/D and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/D mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/D mice elicited a strong CD8 T-cell response toward the immunodominant D-restricted TMEV-derived peptide, VP2, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2D class I molecule.

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“…For CNS-infiltrating immune cells, brains were homogenized and digested with collagenase type IV (Worthington Biochemical #9001-12-1) or manual dounce homogenization (Thomas Scientific, #7722–15) and centrifuged against a percoll density gradient at 7846g for 30 minutes to isolate immune cells. Peptide:MHC tetramers were constructed as previously described [ 31 , 32 ]. Antibodies against CD45, CD8α, and CD44 at a 1:100 dilution (BD Biosciences, #557235, #561097, #553133), as well as ghost dye to assess viability at a 1:1000 dilution (Tonbo Biosciences, #13-0871-T500) were used for staining.…”

Section: Methodsmentioning

confidence: 99%

The Effect of Vector Silencing during Picornavirus Vaccination against Experimental Melanoma and Glioma

et al. 2016

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Virus vector-based vaccination against tumor-specific antigens remains a promising therapeutic approach to overcome the immune suppressive tumor microenvironment. However, the extent that the desired CD8 T cell response against the targeted tumor antigen is impacted by the CD8 T cell response against the virus vector is unclear. To address this question, we used picornavirus vaccination with Theiler’s murine encephalomyelitis virus (TMEV) as our vector against tumor-expressed ovalbumin (OVA257-264) antigen in both the B16-OVA murine melanoma and GL261-quad cassette murine glioma models. Prior to vaccination, we employed vector silencing to inhibit the CD8 T cell response against the immunodominant TMEV antigen, VP2121-130. We then monitored the resulting effect on the CD8 T cell response against the targeted tumor-specific antigen, ovalbumin. We demonstrate that employing vector silencing in the context of B16-OVA melanoma does not reduce tumor burden or improve survival, while TMEV-OVA vaccination without vector silencing controls tumor burden. Meanwhile, employing vector silencing during picornavirus vaccination against the GL261-quad cassette glioma resulted in a lower frequency of tumor antigen-specific CD8 T cells. The results of this study are relevant to antigen-specific immunotherapy, in that the virus vector-specific CD8 T cell response is not competing with tumor antigen-specific CD8 T cells. Furthermore, vector silencing may have the adverse consequence of reducing the tumor antigen-specific CD8 T cell response, as demonstrated by our findings in the GL261-quad cassette model.

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A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents

Cited by 6 publications

References 19 publications

Validation of ligand tetramers for the detection of antigen-specific lymphocytes

Validation of ligand tetramers for the detection of antigen-specific lymphocytes

Brain atrophy in picornavirus‐infected FVB mice is dependent on the H‐2D^bclass I molecule

The Effect of Vector Silencing during Picornavirus Vaccination against Experimental Melanoma and Glioma

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A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents

Cited by 6 publications

References 19 publications

Validation of ligand tetramers for the detection of antigen-specific lymphocytes

Validation of ligand tetramers for the detection of antigen-specific lymphocytes

Brain atrophy in picornavirus‐infected FVB mice is dependent on the H‐2Dbclass I molecule

The Effect of Vector Silencing during Picornavirus Vaccination against Experimental Melanoma and Glioma

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Brain atrophy in picornavirus‐infected FVB mice is dependent on the H‐2D^bclass I molecule