Augmentation Of Immune Response Research Articles

Expression of VISTA regulated via IFN-γ governs endogenous T-cell function and exhibits correlation with the efficacy of CD19 CAR-T cell treated B-malignant mice

BackgroundDespite continuous improvements in the new target and construction of chimeric antigen receptor (CAR)-T, relapse remains a significant challenge following CAR-T therapy. Tumor microenvironment (TME) strongly correlates with the efficacy...

Relationship between the Number of Repeats in the Neck Regions of L-SIGN and Augmented Virus Replication and Immune Responses in Dengue Hemorrhagic Fever.

C-type lectins play a crucial role as pathogen-recognition receptors for the dengue virus, which is responsible for causing both dengue fever (DF) and dengue hemorrhagic fever (DHF). DHF is a serious illness caused by the dengue virus, which exists in four different serotypes: DEN-1, DEN-2, DEN-3, and DEN-4. We conducted a genetic association study, during a significant DEN-2 outbreak in southern Taiwan, to explore how variations in the neck-region length of L-SIGN (also known as CD209L, CD299, or CLEC4M) impact the severity of dengue infection. PCR genotyping was utilized to identify polymorphisms in variable-number tandem repeats. We constructed L-SIGN variants containing either 7- or 9-tandem repeats and transfected these constructs into K562 and U937 cells, and cytokine and chemokine levels were evaluated using enzyme-linked immunosorbent assays (ELISAs) following DEN-2 virus infection. The L-SIGN allele 9 was observed to correlate with a heightened risk of developing DHF. Subsequent results revealed that the 9-tandem repeat was linked to elevated viral load alongside predominant T-helper 2 (Th2) cell responses (IL-4 and IL-10) in K562 and U937 cells. Transfecting K562 cells in vitro with L-SIGN variants containing 7- and 9-tandem repeats confirmed that the 9-tandem repeat transfectants facilitated a higher dengue viral load accompanied by increased cytokine production (MCP-1, IL-6, and IL-8). Considering the higher prevalence of DHF and an increased frequency of the L-SIGN neck's 9-tandem repeat in the Taiwanese population, individuals with the 9-tandem repeat may necessitate more stringent protection against mosquito bites during dengue outbreaks in Taiwan.

Augmented antitumor immune responses of HER2-targeted pyroptotic induction by long-lasting recombinant immunopyroptotins

Pyroptosis is a well-documented form of programmed cell death caused by the gasdermin-driven perforation of cell membranes. Selective induction of pyroptosis in tumor cells represents a promising antitumor strategy to enhance the efficacy of immunotherapy. In this study, we established a recombinant protein-based immunopyroptotin strategy that led to the intratumoral induction of pyroptosis for HER2-directed therapy. Long-lasting immunopyroptotins were constructed by sequentially fusing the humanized anti-HER2 single-chain antibody P1h3, albumin-binding peptide (ABD035 or dAb7h8), cathepsin B-cleavable peptide B2, endosome-disruptive peptide E5C3, and active pyroptotic effector gasdermin D-N fragment (GN). After purification, we evaluated the cytotoxicity and antitumor immune responses primarily induced by the immunopyroptotins in HER2-overexpressing breast cancer cells. The resulting ABD035-immunoGN and dAb7h8-immunoGN showed improved in vitro cytotoxicity in HER2-overexpressing cancer cells compared with that in the immunotBid that we previously generated to induce tumor cell apoptosis. The binding of long-lasting immunopyroptotins to albumin increased the half-life by approximately 7-fold in nude mice. The enhanced antitumor efficacy of long-lasting immunopyroptotins was confirmed in both N87 tumor-bearing T cell-deficient mice and 4T1-hHER2 bilateral tumor-bearing immunocompetent mice. Immunopyroptotin treatment elicited systemic antitumor immune responses involving CD8+ T cells and mature dendritic cells and upregulated the expression of proinflammatory cytokines, leading to sustained remission of non-injected distant tumors. This study extends the repertoire of antibody-based therapeutics through the tumor-targeted delivery of a constitutively active pore-forming gasdermin-N fragment, which shows great potential for pyroptosis-based antitumor therapy.

