Recombinant anti-mouse CD8 antibody

Recombinant murinized CD8α antibody for in vivo use

InvivoGen’s engineered Anti-mCD8-mIgG2a InvivoFit™ antibody

Anti-mCD8-mIgG2a InvivoFit™ is a murinized anti-mouse monoclonal antibody (mAb) featuring the variable region of the previously described anti-mCD8 YTS 169.4 clone [1]. Using recombinant technology, the original YTS 169.4 rat IgG2b constant region has been replaced with a murine IgG2a format which mediates potent cytotoxic functions [2].

CD8 is a transmembrane protein primarily expressed on most thymocytes and highly expressed by the peripheral mature CD8+ T cell population [3, 4].  Two isoforms CD8α (Lyt-2) and CD8β (Lyt-3) are usually co-expressed and form a CD8α/β heterodimer that plays a critical role in CD8+ T cell development and activation. Other immune cells, such as Natural Killer cells and subsets of dendritic cells also express CD8α [5]. 

The anti-mCD8 YTS 169.4 mAb is commonly used for in vivo depletion of the CD8+ T cell population to study its role in various immune responses, including in a viral or tumoral context [6-8]. Depending on the nature of the experiment, extended treatment schedules (up to several months) may be required. Upon repeated injection of a xenogeneic mAb, mice produce anti–species antibodies, causing the removal of the administered mAb from circulation, thereby reducing its in vivo efficacy. Moreover, this immunogenicity can lead to fatal hypersensitivity reactions [9-10] which can be reduced by mAb murinization [11].
Anti-mCD8-mIgG2a is provided in an InvivoFit™ grade, a high-quality standard specifically adapted to in vivo studies.
 

Key features of Anti-mCD8-mIgG2a InvivoFit™:

• Derives from the YTS 169.4 clone, rat IgG2b, κ
• Features the mIgG2a isotype (constant region)
• Filter-sterilized (0.2 µm), endotoxin level < 1 EU/mg
• Suitable for parental delivery in mice (azide-free)
• Low aggregation < 5%
• Produced in animal-free facilities and defined media

Anti-mCD8-mIgG2a InvivoFit™ is produced in Chinese hamster ovary (CHO) cells and purified by affinity chromatography with protein A. The specific binding of this mAb to cell surface mCD8α and its in vivo depleting function have been confirmed (see Figures).

References:

1. Cobbold S.P. et al., 1984. Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature. 312:548-551.
2. Nimmerjahn F. & Ravetch J.V., 2005. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science. 310(5753):1510-2.
3. Fung-Leung W.P. et al., 1991. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell. 65:443-449.
4. Janeway C.A. jr., 1992. The T Cell Receptor as a Multicomponent Signalling Machine: CD4/CD8 Coreceptors and CD45 in T Cell Activation. Annual Rev Immunol. 10:645-674.
5. Laky K. & Kruisbeek AM., 2016. In vivo depletion of T lymphocytes. Current Protocols Immunology. 4.1.1-4.1.9. doi: 10.1002/0471142735.im0401s113.
6. Greczemiel U. et al., 2020. LCMV-specific CD4 T cell-dependent polyclonal B-cell activation upon persistent viral infection is short lived and extrafollicular. Eur J Immunol. 50:396-403.
7. Menares E. et al., 2019. Tissue-resident memory CD8(+) T cells amplify anti-tumor immunity by triggering antigen spreading through dendritic cells. Nat Comm. 10:4401.
8. Vincent-Fabert C. et al., 2019. Reproducing indolent B-cell lymphoma transformation with T-cell immunosuppression in LMP1/CD40-expressing mice. Cell Mol Immunol 16:412–414.
9. Mall C. et al., 2016. Repeated PD-1/PD-L1 monoclonal antibody administration induces fatal xenogenic hypersensitivity reactions in a murine model of breast cancer. Onco Immunol. 5(2):e1075114.
10. Murphy, J.T. et al., 2014. Anaphylaxis caused by repetitive doses of a GITR agonist monoclonal antibody in mice. Blood. 123(14):2172-2180.
11. Belmar N.A. et al., 2017. Murinization and H chain isotype matching of Anti-GITR antibody DTA-1 reduces immunogenicity-mediated anaphylaxis in C57BL/6 mice. J Immunol. 198:4502-4512.

Figures

Cell surface staining of murine CD8 using Anti-mCD8-mIgG2a InvivoFit™ mAb.
~5x105 EL4 (parental) or EL4-mCD8 cells were incubated with 2 μg of Anti‑mCD8‑mIgG2a InvivoFit™ mAb or an isotype control for 1h at 4°C. Cells were then washed and incubated with 0.25 μg of goat anti-mouse κ-light chain coupled to PE for 1h at 4°C. Cell surface staining was analyzed by flow cytometry.

