Significant progress in understanding T cell signalling, particularly with respect to T cell co-receptors such as the co-stimulatory receptor CD28, has been made in recent years. This knowledge has been instrumental in the development of innovative immunotherapies for patients with cancer, including immune checkpoint blockade antibodies, adoptive cell therapies, tumor-targeted immunostimulatory antibodies, and immunostimulatory small-molecule drugs that regulate T cell activation. Following the failed clinical trial of a CD28 superagonist antibody in 2006, targeted CD28 agonism has recently re-emerged as a technologically viable and clinically promising strategy for cancer immunotherapy. In this keynote address, Dr. Olejniczak will provide foundational background for understanding the roles of CD28 co-stimulation in cancer immunotherapy, highlighting the contributions of his lab to this growing field of study. Seminal work linking CD28 to the clinical success of immune checkpoint inhibitor and CAR T cell therapies will be discussed in the context of co-receptor signaling paradigms, as will future prospects for leveraging CD28 co-receptor signaling to improve cancer immunothrapy. 

Learning Objectives:

1. Gain a basic understanding of how CD28 regulates T cell responses.

2. Understand how CD28 co-stimulation contributes to the success of cancer immunotherapy.

3. Appreciate the opportunities and challenges of leveraging CD28 signaling for cancer immunotherapy. 

The success of PD-1 blockade is predicated on the fact that a critical end-effector of anti-tumor immunity is the T cell. We and others have demonstrated the role of CD4+ (helper) and CD8+ (effector) T cells targeting epitopes derived from tumor somatic mutations (neoantigens) in response to anti-PD-1. Additionally, many groups have examined intratumoral characteristics of T cells at the single cell level in human cancers, but these studies suffer from a fundamental limitation: they cannot distinguish true tumor-specific T cells from T cells with irrelevant antigen specificity. In this presentation, I will demonstrate how our lab is developing innovative methods to better detect and study tumor-reactive TIL in the context of resectable lung cancers, and how we have discovered clinically meaningful interactions between tumor-reactive effector T cells and highly suppressive Treg in human tumors treated with neoadjuvant PD-1 blockade. 

Learning Objectives:

1. Understand the heterogeneity of tumor infiltrating Treg as a function of immunotherapy response

2. Discuss therapeutically relevant biology that we are able to discover by querying Treg at single cell resolution

3. Demonstrate how we can apply a mix of ex vivo, in vitro, in vivo, and in silico approaches to discover new therapeutic targets

Aging promotes immune dysfunction which results in suboptimal responses to vaccines, increased susceptibility to infections, and increased cancer risk due to compromised immunosurveillance. Despite these well-established aging-induced immune defects, the effect of aging on the efficacy of immunotherapies remains controversial. In a published study, we have demonstrated that aging-associated T-cell dysfunction in murine and human T-cells could be rejuvenated using recombinant Interleukin-37 (rIL-37) treatment. Furthermore, this beneficial effect extended to aged human CAR T-cells, engineered from healthy donors over the age of 70, where we observed superior protection in murine models of B-cell acute lymphoblastic leukemia.. Based on these results, we designed and patented armored-IL-37-secreting CAR T-cells, which we posited would improve the function of aged CAR T-cells, while also having the added benefit of attenuating aging-associated chronic inflammation. In this presentation, we present data supporting this concept using predominantly aged human T-cells engineered from human donors and older patients with chronic lymphocytic leukemia. In these studies, we demonstrate that IL-37-expressing aged CAR T-cells are, 1) more cytotoxic against malignant B-cells, 2) less exhausted, 3) more stem cell/memory like phenotype, 4) express a more “youthful” T-cell transcriptome, and 5) perform significantly better in pre-clinical models of B-cell malignancies. In conclusion, our patented IL-37-armored CAR constructs potently rejuvenates the function of aged CAR T-cells and increased their anti-tumor capacity.

Learning Objectives:

1. explain the anti-inflammatory role of IL-37.

2. interpret how aging promotes immune dysfunction.

