Cytotoxic T Lymphocytes Research Articles

Abstract C026: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

Abstract Epithelial-to-mesenchymal transition (EMT) plays important roles during development, injury repair, cancer invasion and metastasis. The heterogeneity of EMT programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. Through in vivo selection, we established dormant metastasis models from human and mouse early-stage LUAD cell lines. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers, whereas cells derived from overt metastases express high level of the late-stage progenitor marker Sox9. Using a TGF-β signaling reporter, we showed that in the primitive progenitors, TGF-β induces growth inhibition accompanied by a classic EMT response that subsequently transitions into an atypical mesenchymal state of round morphology as the disseminated cells establish dormancy. Prolonged treatment with TGF-β in vitro recapitulates this transition in these primitive progenitors, but not in the late-stage progenitors. Given this dramatic morphological change, we focused on actin-cytoskeleton regulators and found that TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy. Citation Format: Zhenghan Wang, Yassmin Elbanna, Inês Godet, Lila Peters, George Lampe, Yanyan Chen, Joao Xavier, Morgan Huse, Joan Massagué. TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C026.

Abstract B036: WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer treatment, but resistance limits its clinical effectiveness. Therefore, understanding immune resistance mechanisms and identifying predictive markers are essential. Our transcriptomic analysis of PD-1 blockade-treated patients and non-responsive tumor models identifies WEE1 as a key resistance factor, promoting cancer stem cell (CSC)-like traits and immune resistance phenotypes including low T cell infiltrated into tumor and anti-apoptotic properties to cytotoxic T cells (CTLs). WEE1 drives AKT hyperactivation, enhancing expression of resistance factors such as CYCLIN A which contributes to CSC-like properties, and the anti-apoptotic molecule MCL-1, while also reducing T cell infiltration via CXCL10 downregulation. This signaling axis is conserved across various cancers. Notably, WEE1 inhibition sensitizes tumors to ICB by disrupting these refractory properties and reinvigorating antitumor immunity. These findings underscore the novel role of WEE1 in driving immune resistance and CSC-like characteristics, supporting the use of WEE1 inhibitors as a strategy to overcome resistance to anti-PD-1 therapy. Citation Format: Hyo-Jung Lee, Suyeon Kim, Eunho Cho, Tae Woo Kim. WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B036.

Abstract C046: PARP inhibitors as immune modulators in metastatic ovarian cancer treatment

Abstract Epithelial ovarian carcinoma (EOC) is one of the top five causes of cancer-related death in women. Most patients with EOC achieve initial remission after primary or interval cytoreductive surgery combined with platinum-taxane chemotherapy. However, mutations in BRCA1 or BRCA2 genes, which lead to homologous recombination (HR) defects, play a crucial role in platinum sensitivity and justify the use of poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy, commonly linked to extended progression-free survival (PFS). Besides their direct cytotoxic and cytostatic effects, PARP inhibitors (PARPi) have shown significant immunostimulatory properties by disrupting DNA repair in cancer cells, and opening possibilities for synergy with immune checkpoint inhibitors (ICIs). In this study, we investigate the immunomodulatory effects of PARPi using multiparametric flow cytometry, multiplexed immunolabeling, single-cell transcriptomics, and functional assays in an experimental BR5Brca1-/- syngeneic mouse model and human EOC tumor samples. We examine the molecular and cellular mechanisms that can be exploited to develop more effective combination therapies. PARPi may increase the mutational burden in EOCs due to unresolved DNA damage and the release of damage-associated molecular patterns (DAMPs), thereby increasing T-cell infiltration. In addition, PARPi appear to promote potent type I interferon (IFN) secretion through the activation of cGAMP synthase and the stimulator of the interferon genes (STING) pathway. In combination with ICIs, PARPi showed a beneficial effect on the balance between adaptive anti-tumor immunity and innate myeloid components, leading to an improved cytotoxic T-cell response, as observed in mouse models and HGSOC tumor samples. These observations emphasize the role of strategically designed combinations of PARPi and immunotherapeutic agents, which could be the key to overcoming immunosuppression in the EOC microenvironment, thereby improving clinical outcomes. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Michal Hensler, Romana Mikyskova, Lenka Kasikova, Josef Pasuvka, Jana Drozenova, Katerina Mojzisova, Marek Kovar, Iain McNiesh, Michael Halaska, Lukas Rob, Sandra Orsulic, Milan Reinis, Lorenzo Galluzzi, Radek Spisek, Jitka Palich Fucikova. PARP inhibitors as immune modulators in metastatic ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C046.

