Lancaster, Jessica N.

Abstract Introduction Germinal center (GC) formation in lymph node (LN) follicles is essential for generating high-affinity antibodies following infection or vaccination. This response in aging is impaired, resulting in smaller and fewer GCs that generate less abundant antibodies with reduced antigen affinities as compared to young adults. Early GC formation depends on dendritic cell (DC)-mediated activation of naïve CD4+ T cells, which differentiate into CD4+ T follicular helper (TFH) cells that drive B cell responses. Antigen-specific TFH induction is mediated by type 2 conventional DCs, which localize to the T:B cell interfollicular boundaries. Live-cell 2-photon microscopy has previously revealed that T cell priming by DCs occurs in distinct phases of lasting versus intermittent interactions, though the quality and duration of DC:CD4+ T cell interactions has not been characterized in aged hosts. We hypothesize that age-associated alterations in LN microenvironments disrupt these interactions, leading to impaired GC formation. Methods Young and aged mice were subcutaneously immunized in the flank with nitrophenyl hapten conjugated to ovalbumin in alum adjuvant, and naïve OT-II CD4+ T cells were adoptively transferred via retroorbital injection. 48 hours after immunization, draining LNs and naïve controls were analyzed by multiplex immunofluorescence microscopy to map the spatial organization of DCs and stromal elements. Results Structural mapping showed disrupted alignment of stromal networks, suggesting impaired spatial compartmentalization of the aged microenvironment. Moreover, within aged immunized mice the localization of DCs within the interfollicular region and T:B cell zone borders of the LNs were altered. Conclusion Our findings suggest age-associated disruption of the LN microarchitecture diminishes the quality of DC:CD4+ T cell interactions essential for initiating GCs. These insights highlight tissue microenvironmental remodeling as a key contributor to age-related immune decline. Funding Source NIH Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract In human melanoma, tumor cell-specific MHC class II expression is associated with improved response to programmed death 1 and programmed death-ligand 1 blockade; yet the direct effect of melanoma cell-specific MHC class II expression on anti-melanoma T cell responses remains largely unknown. In the clinically relevant Yale University Mouse Melanoma Exposed to Radiation (YUMMER) cell lines, MHC class II expression was IFN-γ-inducible in a subset of cells. Transduction of melanoma cells with CIITA resulted in uniformly high, constitutive expression of MHC class II. YUMMER.G cells transduced with CIITA vector had increased in vivo tumor growth in wild-type mice compared to melanoma cells transduced with empty vector. YUMMER.G cells transduced with empty or CIITA vectors had the same in vivo tumor growth in RAG−/− mice, indicating that the difference in tumor growth between CIITA- and empty-transduced cells was dependent on an intact adaptive immune system. CIITA-transduced YUMMER.G tumors exhibited an increased frequency and infiltration depth of CD4+ T cells, an increased frequency of T regulatory cells, and a decreased frequency and infiltration depth of CD8+ T cells, compared to empty-transduced tumors. Antibody depletion revealed that CIITA- and empty-transduced YUMMER.G tumors were constrained by CD4+ and CD8+ T cells, but only tumor growth of CIITA-transduced cells was decreased by CD25 blockade, which primarily blocks T regulatory cell function. Overall, this work reveals a potential pro-tumorigenic role of melanoma cell-specific overexpression of MHC class II.

Abstract Macrophages in the tumor microenvironment are known to upregulate PD-L1 expression, thereby suppressing T cells through PD-1 ligation. However, the manner in which PD-L1 expression intrinsically impacts macrophages and their immunomodulatory phenotype is less clear. Clarifying this knowledge gap would yield insight into the mechanisms of immunosuppression within the tumor microenvironment. To characterize the macrophage intrinsic role of PD-L1, we used complementary genetic and pharmacological approaches by analyzing primary murine bone marrow-derived macrophages (BMDMs) with complete genetic PD-L1 deletion and wildtype BMDMs treated with anti-PD-L1 blocking antibodies. Macrophages were evaluated across naïve, pro-inflammatory (M1), and tumor conditioned (TCM) polarization states in vitro. Unlike prior reports, neither genetic deletion nor antibody blockade dramatically altered the expression of macrophage polarization markers or in vitro phagocytic capacity. Both conditions consistently reduced surface levels of the M1-associated costimulatory molecule CD80, prompting further analysis of T cell interacting and antigen presenting proteins, in which we revealed disparate effects of genetic deletion and antibody blockade on the surface levels of MHCI, MHCII, PD-1, and PD-L2. These findings suggest that PD-L1 deletion and antibody-mediated blockade contribute to macrophage immune regulatory profiles in distinct manners. This difference supports a model in which PD-L1 functions in macrophages beyond its canonical role as a ligand for PD-1, influencing antigen presentation and checkpoint molecule levels and playing a broader role in immune regulation in the tumor microenvironment.

