Current asthma treatments manage disease symptoms but fail to address the underlying cause of allergic disease. Allergen immunotherapy holds the promise for durable disease control by establishing allergen-specific tolerance through repeated introduction of native allergens; however, its efficacy can be limited by long interventions, reactions upon administration, and poor patient compliance. Here, we developed a rapid, safe, and effective liver-targeted allergen immunotherapy (LIT) to provide long-term disease control. We developed LIT tolerogens from native respiratory allergens, which induced antigen-specific regulatory T (Treg) cells in vivo with only two interventions. Synthetic mannosylation of native allergens prevented antibody-mediated recognition and subsequent life-threatening anaphylaxis upon administration. Protein engineering prevented sensitization events that occurred because of the proteolytic activity of native respiratory allergens, which limit the effectiveness of allergen immunotherapy. In preclinical models of allergic asthma, LIT ameliorated clinical, pathological, and serological features, and protection was dependent on antigen-specific Treg cells. In sensitized mice, LIT provided a year-long control of allergic asthma symptoms in the absence of additional intervention. Last, LIT induced antigen-specific Treg cells against Der p 1, a major protein in the clinically relevant house dust mite (HDM) respiratory allergen. In mice with established HDM allergy, LIT was well tolerated and provided allergic symptom relief. Together, our data provide a proof of concept that LIT with synthetically mannosylated tolerogens provides a rapid, safe, and effective approach to allergen immunotherapy and holds promise for durable control of allergic asthma.

Tumor-associated lymphatic vessels are clinically linked to aggressive tumor progression and metastasis. Their expansion is driven by vascular endothelial growth factor-C (VEGF-C), which is highly expressed in the tumor microenvironment (TME) in melanoma and other solid tumors. Although elevated VEGF-C promotes an immunosuppressive TME, it paradoxically also enhances the response to immunotherapy in mouse models and correlates with improved survival after checkpoint blockade in patients. This enhanced response was associated with VEGF-C-induced alterations in the tumor microenvironment before immunotherapy, characterized by increased infiltration of naïve T cells in VEGF-C-overexpressing tumors, which creates favorable conditions for local T cell activation. Here, we investigated how VEGF-C reshapes the tumor microenvironment during and after immunotherapy response to improve immunotherapy outcome in melanoma, focusing on immune cell trafficking and memory T cell development. We utilized a B16F10 melanoma model overexpressing VEGF-C (B16-VEGFC) or a control vector (B16-Control) in conjunction with the pmel adoptive cell transfer and peptide vaccination model. Our data demonstrate that B16-VEGFC tumors harbor increased transferred antigen-specific T cells, including more proliferating effector and central memory T cells, along with enhanced endogenous T and B cell populations. In parallel, B16-VEGFC tumors and their draining lymph nodes (tdLN) contain higher fractions of total memory T cells, and Tcf1+ stem-like memory T cells in lymphangiogenic tumors localize near lymphatic vessels. In addition to T cell memory, B16-VEGFC tumors and tdLN exhibit enhanced CD103+ dendritic cell trafficking. Moreover, bystander activation and proliferation of non-targeted CD8 T cells increase in B16-VEGFC tumors, indicating robust immune activation through antigen spreading. Collectively, our study establishes that VEGF-C is pivotal in shaping the anti-tumor immune response during immunotherapy by altering both the TME and the systemic immune response. By promoting T cell memory, boosting DC trafficking, and facilitating T cell recruitment, VEGF-C drives a more robust and sustained immune response against melanoma. Future research using photoconvertible mice will further elucidate the trafficking dynamics between the tumor and its draining lymph nodes post-immunotherapy, providing a more comprehensive understanding of VEGF-C's impact on the tumor microenvironment before and during immunotherapy. Colleen Foley, Lambert Potin, Casey Propst, Melody A. Swartz. VEGF-C remodels the tumor microenvironment during immunotherapy response in melanoma and promotes T cell memory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB075.