The CML experience to elucidate the role of innate T-cells as effectors in the control of residual cancer cells and as potential targets for cancer therapy

Considering the general view that unconventional immune effectors play a major role in antitumor immunity, we recently postulated that the distinct new innate CD8 T-cell pool (co-expressing the transcription factor Eomesodermin and innate markers such as KIR/NKG2A) may counteract tumor cells, and thereby be potential target for cancer therapy. Here, to test this assumption, we used successfully targeted anti-leukemic therapy discontinuation (TFR) in chronic myeloid leukemia (CML). Numerical and functional status of innate CD8 T-cells, iNKT cells and γδ T-cells, in comparison with NK cells, was compared longitudinally between non-relapsed patients (i.e., with > 12 months TFR) and relapsed patients (i.e., who experienced molecular recurrence during the first 12 months after TKI cessation) in a prospective pilot cohort (n=32), starting from treatment discontinuation (D0). Perforin, a key cytotoxic immune player, was expressed in a significantly higher proportion of both innate CD8 T-cell and NK-cell subsets in non-relapsed patients, compared with relapsed patients at D0. In parallel, we assessed the expression of PD-1, an exhaustion marker used as target in cancer therapy. For all T-cell subsets, surface-expression level of PD-1 decreased in non-relapsed patients compared with relapsed patients at D0. This was particularly the case when considering iNKT cells for which surface-expression level of PD-1 even decreased relative to healthy control subjects. Lastly, we found a negative correlation between the proportion of innate CD8 T-cells expressing PD-1 and those expressing perforin in non-relapsed patients at D0. The fact that this was not the case in conventional CD8 T-cells is compatible with a reprogrammed effector profile preferentially targeting innate CD8 T-cells in non-relapsed patients. All in all, our results highlight NK cells and innate CD8 T-cells harboring cytotoxic content, as well as global downregulation of PD-1-expression on effector T-cells, as potential predictive functional signatures for successful TFR in CML. Considering innate CD8 T-cells, further investigations are needed to determine whether their possible contributory role in cancer surveillance in CML could be extended to other cancers, and also whether their targeting by immune cheek-point inhibitors could enhance their anti-tumoral functions.

Tuberculosis Preventive Treatment for Pregnant People With Human Immunodeficiency Virus in South Africa: A Modeling Analysis of Clinical Benefits and Risks.

Although prior studies of tuberculosis-preventive treatment (TPT) for pregnant people with human immunodeficiency virus (PPWH) report conflicting adverse pregnancy outcome (APO) risks, international guidelines recommend TPT for PPWH. We used a microsimulation model to evaluate 5 TPT strategies among PPWH receiving antiretroviral therapy in South Africa: No TPT; 6 months of isoniazid (6H) or 3 months of isoniazid-rifapentine (3HP) during pregnancy (Immediate 6H or Immediate 3HP) or post partum (Deferred 6H or Deferred 3HP). The primary outcomes were maternal, fetal/infant, and combined deaths from causes potentially influenced by TPT (maternal tuberculosis, maternal hepatotoxicity, stillbirth, low birth weight [LBW], and infant tuberculosis). Tuberculosis during pregnancy confers 250% and 81% higher modeled risks of stillbirth and LBW, respectively. In lower-risk or higher-risk scenarios, immediate TPT confers 38% lower or 92% higher risks of stillbirth and 16% lower or 35% higher risks of LBW. Immediate TPT would minimize deaths among PPWH. When TPT confers higher stillbirth and LBW risks, immediate TPT would produce the most combined maternal and fetal/infant deaths, even with low maternal CD4 cell count and high tuberculosis incidence. If immediate TPT yields a <4% or <20% increase in stillbirth or LBW, immediate TPT would produce fewer combined deaths than deferred TPT (sensitivity analysis range, <2%-22% and <11%-120%, respectively). If APO risks are below identifiable thresholds, TPT during pregnancy could decrease combined maternal and fetal/infant deaths. Given uncertainty around isoniazid's risks, and the low threshold at which APO risks could outweigh benefits from tuberculosis deaths averted, studies of newer TPT regimens among PPWH are warranted to inform guidelines.

Systemic chemokine-modulatory regimen combined with neoadjuvant chemotherapy in patients with triple-negative breast cancer

BackgroundHigher cytotoxic T lymphocyte (CTL) numbers in the tumor microenvironment (TME) predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) and positive long-term outcomes in triple-negative breast cancer (TNBC). pCR...

Precision Oncology in Melanoma: Changing Practices.