Abstract 1353: Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (anti-PDL1 or anti-CTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of anti-PDL1 and anti-CTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of anti-PDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and anti-CTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. mice. Lymph nodes and tumors from mice treated with IR and anti-CTLA4 vs. IR and isotype control demonstrated differential expression of genes in T cell functions and enrichment in both CD4+ and CD8+ T cell populations. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining anti-CTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Linsey Gong, Xiaoyong Huang, Stephanie D. White, Hanz T. Chung, Danny Vesprini, Tera N. Petchiny, Emmanouil Fokas, Hansen He, Robert S. Kerbel, Stanley K. Liu. Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1353.

Multifunctional iron-apigenin nanocomplex conducting photothermal therapy and triggering augmented immune response for triple negative breast cancer

Triple negative breast cancer (TNBC) presents a formidable challenge due to its low sensitivity to many chemotherapeutic drugs and a relatively low overall survival rate in clinical practice. Photothermal therapy has recently garnered substantial interest in cancer treatment, owing to its swift therapeutic effectiveness and minimal impact on normal cells. Metal-polyphenol nanostructures have recently garnered significant attention as photothermal transduction agents due to their facile preparation and favorable photothermal properties. In this study, we employed a coordinated approach involving Fe3+ and apigenin, a polyphenol compound, to construct the nanostructure (nFeAPG), with the assistance of β-CD and DSPE-PEG facilitating the formation of the complex nanostructure. In vitro research demonstrated that the formed nFeAPG could induce cell death by elevating intracellular oxidative stress, inhibiting antioxidative system, and promoting apoptosis and ferroptosis, and near infrared spectrum irradiation further strengthen the therapeutic outcome. In 4T1 tumor bearing mice, nFeAPG could effectively accumulate into tumor site and exhibit commendable control over tumor growth. Futher analysis demonstrated that nFeAPG ameliorated the suppressed immune microenvironment by augmenting the response of DC cells and T cells. This study underscores that nFeAPG encompasses a multifaceted capacity to combat TNBC, holding promise as a compelling therapeutic strategy for TNBC treatment.

Targeting refractory/recurrent neuroblastoma and osteosarcoma with anti-CD3×anti-GD2 bispecific antibody armed T cells

BackgroundThe survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits...

Novel UV-B Phototherapy With a Light-Emitting Diode Device Prevents Atherosclerosis by Augmenting Regulatory T-Cell Responses in Mice.

Ultraviolet B (UV-B) irradiation is an effective treatment for human cutaneous disorders and was shown to reduce experimental atherosclerosis by attenuating immunoinflammatory responses. The aim of this study was to clarify the effect of specific wavelengths of UV-B on atherosclerosis and the underlying mechanisms focusing on immunoinflammatory responses. Based on light-emitting diode technology, we developed novel devices that can emit 282 nm UV-B, which we do not receive from natural sunlight, 301 nm UV-B, and clinically available 312 nm UV-B. We irradiated 6-week-old male atherosclerosis-prone Apoe-/- (apolipoprotein E-deficient) mice with specific wavelengths of UV-B and evaluated atherosclerosis and immunoinflammatory responses by performing histological analysis, flow cytometry, biochemical assays, and liquid chromatography/mass spectrometry-based lipidomics. Irradiation of 282 nm UV-B but not 301 or 312 nm UV-B significantly reduced the development of aortic root atherosclerotic plaques and plaque inflammation. This atheroprotection was associated with specifically augmented immune responses of anti-inflammatory CD4+ Foxp3 (forkhead box P3)+ regulatory T cells in lymphoid tissues, whereas responses of other immune cells were not substantially affected. Analysis of various lipid mediators revealed that 282 nm UV-B markedly increased the ratio of proresolving to proinflammatory lipid mediators in the skin. We demonstrated that 282 nm UV-B irradiation effectively reduces aortic inflammation and the development of atherosclerosis by systemically augmenting regulatory T-cell responses and modulating the balance between proresolving and proinflammatory lipid mediators in the skin. Our findings indicate that a novel 282 nm UV-B phototherapy could be an attractive approach to treat atherosclerosis.