The use of a patient’s own immune or tumor cells, manipulated ex vivo, enables Ag- or patient-specific immunotherapy. Despite some clinical successes, there remain significant barriers to efficacy, broad patient population applicability, and safety. Immunotherapies that target specific tumor Ags, such as chimeric Ag receptor T cells and some dendritic cell vaccines, can mount robust immune responses against immunodominant Ags, but evolving tumor heterogeneity and antigenic downregulation can drive resistance. In contrast, whole tumor cell vaccines and tumor lysate-loaded dendritic cell vaccines target the patient’s unique tumor antigenic repertoire without prior neoantigen selection; however, efficacy can be weak when lower-affinity clones dominate the T cell pool. Chimeric Ag receptor T cell and tumor-infiltrating lymphocyte therapies additionally face challenges related to genetic modification, T cell exhaustion, and immunotoxicity. In this review, we highlight some engineering approaches and opportunities to these challenges among four classes of autologous cell therapies.

BACKGROUND:The use of immune checkpoint inhibitors (CPIs) has become a dominant regimen in modern cancer therapy, however immune resistance induced by tumor-associated macrophages (TAMs) with immune suppressive and evasion properties limits responses. Therefore, the rational design of immune modulators that can control the immune suppressive properties of TAMs and polarize them, as well as dendritic cells (DCs), toward a more proinflammatory phenotype is a principal objective in cancer immunotherapy. METHODS:Here, using a protein engineering approach to enhance cytokine residence in the tumor microenvironment, we examined combined stimulation of the myeloid compartment via tumor stroma-binding granulocyte-macrophage colony-stimulating factor (GM-CSF) to enhance responses in both DCs and T cells via stroma-binding interleukin-12 (IL-12). We evaluated tumor responses at the levels of outcome, cellular responses, and cytokine responses in both the tumors and the tumor-draining lymph nodes. We further investigated the potentiation of DC response to IL-12 by GM-CSF stimulation ex vivo. RESULTS:Engineered GM-CSF restored an antitumorigenic tumor myeloid microenvironment otherwise suppressed by TAMs, while engineered IL-12 provided effector signals to T cells, thereby boosting both tumor-resident antitumor macrophage and CD8+ T cell populations. Furthermore, engineered GM-CSF potentiated DC response to IL-12, upregulating DC expression of IL-12 receptor and enhancing their expression of proinflammatory cytokines and chemokines on IL-12 exposure. This resulted in remarkable synergistic efficacy in multiple solid tumor models treated with the dual cytokine combination. The combination therapy also improved the efficacy of CPI in a CPI-resistant genetically-engineered melanoma model and exhibited synergistic antitumor efficacy in a pulmonary metastasis model. CONCLUSION:Our strategy provides a rational design for combination immunotherapy targeting both myeloid and lymphoid compartments through complementary mechanisms.

Supplementary Movie 3 from Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts

<p>In vitro responsiveness of Tumor and Allo/Y-redirected T cells.</p>

<p>LIMMA modeling of expression data.</p>

<p>Assessing Disease State in ACT treated TRAMP mice.</p>

Supplemental Methods and Legends PDF file - 97K, Supplemental methods and figure legends for the supplemental figures

Lymphatic vessels are the main transport routes for fluid, antigens, exosomes, and immune cells from peripheral tissues to the lymph nodes (LNs). In addition to being pervasive through tissue, lymphatic vessels are also abundant within the LN, where they route lymph and cells around the B cell follicles and throughout the paracortical zone; thus, lymphatic endothelial cells (LECs) have unique access to lymph-borne molecules and interactions with immune cells. Upon acute inflammation or vaccination, LN LECs undergo expansion along with lymphocyte proliferation, a process dependent on the VEGF-C/VEGFR-3 signaling axis; however, it is not well understood how LN lymphatics and their expansion contribute to acute adaptive immune responses. Here, we asked how altering VEGFR-3 signaling LN LECs alters the adaptive immune response, using OVA + CpG (a TLR9 agonist) as a model for vaccination. We found that delivering VEGF-C156S, a VEGFR-3 specific agonist, multiple times prior to and concurrent with the inflammatory stimulus led to increased Th2 responses in terms of higher plasma levels of OVA-specific IgG1, more IgG1+ germinal center B cells, and increased production of Th2 cytokines (IL-4, IL-5, and IL-13) by CD4+ T cells upon restimulation. In loss-of-function experiments using a tamoxifen-inducible LEC-specific deletion of VEGFR-3, vaccination led to reduced plasma levels of OVA-specific IgG1 compared to tamoxifen-treated control littermates. Together, these findings suggest that in response to vaccination, VEGFR-3 signaling in LN LECs bias adaptive immune responses towards type 2 immunity. This highlights the role of lymphatics not just as passive conduits for fluid transport, but as having direct immunomodulatory function.