Over the last 2 decades, significant progress has been made in our understanding of the genomics, tumor immune microenvironment, and immunogenicity of malignant melanoma. Historically, the prognosis for metastatic melanoma was poor because of limited treatment options. However, after multiple landmark clinical trials displaying the efficacy of combined BRAF/MEK inhibition for BRAF-mutant melanoma and the application of immune checkpoint inhibitors targeting the programmed death-1, cytotoxic T-lymphocyte antigen-4, and lymphocyte activation gene-3 molecules, overall survival rates have dramatically improved. The role of immune checkpoint inhibition has since expanded to the neoadjuvant and adjuvant settings with multiple regimens in routine use. Personalized therapies, including tumor-infiltrating lymphocytes that are extracted from a patient's melanoma and eventually reinfused into the patient, and messenger RNA vaccines used to target neoantigens unique to a patient's tumor, show promise. Improvements in accompanying imaging modalities, particularly within the field of nuclear medicine, have allowed for more accurate staging of disease and assessment of treatment response. Continued growth in the role of nuclear medicine in the evaluation of melanoma, including the incorporation of artificial intelligence into image interpretation and use of radiolabeled tracers allowing for intricate imaging of the tumor immune microenvironment, is expected in the coming years.

Rapid response of lichen planus to baricitinib associated with suppression of cytotoxic CXCL13+ CD8+ T-cells.

Cutaneous lichen planus (LP) is a recalcitrant, difficult-to-treat, inflammatory skin disease characterized by pruritic, flat-topped, violaceous papules on the skin. Baricitinib is an oral Janus kinase (JAK) 1/2 inhibitor that interrupts the signaling pathway of interferon gamma (IFN)-γ, a cytokine implicated in the pathogenesis of LP. In this phase II trial, twelve patients with cutaneous LP received baricitinib 2 mg daily for 16 weeks, accompanied by in-depth spatial, single-cell, and bulk transcriptomic profiling of pre- and post-treatment samples. An early and sustained clinical response was seen, with 83.3% of patients responsive at week 16. Our molecular data identified a unique, oligoclonal IFN-γ, CD8+, CXCL13+ cytotoxic T-cell population in LP skin and demonstrated a rapid decrease in IFN signature within 2 weeks of treatment, most prominently in the basal layer of the epidermis. This study demonstrates the efficacy and molecular mechanisms of JAK inhibition in LP. NCT05188521.ROLE OF FUNDING SOURCE. Eli Lilly, Appignani Benefactor Funds, 5P30AR075043, Mayo Clinic Clinical Trials Stimulus Funds.

"Towards melanoma in situ vaccination with multiple ultra-narrow X-ray beams".

Despite the recent progress, current treatment modalities are not able to eradicate cancer. We show that Microbeam Radiotherapy (MRT), an innovative type of Spatially Fractionated Radiotherapy, can control murine melanoma by activating the host's own immune system. The beneficial effects are very pronounced in comparison to uniform radiotherapy, traditionally employed in the clinic. Our results displayed that MRT increased antigen presentation, activating Cytotoxic T Lymphocytes (CTLs) which are essential to MRT's treatment efficacy in melanoma. Depletion of CTLs abrogated treatment response. Multiplex nucleic acid hybridization technology revealed key features of lymphocyte populations such as proliferation, differentiation, and ligand-receptor interactions. In addition, CTLs were shown to be essential for locoregional metastatic control and systemic abscopal effects confirmed by activation of antigen presenting cells and CTL trafficking in the tumour-draining lymph nodes. MRT induces a robust antitumour immune response, matching the characteristics of in situ vaccination, that could be exploited to treat a variety of treatment-resistant malignancies.

Autophagy-activating aluminum hydroxide nanovaccine for enhanced antigen presentation and anti-tumor immunity.

Lymph node (LN) targeting and antigen presentation by antigen-presenting cells (APCs) are critical factors affecting the immune responses induced by tumor vaccines. Autophagy activation promotes MHC class I and II antigen presentation in APCs. To enhance antigen presentation in LNs, we developed an aluminum hydroxide nanovaccine that simultaneously incorporates the autophagy-activating peptide Beclin-1 and the antigenic protein OVA (B/O@AN nanovaccine) through layer-by-layer electrostatic interaction. B/O@AN has a particle size of approximately 80 nm and efficiently targets lymph nodes following subcutaneous administration. The combination of the Beclin-1 peptide with the aluminum hydroxide nanovaccine promotes dendritic cell (DC) maturation. More importantly, B/O@AN facilitates antigen cross-presentation by promoting lysosomal escape and autophagy induction. After immunization, compared to O/@AN without Beclin-1, B/O@AN significantly augments antigen-specific cellular immune responses, leading to substantial increases in cytotoxic T lymphocytes (CTLs), T-helper 1 (Th1) cells, as well as serum antibody levels, thereby impeding melanoma development and progression in both prophylactic and therapeutic settings. These results provide evidence that autophagy activation strengthens antigen presentation and augments the antigen-specific immune responses of the aluminum hydroxide nanovaccine.