3. review how IL-37 impacts the function of aged T-cells.

In pharmaceutical research and development as well as basic scientific research, recombinant proteins serve not only as essential research tools but also as potential therapeutic agents or key factors. However, as an inherent component of the Gram-negative bacterial cell wall, endotoxin (lipopolysaccharide/LPS) frequently acts as an "invisible contaminant" in protein products, leading to severe experimental interference. Even trace amounts of residual endotoxin are sufficient to activate immune cells and trigger non-specific inflammatory responses, ultimately resulting in "false-positive" or irreproducible experimental data. To prevent adverse effect induced by endotoxins, food and drug administrations and health regulatory agencies worldwide have established strict threshold standards to ensure the safety of injectable drugs, medical devices, and other products that come into contact with the human body.

This talk will delve into the potential risks associated with endotoxin contamination in immunological and cellular assays, and further introduce Sino Biological’s ultra-low endotoxin protein solutions designed to meet the most stringent experimental demands.

Learning Objectives:

1. Describe the sources and properties of endotoxins.

2. Analyze the risks of endotoxin contamination in experiments.

3. Discuss industry standards and ultra-low endotoxin solutions.

As translational immunology moves toward increasingly complex and clinically driven questions, flow cytometry must deliver both speed and analytical depth. This presentation highlights how the combination of high-speed and spectral flow cytometry meets these evolving demands and supports more efficient immune profiling in real-world research environments. High-speed acquisition allows to process large, complex sample sets within practical timeframes, while spectral flow cytometry enhances multiparametric resolution and experimental flexibility—particularly valuable when working with limited, heterogeneous clinical material.

Through two applied case studies, this talk emphasizes how these technologies impact everyday laboratory work. An example from HIV research will illustrate the importance of rapid, high-dimensional profiling for preserving sample integrity and extracting actionable insights. A second example from cancer immunology will show how this approach enables robust analysis of challenging tumor-derived samples. Together, these use cases demonstrate how combining speed and spectral capabilities not only advances translational discovery but also improves workflow efficiency and confidence in experimental outcomes.

Learning Objectives: 

1. Explain how pairing rapid acquisition with spectral detection increases analytical depth, supports multiparametric immune profiling, and addresses constraints of limited, heterogeneous clinical samples in translational studies. 

2. Describe workflow strategies that leverage high-speed spectral cytometry in HIV research to minimize processing delays, maintain data quality, and translate complex phenotyping into decision-relevant outputs. 

3. Outline panel design, compensation/unmixing, and QC approaches that enable reliable characterization of diverse cell populations in cancer immunology, improving efficiency, reproducibility, and confidence in experimental outcomes.

Despite ongoing success of immunotherapy approaches, including CAR T therapy and immune checkpoint blockade therapies, in many cancers, application of these successful strategies for cancers that affect childhood and the adolescents & young adults (AYA) age group have been largely disappointing. In particular, progress in the effective management of relapsed, recurrent or metastatic sarcoma, including Osteosarcoma (OS) and Rhabdomyosarcoma (RMS), have been stagnant. New therapeutic development and additional insights into understanding the immune-hostile sarcoma tumor microenvironment are desperately needed. We have recently demonstrated antitumor activities of a small molecular inhibitor targeting  TGF-beta signaling in preclinical OS models that has led to an ongoing Phase I/II clinical trial (NCT05588648). In addition, we have also interrogated molecular and cellular mechanisms driving immune-related abscopal effects in mouse models of RMS using cryoablation, a technique commonly used in clinical management of bulky solid tumors with inconsistent clinical outcome. This presentation will present these preclinical evidence and insights gained from such exploration and offer potential future clinical translational impact in the management of these devastating diseases.