Abstract A025: Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (αPDL1 or αCTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects[SL1]. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of αPDL1 and αCTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of αPDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and αCTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. NanoString analysis of lymph nodes from mice treated with αCTLA4 and IR vs. IR alone demonstrated differential expression of genes regulating CD molecules, interleukins, and T-cell functions. Furthermore, enrichment of CD45 and CD8a RNA following αCTLA4 and IR was observed. Validation by flow cytometry is currently ongoing. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining αCTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Xiaoyong Huang, Stepahnie D White, Linsey Gong, Tera N Petchiny, Hans Chung, Emmanouil Fokas, Robert S Kerbel, Stanley K Liu. Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A025.

Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE

AbstractImmunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G–Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

Beyond CTLA-4 and PD-1 Inhibition: Novel Immune Checkpoint Molecules for Melanoma Treatment.

More than ten years after the approval of ipilimumab, immune checkpoint inhibitors (ICIs) against PD-1 and CTLA-4 have been established as the most effective treatment for locally advanced or metastatic melanoma, achieving durable responses either as monotherapies or in combinatorial regimens. However, a considerable proportion of patients do not respond or experience early relapse, due to multiple parameters that contribute to melanoma resistance. The expression of other immune checkpoints beyond the PD-1 and CTLA-4 molecules remains a major mechanism of immune evasion. The recent approval of anti-LAG-3 ICI, relatlimab, in combination with nivolumab for metastatic disease, has capitalized on the extensive research in the field and has highlighted the potential for further improvement of melanoma prognosis by synergistically blocking additional immune targets with new ICI-doublets, antibody-drug conjugates, or other novel modalities. Herein, we provide a comprehensive overview of presently published immune checkpoint molecules, including LAG-3, TIGIT, TIM-3, VISTA, IDO1/IDO2/TDO, CD27/CD70, CD39/73, HVEM/BTLA/CD160 and B7-H3. Beginning from their immunomodulatory properties as co-inhibitory or co-stimulatory receptors, we present all therapeutic modalities targeting these molecules that have been tested in melanoma treatment either in preclinical or clinical settings. Better understanding of the checkpoint-mediated crosstalk between melanoma and immune effector cells is essential for generating more effective strategies with augmented immune response.

Clinical characteristics and management of immune checkpoint inhibitor-related cardiotoxicity: A single-center experience.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy in the past decade and amplify T-cell-mediated immune responses by disrupting immunoinhibitory signals. The augmented T-cell immune response has led to a range of immune-related adverse effects (irAEs). Immune-related cardiotoxicity has been reported in case series but has been underappreciated due to difficulties in diagnosis. This article describes epidemiological, clinical presentation, subtype, and treatment data and a new systematic framework for the clinical management of cardiotoxicity. Data were extracted for cancer patients who received ICIs in a single center between January 1, 2020, and February 28, 2022. ICI-associated cardiotoxicity was clinically diagnosed based on clinical presentations, biochemical biomarkers, and imaging features. We identified a total of 12 (2.46%) cases of ICI-related cardiotoxicity from 487 patients who received PD-1 or PD-L1 inhibitors. All patients were diagnosed with advanced or metastatic solid tumors. The severity of ICI-related cardiotoxicity ranged from subclinical cardiac abnormalities (subclinical type) with only asymptomatic troponin-I (TnI) elevations (25.0%) to symptomatic cardiac abnormalities (clinical type) (75.0%). Patients with symptomatic cardiac abnormalities had several manifestations, including tachyarrhythmia (16.7%), bradyarrhythmia (41.7%), or cardiac failure (8.3%). The median immunotherapy exposure time was 1.5 doses (range: 1 to 5), and the median time from the initial immunotherapy to the onset of ICI-related cardiotoxicity was 33.5 days (IQR: 20.3 to 46.8). Most patients, including those with subclinical cardiac abnormalities, were administered systemic corticosteroids (58.3%). One (8.3%) patient was put on mechanical ventilation, one (8.3%) received plasma exchange therapy, one (8.3%) was implanted with a pacemaker, and one (8.3%) was admitted to the ICU. Three patients with symptomatic cardiac abnormalities (25.0%) died, and other patients presented with significant clinical improvement with good outcomes. ICI-related cardiotoxicity is uncommon but critical with a high mortality rate and poor prognosis, especially for a small group of patients with symptomatic cardiac abnormalities. More attention should be given to cardiotoxicity associated with ICIs, and these patients should be given baseline examinations and biochemical analyses before and after the initiation of immunotherapy, intensive cardiac assessments, an accurate and rapid diagnosis, and timely multidisciplinary management with immunosuppressive agents and other necessary clinical interventions.