Implications of glioblastoma-derived exosomes in modifying the immune system: state-of-the-art and challenges.

Glioblastoma is a brain cancer with a poor prognosis. Failure of classical chemotherapy and surgical treatments indicates that new therapeutic approaches are needed. Among cell-free options, exosomes are versatile extracellular vesicles (EVs) that carry important cargo across barriers suchas the blood-brain barrier (BBB) to their target cells. Thismakes exosomes an interesting option for the treatment ofglioblastoma. Moreover, exosomes can comprise many therapeutic cargos, including lipids, proteins, and nucleic acids,sampled from special intercellular compartments of their origin cell. Cells exposed to various immunomodulatory stimulican generate exosomes enriched in specific therapeutic molecules. Notably, the secretion of exosomes could modify theimmune response in innate and adaptive immune systems. For instance, glioblastoma-associated exosomes (GBex) uptake bymacrophages could influence macrophage dynamics (e.g., shifting CD markers expression). Expression of critical immunoregulatory proteins such as cytotoxic T-lymphocyte antigen-1 (CTLA1) and programmed death-1 (PD-1) on GBex indicates the direct crosstalk of these nano-size vesicles with the immune system. The present study reviews the role of exosomes in immune system cells, including Bcells, Tcells, natural killer (NK) cells, and dendritic cells (DCs), as well as novel technologies in the field.

Abstract B031: Immune cell composition in surgical drain fluid as a novel prognostic marker in head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy globally, and the vast majority of cases are treated with primary surgical resection and adjuvant therapy. Our group has pioneered the use of HNSCC circulating tumor DNA (ctDNA) in surgical effluent collected postoperatively from surgical drains (surgical drain fluid, SDF) as a novel proximal bioanalyte for measuring minimal residual disease after surgery. We now profile the immune cell composition of SDF compared to paired peripheral blood mononuclear cells (PBMCs), and correlate SDF immune cell profiles with clinical variables and oncologic outcomes in HNSCC. Method: SDF and peripheral venous blood were collected 24 hours postoperatively from 58 HNSCC patients, including 11 HPV (+) and 47 HPV (-) patients undergoing standard of care surgical resection, lymph node dissection, and adjuvant therapy. Freshly collected SDF cells and PBMCs were cryopreserved until profiling by multicolor flow cytometry for respective leukocyte compositions. Analyses were used to define the following viable CD45+ leukocyte subsets: total T cells, CD4 T-cells CD8 T-cells, Treg cells, B-cells, non- classical monocytes, classical monocytes, natural killer (NK) cells, classical dendritic cells (cDC), plasmacytoid DC (pDC), macrophages, and monocytic myeloid-derived suppressive cells (MDSCs). Immune cell percentages were correlated with clinical characteristics and cancer recurrence. Results: CD45+ leukocytes comprised &amp;gt;99.5% of SDF cells, with very few EPCAM+ tumor cells (&amp;lt; 10^-4). HPV status did not significantly affect SDF immune cell composition, and no significant differences were noted by tumor site (larynx, oral cavity, oropharynx), or extracapsular lymph node extension. Among 13 HPV (-) HNSCC patients with paired peripheral blood drawn concurrently, SDF had a higher proportion of B-cells(16.8% vs. 9%, p=0.044), NK cells(56% vs. 12%, P&amp;lt;0.001), monocytes(14% vs. 1%, P&amp;lt;0.001), and macrophages(3.1% vs. 0.46%, P&amp;lt;0.001) compared to PBMCs, and a lower proportion of CD4+ T cells(1% vs. 24%, P&amp;lt;0.001), CD8+ T cells(0.6% vs. 18%, P&amp;lt;0.001), MDSCs(2.1% vs. 3.4%, P=0.029), and pDCs(0.1% vs. 0.2%, P=0.014). HPV (-) HNSCC patients that went on to recur (n=9) had a significantly higher proportions of SDF monocytes (total, classical, and non- classical) and CD56dimCD16+ NK cells (all P&amp;lt;0.001) compared to non-recurrent patients (n=38). Patients who went on to recur also had a significantly lower proportion of SDF B-cells (P&amp;lt;0.001), CD56bright NK cells(P=0.01), and macrophages (P=0.018) compared to non- recurrent cases. No significant differences were noted with respect to proportions of CD3+, CD4+, CD8+ T-cells, DC, and MDSC cells. Conclusion: SDF from HNSCC patients contains unique immune cell populations and may provide new insights into postoperative tumor immunity. Differences in SDF immune cell composition by oncologic outcome in patients that go on to recur, highlighting potential prognostic and predictive applications of SDF immune cell profiling for adjuvant therapy decision-making. Citation Format: Zhongping Xu, Noah Earland, Lucien Khalil, Lazar Vujanovic, Danny Azmi Elias, Riyue Bao, Jason J. Luke, Aadel A. Chaudhuri, Jose P. Zevallos. Immune cell composition in surgical drain fluid as a novel prognostic marker in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B031.