The major barrier to clinical translation of αCD137 immunotherapy is separating antitumor efficacy from hepatotoxicity driven by IFN-γ–producing CD8 T cells. We propose a strategy to limit toxicity by promoting contraction of excessively expanded CD8 T cells. We identify CD11c + KLRG1 + effector CD8 T cells (CD11c + T E ) as the primary source of IFN-γ, recirculating between blood and secondary lymphoid organs (SLOs), where they undergo apoptosis during contraction. We show that lymphotoxin β receptor (LTβR) signaling restrains this contraction. Mechanistically, lymphotoxin-expressing B cells activate LTβR in fibroblastic reticular cells (FRCs), suppressing apoptosis of CD11c + T E cells in the spleen and sustaining their systemic circulation and liver infiltration. Pharmacological LTβR blockade abrogates hepatotoxicity by reducing the accumulation of IFN-γ–producing CD11c + T E cells while preserving tumor-specific CD8 T cell responses. These findings identify LTβR as a key regulator of effector CD8 T cell persistence and support the use of LTβR antagonists to improve the safety of αCD137-based immunotherapy.

Abstract Introduction: The prostate cancer (PCa) tumor microenvironment (TME) is immunosuppressive and resistant to immune checkpoint blockade (ICB). Based on multi-omic single cell correlatives of response to neoadjuvant Fc-enhanced (FcE) αCTLA-4 in high-risk localized PCa (NCT04301414), we explored how modulating the balance between regulatory T cells (Tregs) and dendritic cells (DCs) in the PCa TME affects sensitivity to ICB. Methods: We leveraged three single cell transcriptomic atlases of PCa to assess how Tregs and DC frequencies are dynamically regulated during PCa progression. To investigate responses to Treg depletion (via FcE-αCTLA-4) alongside DC expansion (via Flt3L-Ig) and stimulation (via STING agonist ADU-S100) in PCa, we employed the castration-sensitive MycCaP PCa model and 45-parameter spectral flow cytometry. To investigate novel direct mechanisms of DC stimulation by FcE-αCTLA-4 via Fcγ receptor (FcγR) engagement, we optimized reductionist in vitro assays involving bone marrow-derived DCs (BMDCs) from wild-type (WT) mice or mice deficient in activating FcγRs (Fcer1g-/-) or inhibitory FcγRIIb (Fcgr2b-/-). To validate this effect in vivo we established a tumor antigen-specific T cell priming assay with both flow cytometry and ex vivo 2-photon live cell imaging readouts. Results: In three independent single cell transcriptomic PCa atlases, we find cDC2 frequencies are significantly decreased in advanced PCa while effector Tregs (TNFRSF9+) increase in proportion. Hypothesizing that excessive Treg function and DC insufficiency drive ICB resistance in PCa, we find in vivo DC expansion (Flt3L-Ig) prior to hormonal therapy, Treg depletion (FcE-αCTLA-4), and in situ DC stimulation (ADU-S100) drives robust control of established MycCaP tumors, with 87% of animals tumor-free 40-days post-treatment (27/31). Using 45-parameter spectral flow cytometry we find FcE-αCTLA-4 drives DC activation prior to Treg depletion in the TME, supporting a novel direct mechanism of DC modulation by FcE-αCTLA-4. In vitro, we find cell-associated FcE-αCTLA-4 is sufficient to drive BMDC maturation in an FcγR-dependent manner. In vivo, FcE-αCTLA-4 augments DC-mediated T cell priming in an FcγR-dependent manner. Ongoing experiments are investigating how FcE-αCTLA-4 shapes DC-T cell interactions via FcγR engagement and how DC/Treg axis modulation can be optimized to improve long-term curative response rates in the MycCaP model. Conclusions: DC insufficiency and effector Treg differentiation are hallmarks of advanced PCa. Enhancing DC number and function concomitant with Treg depletion can drive effective antitumor immunity in a pre-clinical PCa model. Direct DC modulation via FcγR engagement represents a novel mechanism of response to CTLA-4-targeted ICB. These findings will inform ongoing translation of FcE-αCTLA-4 agents and development of novel immunotherapy strategies for PCa. Citation Format: Jessica C. Hill, Kade R. Copple, Collin Jugler, Phuong Nguyen, Nilika Bhattacharya, Jessica N. Lancaster, Casey R. Ager. Rewiring the dendritic cell: Regulatory T cell axis sensitizes prostate cancer to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 166.

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, and despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4+ T cells (CD4+ Teff) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Treg), which accumulate in lung tumors and mediate suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Herein, we identify the extracellular adenosine 5-triphosphate receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell–specific P2RX7-knockout (P2RX7-KO) mice, reduced Treg infiltration was accompanied by increased CD4+ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared with P2RX7-KO Tregs, which failed to inhibit type 1 and follicular helper T–like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared with P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