Abstract 5104: Targeting IL-1B synergizes with PD-1 blockade for enhanced T and B cell immune responses and inhibition of early lung cancer development

Abstract Early immunotherapy with inhibitors of immune checkpoints such as PD-1 has revolutionized lung adenocarcinoma (LUAD) treatment. Still, many patients do not respond or relapse following PD-1 blockade. Tumor-promoting inflammation, such as that mediated by the pleiotropic cytokine interleukin 1 beta (IL-1B), fosters immunosuppression in the tumor immune microenvironment (TIME). Our group and others showed that IL-1B blockade inhibits lung cancer development. We thus hypothesized that addition of IL-1B blockade to anti-PD-1 treatment may enhance outcomes against LUAD. Using a human-relevant, tobacco-associated, mouse model of LUAD development, we compared the effects of combined PD-1 and IL-1B blockade relative to treatment with single-agents (anti-IL-1B or anti-PD-1) and control antibody on early lung tumor development and the TIME. Drugs (anti-PD-1 + anti-IL-1B, anti-PD-1, anti-IL-1B, control IgG) were administered at end of exposure to the tobacco-specific carcinogen NNK, to evaluate effects on formation of early lesions (preventive), or at 3 months post-NNK (prophylactic) to interrogate LUAD development (8 groups). Comprehensive interrogation of the lung ecosystem and the TIME was performed using deep single-cell RNA-sequencing (scRNA-seq) analysis in a subset of the mice (n = 3 to 4) from each of the 8 groups (n = 31 total; 143,897 cells after stringent quality control). Mice treated with combined PD-1 and IL-1B blockade displayed reduced development of lung tumors when compared to animals treated with anti-IL-1B, anti-PD-1, or control antibodies. Fractions of cytotoxic Cd8+ T cells were conspicuously higher and those of tumor cells and exhausted Cd8+ T cells evidently lower in lungs of mice treated with combined PD-1 and IL-1B blockade relative to monotherapy- or control antibody-treated animals. Igha+ plasma cells were strikingly highest in lungs of mice treated with combined PD-1 and IL-1B blockade and nearly absent in monotherapy- and control-treated groups. Lungs of mice treated with combined PD-1 and IL-1B blockade showed higher fractions of Cd80+/Cd86+ memory B cells and, consistently, T follicular helper T cells, while exhibiting reduced fractions of naïve B and Cd24a+/Tgfb1+ B cells suggestive of enhanced activation of B cell responses by the combinatorial treatment. These effects were, overall, present, or much more pronounced, in animals that were prophylactically treated. Flow cytometry analysis of lung tissues and immune profiling of bronchioalveolar lavage fluid overall confirmed augmented immune cell responses by combined PD-1 and IL-1B blockade. Our findings show that blocking IL-1B synergizes with anti-PD-1 in regression of early tumor cells and reversal of immunosuppression. Combined blockade of PD-1 and IL-1B may be a promising strategy for early treatment of lung cancer that warrants further clinical studies. Citation Format: Warapen Treekitkarnmongkol, Guangchun Han, Zahraa Rahal, Jiping Feng, Ansam Sinjab, Tina Cascone, Christopher S. Stevenson, Cheryl Sweeney, Matt Edwards, Avrum Spira, Junya Fujimoto, Seyed Javad Moghaddam, Linghua Wang, Humam Kadara. Targeting IL-1B synergizes with PD-1 blockade for enhanced T and B cell immune responses and inhibition of early lung cancer development. [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 5104.