IGF2BP1 accelerates the aerobic glycolysis to boost its immune escape in hepatocellular carcinoma microenvironment

IntroductionEnergy metabolism abnormity emerges as a crucial factor that facilitates tumorigenesis by accelerating aerobic glycolysis. However, the function of N6-methyladenosine (m6A) on hepatocellular carcinoma (HCC) aerobic glycolysis and immune escape is still unclear. Here, this investigation was intended to elucidate the regulation of m6A ‘reader’ IGF2BP1 involved in HCC aerobic glycolysis and immune escape.MethodsThe aerobic glycolysis was tested by glucose uptake, lactate, ATP generation and ECAR. The CD8+ T cell-mediated killing effect was tested by cytotoxicity, IFN-γ and granzyme B. The molecular interaction was confirmed by luciferase reporter assay, immunoprecipitation assay and chromatin immunoprecipitation (ChIP)-PCR.ResultsElevated IGF2BP1 expression was associated with poor prognosis in HCC patients. Functionally, IGF2BP1 emerged as an oncogenic factor that accelerated HCC aerobic glycolysis (glucose uptake, lactate, ATP generation and ECAR) and oxaliplatin resistance. Meanwhile, IGF2BP1 repressed the activated CD8+ T cell-mediated killing effect (cytotoxicity, IFN-γ and granzyme B) and apoptosis of HCC cells, indicating a suppressed cytotoxic T-cell response. By recognizing and binding to the m6A-modified sites on c-Myc mRNA, IGF2BP1 enhanced the stability of c-Myc mRNA, consequently upregulating c-Myc expression. In addition, transcription factor c-Myc targeted the programmed death ligand 1 (PD-L1) promoter region to strengthen its transcription.DiscussionTaken together, this study illustrates IGF2BP1 as a potential therapeutic target in HCC, aiming to disrupt the interplay between aberrant metabolism and immune escape.

Abstract 4118086: Doxorubicin Induces a Cytotoxic T-cell Inflammatory Response That Contributes to Cardiac Fibrosis and Systolic Dysfunction

Background: Doxorubicin (DR), the most commonly used chemotherapy, results in dose-dependent cardiotoxicity in part by causing oxidative stress, cardiomyocyte death, and cardiac fibrosis. Whether these elicit a cardiac immune response that contributes to DR cardiomyopathy and could be targeted for cardio-protection is unknown. We hypothesized that an aberrant T-cell immune response secondary to cardiac damage contributes to DR cardiomyopathy. Methods: We treated wild type (WT) mice and CD8 + T-cell antigen restricted OTI mice with 5.0 mg/kg of DR or PBS weekly for 4 or 8 weeks and performed targeted antibody depletion of CD8 + and CD4 + T-cells. Primary mouse T-cells and cardiac fibroblasts (CFB) were used for mechanistic studies. Whole blood was analyzed by flow cytometry and proteomics in human and canine lymphoma patients before and after DR initiation. Results: DR treated mice exhibited cardiac CD8 + T-cell infiltration alongside hallmarks of DR cardiomyopathy, including declined ejection fraction (EF) measured by echocardiography, cardiac cell death, and fibrosis determined by TUNEL and picrosirius red stain. Cardiac CD8 + T-cells co-localized with αSMA + CFB and had enhanced expression of the degranulation marker CD107a and IFNγ in DR compared to PBS treated mice. DR hearts also showed increased gene and protein expression of the IFNγ inducible chemokines Cxcl9 and Cxcl10 . DR resulted in increased circulating CD8 + T-cells and the frequency of activated CD8 + cells in the cardiac draining lymph nodes. Targeted antibody depletion of CD8 + but not CD4 + T-cells in the onset of DR treatment improved EF and decreased cardiac fibrosis. OTI mice were protected from DR induced EF decline, cardiac T-cell infiltration, and cardiac fibrosis, indicating an antigen-specific CD8 + T-cell response . Mechanistically, DR induced ICAM-1 expression on CFB and ICAM-1 dependent adhesion of activated CD8 + T-cells, which in turn increased CFB αSMA expression. Contact between CD8 + T-cells and DR-treated CFB caused CD8 + T-cell degranulation and Granzyme B release that contributed to CFB activation. Human and canine cancer patients followed longitudinally had increased circulating CD8 + T-cells after anthracycline treatment, and human plasma levels of CXCL9/CXCL10 correlated with decreased EF post DR. Conclusion: Our data demonstrate a novel role for cytotoxic T-cells in DR cardiomyopathy across 3 species, through CXCL9/10-dependent cardiotropism and CD8 + T-cell dependent fibrosis.