Learning Objectives:

1. Discuss challenges associated with pediatric and young adult sarcoma management

2. Demonstrate potential impact of direct targeting of an immune suppressive factor in sarcoma therapy

3. Clarify relative contributions of innate sensor activation in sarcoma TME in response to cryotherapy

Immunotherapy (IO) has transformed treatment of Head and Neck Squamous Cell Carcinoma (HNSCC), yet many patients do not benefit. While scRNAseq has advanced our understanding of tumor, stromal, and immune cell states, the spatial organization of the tumor microenvironment is poorly defined. We hypothesize that a distinct cellular neighborhood at the tumor margin contributes to immune exclusion and IO resistance. To investigate, we performed spatial transcriptomics (ST) on a tumor microarray (TMA) of 20 treatment-naïve HNSCC patients using Brüker’s CosMx platform. We integrated an HNSCC scRNAseq atlas with the ST data to enable robust cell identification. Neighborhoods were identified by performing clustering on a cell adjacency matrix. We identified 6 recurrent spatially organized neighborhoods. Neighborhood 5 (N5) represented the tumor core and contained more exhausted CD8+ T cells versus other neighborhoods. Neighborhood 3 (N3) at the tumor-stromal interface displayed immunosuppressive signatures (e.g., TGFβ signaling) and extracellular matrix remodeling, suggesting a role in immune exclusion. Cox regression analysis showed that a high enrichment of a N3 signature correlated with worse outcomes following IO. Exhausted CD8+ T cells were enriched in N5, suggesting that adjacent N3 may restrict access to tumor antigens and limit response to IO. To explore neighborhood function, we developed 3D spheroids with HNSCC tumor cells, fibroblasts, endothelial cells, and peripheral blood immune cells to model immune infiltration in vitro. This spheroid platform enables mechanistic exploration of immune exclusion and allows for identification of strategies to enhance IO response. In short, ST revealed novel immune exclusionary features that may be targeted to improve antitumor immunity. 

Learning Objectives:

1. Discuss the strengths and weaknesses of scRNA-seq vs spatial transciptomics 

2. Summarize the knowledge that can be gained from spatial transcriptomics data

3. Explore a novel cellular neighborhood which may limit anti-cancer immune response in HNSCC

Spatial transcriptomics is revolutionizing the study of tissue architecture, cellular states, and tumor-immune interactions in clinical specimens. This presentation introduces the principles and workflows of spatial single-cell transcriptomics using NanoString’s GeoMx Digital Spatial Profiler and CosMx Spatial Molecular Imaging platforms. The talk contrasts pseudo-spatial region-of-interest profiling with true single-cell, in situ measurement, then follows the analytical pipeline from assay design and field-of-view selection through segmentation, cell typing, and downstream spatial analyses. Using a non-small cell lung cancer case study, the session illustrates how spatial data can identify cell types, define cellular neighborhoods and tumor microenvironment niches, and quantify tumor-immune ligand-receptor interactions, including PD-L1/PD-1, across samples. Particular emphasis is placed on cell segmentation as the primary determinant of data quality and on the computational scale of image-rich datasets. The session concludes by highlighting the translational and clinical relevance of spatial transcriptomics for tumor immune phenotyping and the potential assessment of immunotherapy response.

Learning Objectives:

1. Compare the operating principles, spatial resolution, and analytical outputs of GeoMx DSP and CosMx SMI for spatial transcriptomic profiling.

2.  Explain how cell segmentation, cell typing, neighborhood and niche analysis, and ligand-receptor modeling influence the interpretation of spatial single-cell data in a case study using data from non-small cell lung cancer (NSCLC)

3. Describe how spatial transcriptomics can support tumor immune phenotyping and the translational assessment of immunotherapy response in clinical research.