Abstract OT1-19-01: A Single Arm Phase 2 Study of Peri-Operative Checkpoint-Mediated Immune Therapy and Cryoablation in Women with Hormone Receptor-Negative, HER2-Negative Early Stage/Resectable Breast Cancer

Abstract Background: Local tumor destruction with cryoablation (cryo) induces inflammation and releases antigens that can activate tumor-specific immune responses. Pre-clinically, cryo with immune checkpoint inhibition (ICI)-augmented tumor-specific immune responses and prevented recurrence. Clinically, we established that peri-operative (peri-op) cryo with ipilimumab (ipi) +/- nivolumab (nivo) was not only safe in patients (pts) with operable, early stage breast cancer (ESBC) but also generated robust intra-tumoral and systemic immune responses. In this phase 2 study, we evaluate the disease specific impact of peri-op ICI in women with residual triple negative breast cancer (TNBC) after neoadjuvant chemotherapy (NAC), a subset at high risk of early relapse. Methods: Eligible pts are ≥18y, with ER < 10%, PR < 10%, HER2 negative (per ASCO/CAP definition), ≥ 1.0 cm, residual operable disease after taxane-based NAC. Approximately 80 pts will be enrolled and treated with ipi/nivo/cryo followed by breast surgery and adjuvant nivo across multiple institutions. Pts undergo percutaneous, image-guided cryo with concurrent research core biopsy 7-10 days prior to surgery and will receive ipi (1mg/kg IV) with nivo (240mg IV) 1 to 5 days prior to cryo. After surgery, pts will receive 3 additional doses of nivo at 240mg IV Q2 weeks. Adjuvant capecitabine is recommended for all patients per local standard-of-care. Patients will be stratified by NAC platinum administration, NAC anthracycline administration, and clinical nodal status (positive versus negative). The primary endpoint is 3-year Event Free Survival (EFS). Secondary endpoints include Invasive Disease-Free Survival (IDFS), Distant Disease-Free Survival (DDFS), overall survival (OS) and safety. Exploratory correlative studies will be performed on tumor and serum to characterize the immunologic impact of the intervention and to explore predictors of efficacy and toxicity. Funding sources: Susan G. Komen, ASCO Conquer Cancer Foundation, Breast Cancer Research Foundation, Bristol-Myers Squibb, BTG International Ltd. NCT03546686 Citation Format: Heather McArthur, Elizabeth Comen, Yolanda Bryce, Stephen Solomon, Jorge Henrique Santos Leal, Christina DiLauro Abaya, Cristal Martinez, Reva Basho, Dorothy Park, Philomena McAndrew, Brigid Larkin, William Mills, David B. Page, Staci Mellinger, Nicole Fredrich, Nicole Moxon, Sangeetha Reddy, Meredith Carter, Sujata Patil, Larry Norton. A Single Arm Phase 2 Study of Peri-Operative Checkpoint-Mediated Immune Therapy and Cryoablation in Women with Hormone Receptor-Negative, HER2-Negative Early Stage/Resectable Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-19-01.

NLRP12 is an innate immune checkpoint for repressing IFN signatures and attenuating lupus nephritis progression.

