Jyh Liang Hor

The iterative bleaching extends multiplexity (IBEX) Knowledge-Base is a central portal for researchers adopting IBEX and related 2D and 3D immunofluorescence imaging methods. The design of the Knowledge-Base is modeled after efforts in the open-source software community and includes three facets: a development platform (GitHub), static website, and service for data archiving. The Knowledge-Base facilitates the practice of open science throughout the research life cycle by providing validation data for recommended and non-recommended reagents, such as primary and secondary antibodies. In addition to reporting negative data, the Knowledge-Base empowers method adoption and evolution by providing a venue for sharing protocols, videos, datasets, software, and publications. A dedicated discussion forum fosters a sense of community among researchers while addressing questions not covered in published manuscripts. Together, scientists from around the world are advancing scientific discovery at a faster pace, reducing wasted time and effort, and instilling greater confidence in the resulting data.

The transition from trainee to independent investigator is one of the most challenging and formative phases of a scientific career. It requires not only scientific expertise but also the skills to lead, mentor, manage, and communicate effectively. The Arthur and Sandra Irving Cancer Immunology Symposium serves as a platform for established investigators to mentor trainees and early-career faculty as they navigate this transition to independence. Through sharing personal experiences and lessons from their own careers, senior leaders provide guidance on the scientific, professional, and personal challenges that shape a successful career in cancer immunology—emphasizing how curiosity, persistence, and a translational mindset can make a lasting real-world impact. This commentary highlights key themes, including leadership, communication, recruitment, and fundraising. Altogether, these insightful thoughts provide a framework for the next generation of cancer immunologists as they establish their independent careers as future leaders in the field.

Stem-like progenitors are self-renewing cytotoxic T cells that expand as effector cells during successful checkpoint immunotherapy ^1,2 . Emerging evidence suggests that tumour-draining lymph nodes support the continuous generation of these stem-like cells that replenish tumour sites and are a key source of expanded effector populations ^3–6 , underlining the importance of understanding what factors promote and maintain activated T cells in the stem-like state. Here, using advanced three-dimensional multiplex immunofluorescence imaging, we identify antigen-presentation niches in tumour-draining lymph nodes that support the expansion, maintenance and affinity evolution of TCF-1 ⁺ PD-1 ⁺ SLAMF6 ^high stem-like CD8 ⁺ T cells. Contrary to the prevailing view that persistent T cell receptor (TCR) signalling drives terminal effector differentiation, prolonged antigen engagement days beyond initial priming sustains the proliferation and self-renewal of these stem-like T cells in vivo. The inhibitory PD-1 pathway has a central role in this process through fine-tuning the TCR signal input that enables the selective expansion of high-affinity TCR stem-like clones as a renewable source of effector cells. PD-1 blockade disrupts this tuning, leading to terminal differentiation or death of the most avid anti-tumour stem-like cells. Our results therefore reveal a relationship between TCR ligand affinity recognition, a key negative-feedback regulatory loop and T cell stemness programming. Furthermore, these findings raise questions about whether anti-PD-1 blockade during cancer immunotherapy provides a short-term anti-tumour effect at the cost of diminishing efficacy due to progressive loss of these critical high-affinity precursors.

With the ever-increasing complexity of microscopy modalities, it is imperative to have computational workflows that allow researchers to process and perform in-depth quantitative analysis of the resulting images. However, workflows that enable flexible, interactive and intuitive analysis from raw images to analysed data are lacking. We present Cecelia, a toolbox that integrates various open-source packages into a coherent data management suite to make quantitative image analysis accessible for non-specialists.

Stem-like progenitors are a critical subset of cytotoxic T cells that self-renew and give rise to expanded populations of effector cells critical for successful checkpoint blockade immunotherapy. Emerging evidence suggests that the tumor-draining lymph nodes can support the continuous generation of these stem-like cells that replenish the tumor sites and act as a critical source of expanded effector populations, underlining the importance of understanding what factors promote and maintain activated T cells in the stem-like state. Using advanced 3D multiplex immunofluorescence imaging, here we identified antigen-presentation niches in tumor-draining lymph nodes that support the expansion, maintenance, and affinity evolution of a unique population of TCF-1+PD-1+SLAMF6 ^hi stem-like CD8+ T cells. Our results show that contrary to the prevailing view that persistent TCR signaling drives terminal effector differentiation, prolonged antigen engagement well beyond the initial priming phase sustained the proliferation and self-renewal of these stem-like T cells in vivo . The inhibitory PD-1 pathway plays a central role in this process by mediating the fine-tuning of TCR and co-stimulatory signal input that enables selective expansion of high affinity TCR stem-like clones, enabling them to act as a renewable source of high affinity effector cells. PD-1 checkpoint blockade disrupts this fine tuning of input signaling, leading to terminal differentiation to the effector state or death of the most avid anti-tumor stem-like cells. Our results thus reveal an unexpected relationship between TCR ligand affinity recognition, a key negative feedback regulatory loop, and T cell stemness programming. Furthermore, these findings raise questions about whether anti-PD-1 checkpoint blockade during cancer immunotherapy provides a short-term anti-tumor effect that comes at the cost of diminishing efficacy due to progressive loss of these critical high affinity stem-like precursors.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.

A central question in immunology is what features allow the immune system to respond in a timely manner to a variety of pathogens encountered at unanticipated times and diverse body sites. Two decades of advanced and static dynamic imaging methods have now revealed several major principles facilitating host defense. Suborgan spatial prepositioning of distinct cells promotes time‐efficient interactions upon pathogen sensing. Such pre‐organization also provides an effective barrier to movement of pathogens from parenchymal tissues into the blood circulation. Various molecular mechanisms maintain effective intercellular communication among otherwise rapidly moving cells. These and related discoveries have benefited from recent increases in the number of parameters that can be measured simultaneously in a single tissue section and the extension of such multiplex analyses to 3D tissue volumes. The application of new computational methods to such imaging data has provided a quantitative, in vivo context for cell trafficking and signaling pathways traditionally explored in vitro or with dissociated cell preparations. Here, we summarize our efforts to devise and employ diverse imaging tools to probe immune system organization and function, concluding with a commentary on future developments, which we believe will reveal even more about how the immune system operates in health and disease.