The 5-year survival rate for localized breast cancer (BC) is >99%, due in large part to established screening and surveillance criteria. While this success is undeniable, millions of women survive BC and spend their post-treatment years enduring fatigue that negatively affects their quality of life. The goal of our laboratory has been to develop supportive therapies to alleviate breast cancer-associated muscle fatigue which are complimentary to standard-of-care therapies.  The central research hypothesis that has been driving this goal is that early stage breast cancer is associated with a clinically relevant phenotype of muscle fatigue in the absence of muscle wasting (i.e., cachexia).  Data from our laboratory in the breast cancer patient-derived orthotopic xenograft (BC-PDOX) mouse model has supported this hypothesis.  Specifically, muscles from these mice fatigue at a greater rate during a repeated stimulation protocol, while muscle mass and tumor-free body mass are not different from tumor naive mice.  In addition, bulk RNA-sequencing profiles of muscles from BC-PDOX mice are strikingly similar to the transcriptional profile of skeletal muscles from patients with early stage breast cancer.  Therefore, this model has been used for preclinical studies aimed at restoring dysfunctional molecular patterns within skeletal muscle which are associated with a fatigable phenotype.  However, our recent data analyzing muscle from patients with early stage breast cancer, using spatial transcriptomics and muscle morphological assessments, has caused our laboratory to reconsider the central research hypothesis.  This seminar will include unpublished data to highlight molecular and morphological changes of muscle in early-stage breast cancer and our thoughts on strategies for treating this phenotype clinically. 

Learning Objectives:

1. Interpret the changes in muscle morphology that occur in weight-stable patients with early stable breast cancer.

2. Understand the underlying molecular adaptations that occur in skeletal muscles of patients with early stage breast cancer.

3. Determine practical therapeutic interventions aimed at alleviating muscle fatigue in patients with early stage breast cancer.

The five-year survival rate for ovarian cancer patients remains below 50%, underscoring the need for innovative therapies. One promising approach involves engineering T cells to specifically target proteins uniquely overexpressed in tumors to control tumor growth without toxicity to healthy tissues. Mesothelin (Msln) contributes to the malignant and invasive phenotype in ovarian cancer and has limited expression in healthy cells, making it a candidate immunotherapy target. Our previous results in a mouse model of ovarian cancer demonstrated that T cells engineered to express a T cell receptor (TCR) targeting MSLN (TCRMsln) mediated therapeutic activity, delaying tumor growth and prolonging mouse survival. However, inhibitory ligands expressed in the tumor microenvironment (TME) interacted with inhibitory receptors on activated T cells, suppressing antitumor function. We hypothesized combining engineered T cells with checkpoint blockade would enhance T cell function in the TME and improve therapeutic efficacy, but administration of mono-specific antibodies targeting individual inhibitory pathways had no significant impact on T cell efficacy. By contrast, the combination of PD-1, Tim-3 and Lag-3 blockade with engineered T cells significantly improved T cell function and overall animal survival relative to treatment with antibody alone, engineered T cells alone, or engineered T cells with singlet or doublet antibody combinations. Single-cell RNA sequencing revealed TCRMsln T cells treated with the triplet antibody combination increased expression of genes involved in interferon responses and metabolic function, and reduced expression of genes associated with exhaustion. These results suggest that strategies to disrupt multiple inhibitory pathways simultaneously may be necessary for improved adoptive T cell therapy efficacy in patients.

Learning Objectives:

1. Describe a TCR-T cell therapy strategy with the ability to control advanced stage ovarian cancer growth in a murine model. 