Phenotype of TCR-redirected T cells.

T cells of host and HSCT origin do not participate to tumor recognition.

PDF file - 22K, As the tumor progresses, OVA expression decreases in the tumor.

Supplementary Movie 2 from Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts

Abstract Tumor lymphangiogenesis is typically associated with cancer progression and metastasis. However, recent studies from our group have demonstrated that tumor lymphangiogenesis promotes intratumor T cell infiltration and potentiates the efficacy of a wide range of immunotherapies in melanoma-bearing mice as well as patients undergoing checkpoint blockade. Radiotherapy (RT) can also induce immunogenic tumor cell death, so we investigated whether lymphangiogenic tumors would yield more immunogenic responses to RT than non-lymphangiogenic tumors. Here, we compared effects of RT in mice bearing B16-F10 melanomas expressing either a control vector (B16-Ctrl) or overexpression the lymphangiogenic factor VEGF-C (B16-VC), where lymphatic vessels were significantly increased in number and size compared to B16-Ctrl tumors. First, we find that B16-VC regress faster than B16-CT after RT (either a single 20 Gy dose or two 15 Gy doses), and that this regression was correlated with enhanced CD8+ T cell activity. On the other hand, lymphatic endothelial cells (LECs) can be more sensitive to damage from RT if they are undergoing lymphangiogenesis. We found that irradiated B16-VC tumors showed higher levels of intra-lymphatic fibrin. Consistent with this, LECs in the irradiated B16-VC tumors expressed higher levels of tissue factor and caspase-3, which correlated with the degree of lymphatic clotting. Fibrin clotting was also observed in the subcapsular sinus of the draining lymph nodes of irradiated B16-VC tumors. These findings highlight the complex role of tumor lymphangiogenesis in radiotherapy responses, where they may potentiate acute anti-tumor immune responses but may also cause localized damage of the vasculature, potentially leading to increased metastasis. Further studies are needed to investigate therapeutic targets to inhibit lymphatic coagulation and look at its effects on cancer immunotherapy and metastasis. Citation Format: Anish Mukherjee, Nikolaos Mitrousis, Mari Stella Sasso, Margo MacDonald, Ainhoa Arina, Phillip Ang, Ariana Baginski, J. Emiliano G. Medellin, Ralph R. Weichselbaum, Melody A. Swartz. Tumor lymphangiogenesis sensitizes melanomas to the immunological effects of radiotherapy but also disrupts the local lymphatic vasculature. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4585.

Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)–resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.

<p>H&E analyses of CD3+ tumor-infiltrating cells.</p>

Supplemental Figures PDF file - 346K, Supplementary figures to the main text

Lymphatic endothelial cells (LECs) express MHC class II (MHC-II) upon IFN-γ stimulation, yet recent evidence suggests that LECs cannot activate naive or memory CD4+ T cells. In this article, we show that IFN-γ-activated human dermal LECs can robustly reactivate allogeneic human memory CD4+ T cells (hCD4+ TMs), but only when TGF-β signaling is inhibited. We found that in addition to upregulating MHC-II, IFN-γ also induces LECs to upregulate glycoprotein A repetitions predominant, which anchors latent TGF-β to the membrane and potentially inhibits T cell activation. Indeed, hCD4+ TM proliferation was substantially increased when LEC-CD4+ TM cultures were treated with a TGF-β receptor type 1 inhibitor or when glycoprotein A repetitions predominant expression was silenced in LECs. Reactivated hCD4+ TMs were characterized by their proliferation, CD25 expression, and cytokine secretion. CD4+ TM reactivation was dependent on LEC expression of MHC-II, confirming direct TCR engagement. Although CD80 and CD86 were not detected on LECs, the costimulatory molecules OX40L and ICOSL were upregulated upon cytokine stimulation; however, blocking these did not affect CD4+ TM reactivation by LECs. Finally, we found that human dermal LECs also supported the maintenance of Foxp3-expressing hCD4+ TMs independently of IFN-γ-induced MHC-II. Together, these results demonstrate a role for LECs in directly modulating CD4+ TM reactivation under inflammatory conditions and point to LEC-expressed TGF-β as a negative regulator of this activation.