Efficacy and safety of DTG plus 3TC for prophylaxis of perinatal transmission of HIV infection in pregnant women who have detectable viral load after 14 weeks of gestation: A pilot study (PREGNANCY)

Abstract Background The prevention of perinatal HIV transmission depends on the safe and effective use of antiretroviral therapy (ART). Simplifying treatment reduces drug exposure for both mother and child. We evaluated the safety and efficacy of DTG/3TC for antiretroviral-naïve pregnant women living with HIV (PWLHIV). Methods This proof-of-concept trial enrolled ART-naïve pregnant women over 15 years old with HIV infection and a gestational age between 14 and 28 weeks. Participants received a fixed-dose combination of DTG/3TC. Baseline HIV genotyping was performed. Participants were monitored at baseline, every 4 weeks, and at delivery. Infants were assessed at birth, 4 weeks, and 6 weeks of age. Outcomes included the proportion of women achieving an undetectable HIV-1 plasma viral load (&amp;lt;50 copies/mL) at delivery, therapy modification frequency, perinatal HIV transmission rate, and adverse events. Results Between January 2019 and March 2021, 20 women were enrolled. At baseline, the median CD4 cell count was 401.6 ± 113.6 cells/μL, increasing to 690.2 ± 266 cells/μL at delivery. Median viral load was 9,514 copies/mL. All women achieved an undetectable viral load after an average of 40 days. No cases of perinatal HIV transmission were detected. No therapy modifications were necessary during the study, and no adverse events were related to the ART. Conclusion In this pilot trial, DTG/3TC demonstrated safety and efficacy, with all participants achieving viral suppression before delivery. There were no cases of perinatal HIV transmission and no drug-related adverse events. DTG/3TC can be an option for initial treatment of drug-naïve PWLHIV.

TMIC-58. LEVERAGING VIRO-IMMUNOTHERAPY BY TARGETING IGF2-IGF1R SIGNALING

Abstract While an FDA-approved oncolytic herpes simplex-1 virus (oHSV) has shown therapeutic promise with superior safety, accumulating clinical data revealed that its therapeutic efficacy is observed in a small subset of patients. Here, we show that NFκB-mediated Insulin-like Growth Factor 2 (IGF2)/Insulin-like Growth Factor-1 Receptor (IGF1R) signaling pathway as a key modulator for oHSV therapy-mediated tumor resistance. RNA sequencing on the oHSV-infected primary glioblastoma (GBM) and breast cancer (BC) cells identified IGF2 as one of the top 10 upregulated secretomes. Transcriptomic analysis also revealed significant upregulation/enrichment of genes related to IGF2-IGF1R-MAPK pathway in oHSV-treated tumor cells. Infection with oHSV in GBM and BC cells up-regulated IGF2 transcription via direct binding of NFκB in the IGF2 promoter 3. Compromising the IGF2-IGF1R signaling pathway using IGF2 neutralizing antibody significantly increased oHSV-mediated tumor cell killing in vitro and mice survival in vivo. To circumvent this and promptly translate our findings in clinical settings, we have developed a novel oHSV, oHSV-D11mt, by incorporating modified IGF2R domain 11 into the parental oHSV genome to function as IGF2 decoy receptor as a single-agent modality. oHSV-D11mt specifically bind to IGF2 not IGF1, blocks oHSV-induced IGF2-IGF1R signaling, increases tumor cell killing, decreases oHSV-induced neutrophils/PMN-MDSCs infiltration, immune suppressive/proangiogenic cytokine secretion, and increases CD8+ cytotoxic T lymphocytes (CTLs) activation, resulting in increased virus propagation and mice survival. These findings suggest that IGF2-IGF1R signaling is a novel target to overcome oHSV therapy resistance and oHSV-D11mt could be used for improved viro-immunotherapy.