2. Summarize mechanisms of immune suppression in ovarian tumors.

3. Discuss opportunities to enhance the efficacy of engineered T cell therapies.

"Cold" tumor microenvironments (TME) lack tumor infiltrating cytotoxic T cells (CTLs) against tumor-associated antigens (TAAs and TSAs) and show reduced responsiveness to cancer immuno-, chemo- and radiotherapies. Our group developed "polarized" dendritic cells (DC1) matured in the conditions of acute viral infections (dsRNA, type-1 and type-2 interferons) to selectively induce effector-type immunity (tumor-specific CTLs  and Th1 cells) without enhancing suppressiveTreg responses.  DC1s polarization enhances their ability to cross-resent TAAs and induce NK cell receptor (NKR)-expressing CTLs which recognize cancer cells with enhanced sensitivity and selectivity while sparing healthy cells.
Our complementary platform, the “chemokine-modulating” regimen (CKM) composed of TLR3-ligand and IFN-a selectively targets endogenous cDC1s and other myeloid cells in cancer lesions (which, in contrast to healthy tissues show elevated levels of active NFkB), allowing its systemic application to promote selective CTL infiltration into multiple “cold” tumors. Our pilot trial NCT03403634 demonstrated the safety and immunologic efficacy of systemic (i.v.) CKM composed of IFN-a and selective TLR3-ligand (rintatolimod/Ampligen) in patients with metastatic triple-negative breast cancer (TNBC). We observed its very good tolerability, average of 10.3-fold increases in intratumoral CTL markers and preliminary indications of potential clinical activity (Gandhi et al. JITC 2023;11:e007381). Analogous immunologic efficacy was observed on study NCT04081389 (neoadjuvant CKM plus chemotherapy in primary TNBC), with 7 of 9 patients displaying prognostically-favorable RCB0/RCB1 at tumor resection, associated with lack of recurrence (Gandhi et al. JITC 2024;12: e010058). Our upcoming trials will test combinations of CKM with immune checkpoint inhibitors (ICI), guided by our latest mouse data showing strong synergy between CKM and  PD1 blockade, when both components are applied at the same time (Kokolus et al. JITC 2025;13: e012307).

Learning Objectives:

1. Summarize key elements of tumor immunology and tupes of immune therapies

2. Discuss the factors limiting the effectiveness of cancer therapies

3. Discuss the emergingl approaches to overcome cancer resistance to immunotherapies and chemo-immunotherapies

TLS are often linked to enhanced immunotherapy response and increased overall survival in most solid tumors. However, cues that drive differences in TLS formation and function are not clearly understood. Here, we spatially interrogated TLS in treatment naïve non-small cell lung cancer (NSCLC) patients to delineate distinct functional states. 

We combined multispectral imaging and spatial transcriptomics (GeoMx® whole transcriptomic atlas) to comprehensively profile the tumor microenvironment (TME) in 70 NSCLC patients. 
We identified TLS (herein referred to as TLS type I state) with a distinct transcriptional signature which also correlated with improved prognosis. Importantly, we observed a trajectory of chemokine expression that heightened in the TLS type I state, indicative of TLS development in the TME. Furthermore, the TLS type I state had enhanced B cell receptor signaling pathways that coincided with high expression of CD37 and CD52. CD37 and CD52 were highly abundant in some TLS while lacking in others. Of note, expression of CD37 or CD52 in TLS correlated with enhanced TLS activity, measured by Ki67 expression within the TLS. Patients with a high density of CD37+ or CD52+ TLS or both had a higher probability of survival.

Our work utilizes spatial omics to delineate transcriptional differences that govern TLS function. We describe key pathways that are associated with TLS functional states, highlighting transcriptional changes in TLS state. We identified and validated CD37 and CD52 as important biomarkers for functional TLS in NSCLC. Our findings shed light on key developmental changes governing TLS function that could be used to modulate TLS in solid tumors for improved immunotherapy response.
 

Learning Objectives:

1. Describe how spatial multi-omics approaches (multispectral imaging and spatial transcriptomics) can be used to characterize tertiary lymphoid structures (TLS) in the NSCLC tumor microenvironment.

2. Identify transcriptional and signaling features that distinguish the TLS type I functional state from other TLS states in non-small cell lung cancer.

3. Interpret the prognostic significance of CD37 and CD52 expression within TLS and their relationship to TLS activity and patient survival.

T cell engagers are an established class of immunotherapies with proven survival benefit across multiple hematologic malignancies and certain solid tumors, including small cell lung cancer and uveal melanoma. With multiple phase III trials now underway across cancer types, these agents are rapidly reshaping the treatment landscape in oncology. The learning objectives of this session will be to review the mechanism of action, describe toxicities, and evaluate mechanisms of resistance of T cell engagers. The presentation will apply lessons learned from hematologic malignancies and from past and ongoing trials in solid tumors to inform the continued development of these agents across oncology.

Learning Objectives:

1. review the mechanisms of action of T cell engagers 

2. describe toxicities of T cell engagers 

3. evaluate mechanisms of resistance of T cell engagers