Abstract Description The presence of Foxp3+CD4+ regulatory T cells (Tregs) hinders the protective antitumor T cell responses to lung cancer. The accumulation of Tregs is linked to lung cancer progression, resulting in poorer survival rates. However, the mechanisms behind this accumulation are not fully understood. Tregs express elevated levels of the extracellular ATP (eATP) receptor P2RX7 in both human and mouse lung cancer. Here, we show that P2RX7 is crucial for the accumulation of Tregs in lung tumor, promoting tumor growth in a murine lung cancer model. We observed, in T cell-specific P2RX7-KO mice (CD4-cre P2rx7fl/fl), reduced Treg infiltration and a concomitant increase in tumor CD4+ effector T cells, leading to better tumor control. In contrast, P2RX7-KO Tregs accumulated in mediastinal lymph nodes, suggesting that blocking P2RX7-KO signaling impairs the Tregs migratory ability. P2RX7+ Tregs had increased expression of T-bet and KLRG1, which are associated with greater suppressive function. Finally, Treg-specific P2RX7-KO mice (Foxp3-Cre-ERT2 P2rx7fl/fl) had reduced tumor growth and Treg tumor accumulation, indicating that P2RX7 controls lung tumor Tregs in a cell-intrinsic way. These results suggest that P2RX7 promotes lung tumor Treg infiltration and subsequent loss of cancer control. They also suggest that P2RX7 blockade can be used as a therapy to improve lung cancer control, which we are currently exploring in proof-of-concept experiments. Funding Sources Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and Tfh differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen-specific GC B cells and reduced antibody titers. Aging was associated with accumulated Tfh cells with reduced BCL6 and increased CD39. When activated in vitro by aged antigen-presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, young, antigen-specific B and CD4+ T cells were transferred into aged hosts prior to immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen-specific antibody titers when compared to young hosts. Young CD4+ T cells transferred into aged hosts differentiated into Tfh cells with reduced PD-1 and BCL6, and increased CD39. Recent studies have focused on immune profiling of CD4 T cells in young and aged mice following immunization. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired initiation and maintenance of germinal centers. Funding Sources Research was supported by NIH/NIA R01 AG080037 and the Mayo Clinic Robert and Arlene Kogod Center on Aging Innovation Award UL1R002377 to J.L. Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Description Immunotherapy combinations can improve patient outcomes, yet the interactions within the tumor microenvironment (TME) that drive therapeutic synergy are poorly understood. Tumor establishment drives monocyte recruitment and differentiation into tumor-associated macrophages (TAMs), which have essential roles in coordinating immune responses and are thus attractive targets for therapeutic modulation. In a murine model of combination anti-PD-1 and anti-PD-L1 checkpoint blockade, tumor control was associated with increased infiltration of CD8+ T cells and M1-like repolarization of TAMs. Live-cell imaging of the tumor microenvironment revealed stable contacts between tumor-infiltrating CD8+ T cells and TAMs, in which the extent of the contact interfaces increased with combination immunotherapy. Treatment with anti-PD-L1 was able to increase macrophage expression of pro-inflammatory factors and phagocytic activity, suggesting a role for TAMs to reactivate CD8+ T cells in the TME. However, co-treatment with anti-PD-1 was ultimately necessary for tumor control, indicating the need for combination targeting of the TME. Funding Sources Mayo Clinic David and Margaret Grohne Cancer Immunotherapy Pilot Award Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background Immune checkpoint therapies targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) have been met with limited clinical responses in the treatment of patients of varying cancer types. Current US Food and Drug Administration (FDA)-approved therapies function to prevent PD-1 binding to PD-L1, thereby preventing T-cell suppression. However, these therapies fail to prevent PD-L1 intrinsic signaling and recycling, which can render these treatments ineffective. Moreover, most research primarily focuses on how these antibodies affect tumor cells and their interactions with T cells. Myeloid cells, which also express PD-L1 and influence T-cell responses, are integral to the efficacy of these therapies. However, strategies that can target these cells to boost antitumor responses have not been effective. Methods Our group has developed a new anti-PD-L1 antibody (H1A), which disrupts PD-L1 recycling and redirects it towards degradation. Due to the loss of available PD-L1 protein, H1A effectively prevents PD-L1 intrinsic signaling. Using in vivo humanized PD-1/PD-L1 mouse tumor models, we evaluated the therapeutic efficacy of H1A against current FDA-approved PD-L1 blocking antibodies. Using human-derived immune cell in vitro assays, we pinpoint the biological consequences of H1A-induced PD-L1 degradation in myeloid cells. Results H1A demonstrated improved tumor control and established immunological memory responses in vivo in the treatment of tumors with moderate immunogenicity that are less responsive to current immunotherapies. This was supported by enhanced activation of myeloid cells (major histocompatibility complex (MHC)-II and CD80) and frequencies of effector T-cell populations found intratumorally. Human myeloid cells treated with H1A were also observed to have increased activation (MHC-II and CD80) and cytokine secretion. Similar to our in vivo findings, human peripheral blood lymphocyte cultures demonstrated increased frequencies of effector T-cell populations and greater tumor cell killing. Conclusions This work highlights the importance of targeting PD-L1 recycling and intrinsic signaling, specifically in myeloid cells, to boost antitumor responses. Our studies suggest that H1A can provide a solution to the lack of responses seen with current therapeutics, while also revealing previously unknown intrinsic functions of PD-L1 in myeloid cells.