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Updated: Feb 9, 2026
Anti-Nuclear Antibody Screening Using HEp-2 Cells
Published on: June 23, 2014
Dying cells expose a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies
Philipp Metzger1, Sabrina V Kirchleitner1, Lars M Koenig1
1Center of Integrated Protein Science Munich (CIPSM) and Division of Clinical Pharmacology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany.
Abstract:
Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply.
Insights
Programmed death 1 (PD-1) antibodies are vital cancer therapies. This study reveals that PD-1 staining on non-T cells is likely due to cross-reacting nuclear antigens, not actual PD-1 expression.
Area of Science:
- Immunology
- Cancer Biology
- Cellular Biology
Background:
- Programmed death 1 (PD-1) is a key immune checkpoint molecule that regulates T cell activation and maintains immune homeostasis.
- PD-1-specific monoclonal antibodies have transformed cancer therapy by reversing T cell exhaustion and restoring anti-tumor immunity.
- Emerging evidence suggests PD-1 expression on non-T cells, including myeloid and tumor cells, with potential cell-intrinsic functions.
Purpose of the Study:
- To investigate the reported PD-1 expression on non-T cells in murine models.
- To determine if the observed staining on non-T cells represents genuine PD-1 receptor expression or a cross-reacting phenomenon.
- To clarify the mechanisms underlying PD-1 antibody interactions with various cell types.
Main Methods:
- Flow cytometry analysis of murine immune and tumor cells using multiple anti-PD-1 antibody clones.
- CRISPR/Cas9 genome editing to assess PD-1 gene function.
- Confocal imaging to visualize PD-1 localization within cells.
- Analysis of PD-1 expression in B16-F10-derived 3D cultures and ex vivo tumors.
Main Results:
- Positive PD-1 antibody staining was observed on various murine immune and tumor cells, particularly those with low forward scatter characteristics.
- This staining pattern was distinct from genuine PD-1 receptor expression on T cells.
- Evidence suggests the staining is attributed to a nuclear antigen that cross-reacts with anti-PD-1 antibodies, rather than PD-1 itself.
- Lack of PD-1 expression was confirmed in B16-F10-derived cultures and ex vivo tumors.
Conclusions:
- Non-T cell PD-1 expression, as detected by standard antibody staining, is unlikely to be due to the PD-1 receptor.
- The observed staining pattern is likely caused by cross-reactivity with a nuclear antigen.
- These findings suggest that alternative antibody-mediated pathways may be responsible for reported PD-1 functions in tumor cells, warranting further investigation.
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