Signaling driven by nucleic acid sensors participates in interferonopathy-mediated autoimmune diseases. NLRP12, a pyrin-containing NLR protein, is a negative regulator of innate immune activation and type I interferon (IFN-I) production. Peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients expressed lower levels of NLRP12, with an inverse correlation with IFNA expression and high disease activity. NLRP12 expression was transcriptionally suppressed by runt-related transcription factor 1-dependent (RUNX1-dependent) epigenetic regulation under IFN-I treatment, which enhanced a negative feedback loop between low NLRP12 expression and IFN-I production. Reduced NLRP12 protein levels in SLE monocytes was linked to spontaneous activation of innate immune signaling and hyperresponsiveness to nucleic acid stimulations. Pristane-treated Nlrp12-/- mice exhibited augmented inflammation and immune responses; and substantial lymphoid hypertrophy was characterized in NLRP12-deficient lupus-prone mice. NLRP12 deficiency mediated the increase of autoantibody production, intensive glomerular IgG deposition, monocyte recruitment, and the deterioration of kidney function. These were bound in an IFN-I signature-dependent manner in the mouse models. Collectively, we reveal a remarkable link between low NLRP12 expression and lupus progression, which suggests the impact of NLRP12 on homeostasis and immune resilience.

PS-B06-9: SINGLE-CELL TRANSCRIPTOMIC PROFILING OF AORTIC B CELLS DURING EXPERIMENTAL HYPERTENSION

Objective: Inflammation and immunity are known to strongly influence aortic fibrosis leading to stiffening and remodelling. We have shown that genetic and pharmacological ablation of B cells affords protection from the development of aortic stiffening and remodelling associated with hypertension. Whether this protective phenotype is due to systemic effects such as reduced antibody secretion, or local B cell effector functions within the aorta remains unclear. Using single-cell transcriptomics, this study aimed to characterise the B cell populations present in the mouse aorta and identify phenotypes/effector functions which may drive inflammation and aortic remodelling. Design and Methods: Hypertension was induced in 12-week-old C57BL6/J mice by infusion of angiotensin II (0.7 mg/kg/day; s.c.) for 28-days via osmotic minipumps. Normotensive mice received vehicle infusion (0.5% NaCl, 0.1% acetic acid). Weekly systolic blood pressure (SBP) measurements were acquired by tail-cuff plethysmography. High-resolution ultrasound imaging was used for pulse wave velocity (PWV) measurements on day 28. Thoracic and abdominal aortas were then isolated and digested for single-cell transcriptomic analyses. Aortic suspensions were stained with Calcein Blue and Vybrant Ruby to sort for metabolically active, nucleated cells. RNA libraries were generated using the 10x Chromium platform. Results and Conclusions: Angiotensin II caused significant increases in SBPs (∼180 mmHg) compared to vehicle treated mice (∼120 mmHg). Hypertensive (HT) mice had increased aortic PWV (∼4 m/s) compared to normotensive (NT) mice (∼1 m/s). Single-cell transcriptomic analyses revealed a near 2-fold increase in B cells present in the aorta of hypertensive mice. Leiden-based community detection identified 7 clusters within the aortic B cell population, including pro-B cells, pre-BCR-independent B cells, transitional B cell populations, B1 and B2 B cells, as well as antibody secreting cells. A greater number of B2 B cells was observed in the hypertensive aorta (58% HT vs 36% NT), without an observable increase in B1 B cell numbers. Gene ontology (GO) analysis of aortic B2 B cells from angiotensin II-treated mice revealed an upregulation in biological processes associated with B cell proliferation (GO:0042100), B cell activation (GO:0042113) and leukocyte differentiation (GO:0002521) compared to vehicle controls. Interestingly, the majority of antibody secreting cells in the aorta appear to be derived from B1 B cells in both vehicle (∼82%) and angiotensin II-treated mice (∼96%). In summary, hypertension is associated with a divergent aortic B cell transcriptome reflective of an augmented immune response, with observable shifts in composition which may be associated with inflammation and aortic remodelling.

Long-term sialidase-specific immune responses after natural infection with cholera: Findings from a longitudinal cohort study in Bangladesh.