Analysis of CD8+ T cell population in mice peripheral blood upon injection of Anti-mCD8-mIgG2a InvivoFit™ mAb.
Blood samples (20 μl) from SWR/J mice aged 10 weeks were harvested 1 week prior to a series of intraperitoneal (IP) injections with the depleting Anti‑mCD8‑mIgG2a InvivoFit™ mAb (200 μg) or NaCl (control), and 24h after each IP injection. IP injections were performed at day 0, +3, and +7 (arrows). Thereafter, blood samples were harvested once a week. To assess the kinetics of the in vivo depletion of CD8+ T cells, peripheral blood was stained with the following conjugated antibodies: Anti-CD45-Pe-Cy7 (clone 30-F11), Anti-CD3e-PE (clone 145-2C11), Anti-CD4-FITC (clone RMA-4), and Anti-CD8-APC (clone H35-17.2) according to standard procedures.
Cell surface staining was analyzed by flow cytometry. Data are represented as the frequency of CD8+ T cells among the total CD3+ T cell population.

Specifications

Specificity: Targets cells expressing murine CD8α

Formulation: Lyophilized from 0.2 μm filtered solution in 150 mM sodium chloride, 20 mM sodium phosphate buffer with 5% saccharose

Clonality: Monoclonal antibody

Isotype: Murine IgG2a, kappa

Control: mIgG2a  InvivoFit™ isotype control

Source: CHO cells

Purity: Purified by affinity chromatography with protein A

Tested application: Flow cytometry; in vivo depletion

Quality control:

• The binding of Anti-mCD8-mIgG2a InvivoFit™ to mCD8α has been confirmed using Flow cytometry
• Mouse CD8+ T cell in vivo depletion using Anti-mCD8-mIgG2a InvivoFit™ has been confirmed
• The complete sequence of the antibody construct has been verified
• < 5% aggregates (confirmed by size exclusion chromatography)
• Endotoxin level <1 EU/mg (determined by the LAL assay)

Contents

Anti-mCD8-mIgG2a InvivoFit™ is filter-sterilized (0.2 µm), endotoxin-free, azide-free, and lyophilized.  

This product is available in two pack sizes:

• mcd8-mab10-1​: 1 mg
• mcd8-mab10-10​: 10 mg

 The product is shipped at room temperature.

 Store lyophilized antibody at -20 °C.

 Lyophilized product is stable for at least 1 year

 Avoid repeated freeze-thaw cycles.

InvivoFit™

InvivoFit™ is a high-quality standard specifically adapted for in vivo studies. InvivoFit™ products are filter-sterilized (0.2 µm) and filled under strict aseptic conditions in a clean room. The level of bacterial contaminants (endotoxins and lipoproteins) in each lot is verified using a LAL assay and a TLR2 and TLR4 reporter assay.

Details

CD8 background:

The cluster of differentiation 8 (CD8) receptor (formerly named T8) is a 34 kDa transmembrane protein primarily expressed on most thymocytes, and highly expressed by the peripheral mature CD8+ T cell population [1, 2].  Two isoforms CD8α (Lyt-2) and CD8β (Lyt-3) are usually co-expressed and form a CD8α/β heterodimer that plays a critical role in CD8+ T cell development and activation. CD8α is mandatory for the development of MHC class I-restricted cytotoxic T cells (CTLs) [1]. CTLs recognize and eliminate infected cells and tumor cells upon the interaction between the TCR on the T cell and the MHC-peptide complex on the antigen-presenting cell [2-4]. Upon antigen recognition, the proximity association of CD8 and the TCR/CD3 complex on T cells triggers downstream intracellular signaling and participates in the cytotoxic T cell activation [1, 2]. Of note, CD8α/α homodimers have been shown to promote the survival and differentiation of activated CD8α/β T cells into memory CD8+ T cells [5]. Other immune cells, such as Natural Killer cells and subsets of dendritic cells also express CD8α [6]. 

References:

1. Fung-Leung W.P. et al., 1991. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell. 65:443-449.
2. Janeway C.A. jr., 1992. The T Cell Receptor as a Multicomponent Signalling Machine: CD4/CD8 Coreceptors and CD45 in T Cell Activation. Annual Rev Immunol. 10:645-674.
3. Norment A.M. et al., 1988. Cell-cell adhesion mediated by CD8 and MHC class I molecules. Nature. 336:79.
4. Shore D.A. et al., 2008. The Crystal Structure of CD8 in Complex with YTS156.7.7 Fab and Interaction with Other CD8 Antibodies Define the Binding Mode of CD8 αβ to MHC Class I. J Mol Biol. 384:1190-1202.
5. Madakamutil L. et al., 2004. CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors. Science. 304:590-593.
6. Laky K. & Kruisbeek AM., 2016. In vivo depletion of T lymphocytes. Current Protocols Immunology. 4.1.1-4.1.9. doi: 10.1002/0471142735.im0401s113.