EXTH-48. ADENO-ASSOCIATED VIRUS TARGETING OF THE TUMOR MICROENVIRONMENT TO IMPROVE LYMPHOCYTE RECRUITMENT IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment, infiltration, and activation. We have developed a novel recombinant adeno-associated virus (AAV) gene therapy strategy that enables focal and stable reconstitution of the GBM tumor microenvironment (TME) with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for cytotoxic T lymphocytes (CTLs). By increasing lymphocyte recruitment in GBM, we HYPOTHESIZE that these tumors will be more responsive to immunotherapy. METHODS 3D fluorescence microscopy was used to characterize the distribution, tropism, and durability of AAV6 transgene expression in preclinical GBM. Flow cytometry and 3D fluorescence microscopy was used to quantify T cell infiltration and visualize their biodistribution in preclinical GBM treated with AAV-CXCL9 in combination with anti-PD-1 immune checkpoint blockade (ICB). Single-cell RNA sequencing was used to characterize intratumor lymphocyte subsets following treatment. Survival studies with and without lymphocyte depletion were done to evaluate immunogenic responsiveness of therapy. RESULTS Focal delivery of CXCL9 with AAV6 results in stable transduction of peri-tumoral astrocytes. When combined with anti-PD-1 ICB, lymphocyte infiltration was improved, with cells found disseminated across the tumor stroma and within the tumor parenchyma. CXCR3, the chemokine receptor for CXCL9, was expressed across lymphocyte subsets and NKT cells, validating treatment selectivity of AAV6-CXCL9 for lymphocytes. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by CD8-postive cytotoxic lymphocytes, sensitizing GBM to anti-PD-1 ICB. These effects are accompanied by immunologic signatures evocative of an inflamed and responsive TME. These findings support targeted AAV gene therapy as a promising adjuvant strategy for reconditioning GBM immunogenicity given its excellent safety profile, TME-tropism, modularity, and off-the-shelf capability.

RTID-04. A MULTICENTER, RANDOMIZED, CONTROLLED PHASE 2B TRIAL OF SURVIVIN VACCINE SURVAXM PLUS ADJUVANT TEMOZOLOMIDE FOR NEWLY-DIAGNOSED GLIOBLASTOMA (SURVIVE)

Abstract BACKGROUND Glioblastoma cells express high levels of survivin protein, whose presentation by MHC class I gets recognized by antibodies and cytotoxic T-lymphocytes (CTLs). A phase 2a trial (NCT02455557) demonstrated a median overall survival (OS) of 25.9 months in newly-diagnosed glioblastoma (nGBM) patients with 15-amino-acid-peptide-conjugate survivin vaccine (SurVaxM), which also stimulated production of survivin-directed antibodies and anti-tumor CTL. This phase 2b trial aims to further investigate efficacy and safety of SurVaxM plus adjuvant temozolomide (TMZ) for nGBM. METHODS SURVIVE is an ongoing, multicenter, randomized, placebo-controlled, investigator- and patient-blinded, phase 2b trial that randomizes nGBM patients receiving standard-of-care therapies to either SurVaxM (SurVaxM arm) or saline (control arm) in 3:2 ratio at 15 sites. Included patients have age ≥18 years, normal organ function, KPS≥70, and receive surgical resection with ≤1cm3 residual MRI contrast enhancement within 72 hours post-resection plus TMZ. Patients with recurrent, multicentric, brainstem- or cerebellar-origin GBM are excluded as well as patients on TTFields or other immunotherapies. All patients undergo 3 phases: (1) vaccine priming (VP) phase, starting ≤4 weeks after completing chemoradiation – 4 doses of SurVaxM or placebo administered q2weeks; (2) TMZ phase, started after ≥1 VP dose; and (3) vaccine maintenance phase, started 8 weeks after VP, with SurVaxM or placebo given q8weeks. TMZ phase overlaps with VP and VM phases. Primary endpoint is median OS. Assuming 1-year-OS of 75% in SurVaxM arm, based on prior trials, and 60% for control, the required sample size is N=228; 137 in SurVaxM and 91 in control arm. Accounting for dropouts, we aimed to enroll 239 patients. A log-rank test stratified by KPS (70-80 vs 90-100), MGMT methylation status, and IDH mutation status will be used for analysis. Secondary endpoints are OS and PFS at 15, 18, and 24 months, median PFS, and toxicity. A single sequential OS-driven interim analysis is planned when 50% of deaths occur. Trial is fully enrolled (NCT05163080).

