OMAP-17 was developed for IBEX (Iterative Bleaching Extends multi-pleXity) imaging of fixed frozen human pediatric
thymus tissue samples (Radtke et al. 2022; 2020). The panel spans 9 imaging cycles and includes 43 primary antibodies,
6 secondary antibodies, and Hoechst nuclear dye for image alignment and segmentation. It provides spatial context for
7 anatomical structures and over 30 cell types and developmental states (e.g., dividing, immature, precommitted) from
the ASCT+B thymus table v1.4 (Brusko, Beuschel, and Radtke 2023). This is the first OMAP developed specifically for
the thymus and was designed to detect rare thymic epithelial cell (TEC) populations such as myo-TECs and neuro-TECs,
informed by scRNA-seq and CITE-seq studies. OMAP-17 shares markers with other lymphoid tissue OMAPs, including OMAP-1
(lymph node), OMAP-10 (tonsil), and OMAP-11 (spleen). However, it does not include markers for plasma cells or
ionocyte/tuft TECs. CD1a, a key thymocyte marker, was omitted due to steric hindrance during multi-cycle labeling.
Some antibodies were custom conjugated in-house or by suppliers (e.g., Alexa Fluor 532, iFluor 594). Three antibodies
used in this OMAP have since been discontinued: KRT10 (NBP2-54402PE), LY75 (NBP2-75467), and Z25303 from Thermo
Fisher. This panel contributed to the Human Cell Atlas spatial thymus atlas project. Imaging data can be explored on
the Image Data Resource (https://idr.openmicroscopy.org, project-3051), and detailed protocols and examples are
available on the IBEX Knowledge-Base (Radtke et al. 2025; Yaniv et al. 2025). Further information on image analysis,
integration, and the thymus coordinate framework is described in Yayon et al. (2024).

Bibliography:

* Brusko, Maigan, Rebecca T. Beuschel, and Andrea J. Radtke. 2023. “Thymus (v1.4) Graph Data, v1.4.” https://lod.humanatlas.io/asct-b/thymus/v1.4/.
* Image Data Resource. 2025. “Webclient for Image Data Resource.” https://idr.openmicroscopy.org/webclient/?show=project-3051.
* Müller, Werner, Silvia Rüberg, and Andreas Bosio. 2023. “10-Palatine-Tonsil-Mics (v1.0) Graph Data, v1.0.” https://lod.humanatlas.io/omap/10-palatine-tonsil-mics/v1.0/.
* Radtke, Andrea J. 2023a. “1-Human-Lymph-Node-Ibex (v1.3) Graph Data, v1.3.” https://lod.humanatlas.io/omap/1-human-lymph-node-ibex/v1.3/.
* Radtke, Andrea J. 2023b. “11-Spleen-Ibex (v1.0) Graph Data, v1.0.” https://lod.humanatlas.io/omap/11-spleen-ibex/v1.0/.
* Radtke, Andrea J., et al. 2022. “IBEX: An Iterative Immunolabeling and Chemical Bleaching Method for High-Content Imaging of Diverse Tissues.” Nature Protocols 17 (2): 378–401. https://doi.org/10.1038/s41596-021-00644-9.
* Radtke, Andrea J., et al. 2020. “IBEX: A Versatile Multiplex Optical Imaging Approach for Deep Phenotyping and Spatial Analysis of Cells in Complex Tissues.” PNAS 117 (52): 33455–65. https://doi.org/10.1073/pnas.2018488117.
* Yayon, Nadav, et al. 2024. “A Spatial Human Thymus Cell Atlas Mapped to a Continuous Tissue Axis.” Nature 635 (8039): 708–18. https://doi.org/10.1038/s41586-024-07944-6.
* Radtke, Andrea J., et al. 2025. “The IBEX Knowledge-Base: A Central Resource for Multiplexed Imaging Techniques.” PLoS Biol 23(3): e3003070. https://doi.org/10.1371/journal.pbio.3003070.
* Yaniv, Ziv, et al. 2025. “The IBEX Imaging Knowledge-Base: A Community Resource Enabling Adoption and Development of Immunofluorescence Imaging Methods.” eLife 14:RP105737.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre–a T cell receptor (pre-TCRa) expression. Low circulating naive ab T cell counts at birth persisted over time, with normal memory ab and high gd T cell counts. Their TCRa repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue ab T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive ab T cell counts but high gd T cell counts. Although residual pre-TCRa expression drove the differentiation of more ab T cells, autoimmune conditions were more frequent in these patients compared with the general population.

OMAP-17 was designed for IBEX (Iterative Bleaching Extends multi-pleXity) imaging of fixed frozen human pediatric
thymus samples (https://doi.org/10.1038/s41596-921-00644-9; https://doi.org/10.1073/pnas.2018488117). The panel
consists of 9 cycles of 43 primary antibodies, 6 secondary antibodies, and the nuclear label Hoechst for image
alignment and nuclear segmentation. This OMAP provides a spatial context for 7 anatomical structures and more than 30
cell types and cell states (dividing, immature, precommitted) present in the ASCT+B thymus table
(https://doi.org/10.48539/HBM668.QPSF.678). This panel was specifically designed to phenotype unique, and often rare,
populations of thymic epithelial cells (TECs) of the medulla, e.g., myo-TECs and neuro-TECs. Marker selection was
guided by companion studies using advanced sequencing methods (scRNA-seq and CITE-seq) as well as expert review.
OMAP-17 is the first OMAP for the human thymus but targets several stromal and immune cell types included in OMAPs for
other lymphoid organs. OMAP-1 (lymph node; https://doi.org/10.48539/HBM458.NSWR.957), OMAP-11 (spleen;
https://doi.org/10.48539/HBM957.RHVB.545), and OMAP-10 (tonsil; (https://doi.org/10.48539/HBM663.JJMJ.628). This OMAP
does not contain markers for phenotyping plasma cells or ionocytes/tuft TECs. CD1a, a critical marker for developing
thymocytes, was removed from OMAP-17 due to steric hindrance of T cell markers in later cycles. Several antibodies
required custom conjugation by commercial suppliers or antibody conjugation kits (e.g., AF532 and iF594 conjugates).
This work is included in a spatial thymus atlas constructed by the Human Cell Atlas. Additional details on image
analysis, data integration, and the establishment of a common coordinate framework for the thymus can be found in the
accompanying paper (https://doi.org/10.1101/2023.10.25.562925). Note that three (3) of the antibodies have been
discontinued since this OMAP-17 was created: Novus Biologicals (catalog #:NBP2-54402PE) KRT10, Novus Biologicals
(catalog #: NBP2-75467) LY75 and this secondary antibody Thermo Fisher Scientific Z25303 AF532 conjugate.