13-5888-82

antibody from Invitrogen Antibodies

Targeting: PDCD1LG2 B7-DC, bA574F11.2, Btdc, CD273, PD-L2, PDL2

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

13-5888-82

Provider

Invitrogen Antibodies

Product name

CD273 (B7-DC) Monoclonal Antibody (MIH18), Biotin, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: The MIH18 monoclonal antibody reacts with human B7-DC, also known as PD-L2. B7-DC, a recently identified member of the B7 family, has a predicted molecular weight of approximately 25 kDa and belongs to the Ig superfamily. B7-DC is primarily expressed by subpopulations of dendritic cells and monocytes/macrophages. Although B7-DC has structural and sequence similarities to the B7 family, it does not bind CD28/CTLA-4, rather it is a ligand for PD-1. The interactions between PD-1 and B7-DC/PD-L2 have been reported to be involved in costimulation or suppression of T cell proliferation depending on state of cellular activation. MIH18 is a useful tool for studies to elucidate the exact function of B7-DC/PD-L2 in APC/T cell interaction and to characterize the expression pattern of this molecule in humans. MIH18 is a blocking antibody.

Conjugate

Biotin

Antibody clone number

MIH18

Concentration

0.5 mg/mL

References

Submitted references

TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2.

Baruah P, Bullenkamp J, Wilson POG, Lee M, Kaski JC, Dumitriu IE
Frontiers in immunology 2019;10:1644

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Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways.

Ren T, Zheng B, Huang Y, Wang S, Bao X, Liu K, Guo W
Cell death & disease 2019 Mar 18;10(4):261

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PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.

Zheng B, Ren T, Huang Y, Sun K, Wang S, Bao X, Liu K, Guo W
Journal of hematology & oncology 2018 Feb 6;11(1):16

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PD-1 is a regulator of NY-ESO-1-specific CD8+ T cell expansion in melanoma patients.

Fourcade J, Kudela P, Sun Z, Shen H, Land SR, Lenzner D, Guillaume P, Luescher IF, Sander C, Ferrone S, Kirkwood JM, Zarour HM
Journal of immunology (Baltimore, Md. : 1950) 2009 May 1;182(9):5240-9

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Staining of in vitro derived human dendritic cells with Anti-Human CD209 (DC-SIGN) APC (Product # 17-2099-42) and 0.125 µg of Mouse IgG1 kappa Isotype Control Biotin (Product # 13-4714-85) (left) or 0.125 µg of Anti-Human CD273 (B7-DC) Biotin (right) followed by Streptavidin PE (Product # 12-4317-87). Total viable cells were used for analysis.

Conjugate

Biotin

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 2 Effect of PD-L2 expression on the proliferation of osteosarcoma cells. a PD-L2 expression in osteosarcoma cell lines was evaluated by western blotting and real-time PCR. b The osteosarcoma cell lines (red) exhibited differing degrees of PD-L2 expression compared with the isotype control (black) by flow cytometry. c KHOS and U2OS cells were stably transfected with shPD-L2 or shNC lentivirus, following evaluation of PD-L2 expression by western blotting and real-time PCR. d Cell proliferation of KHOS and U2OS cells in response to PD-L2 knockdown determined by cell colony formation assay. e Cell viability of KHOS and U2OS cells after PD-L2 knockdown assayed by CCK-8. All experiments were repeated three times. Data are presented as the mean +- SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 HPV-positive HNSCCs increase PD-L1 and PD-L2 on fibroblasts. PD-L1 and PD-L2 expression on primary BJ human fibroblasts, HPV-positive (SCC154), and HPV-negative (SCC099) HNSCC cell lines (HNSCCs) was detected by flow cytometry. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts (black histograms), HPV-positive (red histograms), or HPV-negative (blue histograms) HNSCCs (A) . Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 5) in fibroblasts, HPV-positive, and HPV-negative HNSCCs (B) . Fibroblasts were cultured alone or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs. Fibroblasts were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 ( C ; red histograms) or HPV-negative SCC099 ( D ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 5) on fibroblasts cultured alone (w/o) or co-cultured directly with HNSCC cells (E) . HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with fibroblasts. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with fibroblasts ( F ; red histograms). Graphs show PD-L1 and PD-

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 PD-L1 and PD-L2 expression in co-cultures of macrophages and HPV-positive or HPV-negative HNSCCs. Macrophages were cultured alone (w/o) or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCC cell lines (HNSCCs). Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone ( A ; black histograms). Macrophages were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 ( B ; red histograms) or HPV-negative SCC099 ( C ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 4) on macrophages cultured alone (w/o) or co-cultured directly with HNSCCs (D) . HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with macrophages. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with macrophages ( E ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 3) on HPV-positive SCC154 cultured alone or co-cultured directly with macrophages (F) . Illustrative histograms show PD-L1 and PD-L2 expression on HPV-negative SCC099 cultured alone (black histograms) or co-cultured directly with macrophages ( G ; blue hist

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 5 Conditioned medium from HPV-positive HNSCCs up-regulates PD-L1 and PD-L2 on fibroblasts. Fibroblasts were cultured alone or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured with conditioned medium from HPV-positive SCC154 ( A ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 13) on fibroblasts cultured alone (w/o), co-cultured directly with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM) (B) . HPV-positive (SCC154) HNSCCs were cultured alone or co-cultured in direct contact with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured with conditioned medium from fibroblasts ( C ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 4) on HPV-positive SCC154 cultured alone (w/o), co-cultured directly with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM) (D) . Macrophages were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 3) on macrophages for the in

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 6 Blockade of IFN-gamma, TNF-alpha, or CD81 does not affect PD-L1 and PD-L2 up-regulation by HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) as indicated. Graphs (A) show IFN-gamma and TNF-alpha levels in culture supernatants (mean +- SEM; n = 4). The dashed red line indicates the lowest value (15.6 pg/ml) of the dynamic range for the ELISA assays used. Neutralizing antibodies anti-IFN-gamma (B,C) , anti-TNF-alpha (D,E) , or anti-CD81 (F,G) were added to the cultures as indicated. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 or with conditioned medium from HPV-positive SCC154 alone (red histograms) or in the presence of blocking antibodies (green histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 3) on fibroblasts for the indicated treatments. ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 7 The TLR9 antagonists ODN TTAGGG and chloroquine inhibit PD-1 ligands up-regulation on fibroblasts co-cultured with HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) in the presence or absence of the TLR9 antagonists ODN TTAGGG (ODN) or chloroquine (CHQ). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 (red histograms) or co-cultured directly with HPV-positive SCC154 in the presence of ODN (A) or CHQ (H) (green histograms). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), cultured with conditioned medium from HPV-positive SCC154 (red histograms) or with conditioned medium from HPV-positive SCC154 in the presence of ODN (B) or CHQ (I) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 6) on fibroblasts for the indicated treatments (C,J) . Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or in the presence of ODN (D) or CHQ (K) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 4) on fibroblasts for the indicated treatments (E,L) . HPV-positive SCC154 were cultured alone or co-cultured in direct contact (direct) with fibroblasts in the presence

Conjugate

Biotin