IMMU-12. TRANSLATING COMBINED PD1 AND LAG3 INHIBITION FROM PRECLINICAL MODELS TO PATIENTS WITH REFRACTORY, DNA REPLICATION REPAIR DEFICIENT (RRD) GLIOBLASTOMA: AN IRRDC STUDY

Abstract BACKGROUND AND AIMS RRD-glioblastoma harbour high tumor mutation burden (TMB) and respond to anti-PD1 immune-checkpoint inhibition (ICI). However, not all respond, and the majority progress, highlighting the need for combinatorial therapies for sustained immune-surveillance. METHODS We performed transcriptomic analyses of human RRD-glioblastoma specimens for immune checkpoint expression, and accordingly, tested combined ICI in immunocompetent murine models. Based on these preclinical data, we treated refractory patients using a combination of anti-PD1+anti-LAG3 through single-patient trial/compassionate access. Complimentary immuno-genomic biomarker analyses including circulating tumor DNA (ctDNA) were performed to investigate mechanisms and track responses. RESULTS Human RRD-glioblastoma (n=80) demonstrated high LAG3 expression, providing a strong rationale for therapeutic targeting. We tested combined anti-PD1+anti-LAG3 inhibition in three immunocompetent RRD-glioblastoma murine models. In the anti-PD1-responsive (Nestin-CreMSH2LoxP/LoxP-POLES459F/+) model, anti-PD1+anti-LAG3 resulted in universal tumor response and superior survival to ICI-monotherapy. In the anti-PD1 resistant models (Mlh1-/-/NestinCre+/Trp53LoxP/LoxP and therapy-induced hypermutant ENU/Trp53-/- gliomas), anti-PD1+antiLAG3 improved survival, overcoming the lack of response to ICI-monotherapy. Biologically, high LAG3 expression and immune-exhaustion observed in CD8 T-cells after treatment with anti-PD1 was ablated following the addition of anti-LAG3. Serially transplanted, post-anti-PD1 treated tumors showed response, confirming, in-vivo, that resistance to anti-PD1 could be abrogated by anti-PD1+anti-LAG3. Seven children with refractory RRD-glioblastoma who had progressed after anti-PD1 treatment were treated using anti-PD1+anti-LAG3, resulting in objective radiological responses and prolonged ongoing survival. Tolerability was better than a previous study of combined CTLA4 and PD1 inhibition for similar patients. Paired immuno-genomic tumor analyses, serial blood flow-cytometry, T-cell receptor clonotype, and CSF ctDNA analyses provided novel insights into the mechanisms of immunological invigoration and first-in-human, radiological responses. CONCLUSIONS LAG3 is an effective target in refractory RRD-glioblastoma. Combined inhibition with anti-PD1 inhibition demonstrated radiological response, prolonged survival and manageable toxicities in patients, and unearthered mechanisms of immune-responses. The combination will now be tested in biomarker-driven clinical trials in RRD-glioblastoma and other immune-inflamed solid tumors.

EPCO-46. SPATIAL TRANSCRIPTOMICS ANALYSIS OF GLIOBLASTOMA REVEALS THREE DISTINCT REGIONAL PROGRAMS OF T-CELL INFILTRATION

Abstract Glioblastoma (GBM) resists T-cell killing by developing an immunosuppressive tumor microenvironment. Transcriptomic technologies, such as single-cell RNA sequencing (scRNA-seq), have characterized immune cell phenotypes and putative cell-cell interactions in GBM. However, the tumor microenvironment surrounding T-cells is still not well characterized, motivating the need for spatial analyses. Two publicly-available GBM spatial transcriptomic datasets were analyzed, with one discovery cohort (N=18) and one validation cohort (N=12). Using cell deconvolution with a scRNA-seq reference dataset, areas of high T-cell infiltration were identified and clustered into regional programs based on transcriptomic signatures. To highlight which ligand-receptor interactions are most prominent in these regional programs, a novel spatial enrichment algorithm was applied. The impact of spatially enriched ligand-receptor interactions on overall survival was evaluated using bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA). Three distinct T-cell regional programs (transitional, immunosuppressive, and infiltrative) were found across both the discovery and validation cohort. The infiltrative T-cell regional program resided on the periphery of the tumor with a neural progenitor phenotype (NRGN, VSNL1, SLC17A7). In contrast, the immunosuppressive T-cell region had a higher proportion of malignant cells and a greater mesenchymal tumor signature (ANXA1, CD44, complement pathway). Furthermore, ligand-receptor enrichment analysis showed that the immunosuppressive region had higher tumor and tumor-associated macrophage (TAM) signaling towards CD4 T-cells along the collagen, midkine, and osteopontin pathways. TCGA survival analysis showed that higher collagen and midkine signals were associated with worse overall and disease free survival. Our results identified three distinct regional programs in GBM and highlight a set of signaling pathways associated with T-cell suppression. Our findings suggest that the disruption of these pathways could reduce immunosuppressive signals from tumor and TAMs to T-cells. Subsequent validation with spatial proteomics data in glioblastoma is ongoing.