Immune responses that target sialidase occur following natural cholera and have been associated with protection against cholera. Sialidase is a neuraminidase that facilitates the binding of cholera toxin (CT) to intestinal epithelial cells. Despite this, little is known about age-related sialidase-specific immune responses and the impact of nutritional status and co-infection on sialidase-specific immunity. We enrolled 50 culture-confirmed Vibrio cholerae O1 cholera cases presenting to the icddr,b Dhaka hospital with moderate to severe dehydration. We evaluated antibody responses out to 18 months (day 540) following cholera. We assessed immune responses targeting sialidase, lipopolysaccharide (LPS), cholera toxin B subunit (CtxB), and vibriocidal responses. We also explored the association of sialidase-specific immune responses to nutritional parameters and parasitic co-infection of cases. This longitudinal cohort study showed age-dependent differences in anti-sialidase immune response after natural cholera infection. Adult patients developed plasma anti-sialidase IgA and IgG responses after acute infection (P<0.05), which gradually decreased from day 30 on. In children, no significant anti-sialidase IgA, IgM, and IgG response was seen with the exception of a late IgG response at study day 540 (p=0.05 compared to adults). There was a correlation between anti-sialidase IgA with vibriocidal titers, as well as anti-sialidase IgA and IgG with anti-LPS and anti-CtxB antibody responses in adult patients, whereas in children, a significant positive correlation was seen only between anti-sialidase IgA and CtxB IgA responses. Stunted children showed significantly lower anti-sialidase IgA, IgG, and IgM antibody responses and higher LPS IgG and IgM antibody responses than healthy children. The anti-sialidase IgA and IgG responses were significantly higher in cases with concomitant parasitic infection. Our data suggest that cholera patients develop age-distinct systemic and mucosal immune responses against sialidase. The stunted children have a lower anti-sialidase antibody response which may be associated with gut enteropathy and the neuraminidase plays an important role in augmented immune response in cholera patients infected with parasites.

Pheophorbide A-Mediated Photodynamic Therapy Potentiates Checkpoint Blockade Therapy of Tumor with Low PD-L1 Expression.

Although the immune checkpoint blockade (ICB) has made a great success in cancer immunotherapy, the overall response rate to the ICB, such as anti-programmed death ligand 1 (PD-L1) therapy, remains only at 20-30%. One major reason is the low expression level of the immune checkpoint in a certain type of tumor cells and its insufficient activation of the host immune system. Herein, we reported a cyclic RGD (cRGD)-modified liposomal delivery system loading the anti-PD-L1 antibody and the photosensitizer pheophorbide A (Pa), allowing a targeting of the low PD-L1 expressing 4T1 mouse breast cancer cells through the recognition of an overexpression of αvβ3 integrin on the tumor cells. The Pa-mediated photodynamic therapy (PDT) elevated the expression of PD-L1 on the tumor cells. PDT, in combination with the anti-PD-L1 therapy, promoted the activation and maturation of dendritic cells as well as the infiltration of cytotoxic T lymphocytes, resulting in the augmented antitumor immune response for the enhanced therapeutic effect. These results demonstrated the combined therapeutic effects of PDT and ICB on the tumor with low PD-L1 levels. Our study suggested that an increase in the PD-L1 expression in tumor cells by PDT would be a promising adjuvant treatment to overcome the ICB irresponsiveness.

Antibody-Mediated Antigen Loss Can Switch an Augmented Immune Response to Antibody-Mediated Immunosuppression

Antibody-Mediated Antigen Loss Can Switch an Augmented Immune Response to Antibody-Mediated Immunosuppression

IMMU-31. PROTEASOME REARRANGED PEPTIDES ARE A NOVEL SOURCE OF ANTIGENS FOR THE PEPTIDE VACCINE IMMUNOTHERAPY OF GLIOBLASTOMAS

Abstract pepVIII, an anticancer vaccine targeting EGFRvIII, has been tested in several trials for glioblastoma including ACT IV. a randomized Phase III trial for newly diagnosed patients. While results from ACT IV were equivocal, available data suggests the elimination of EGFRvIII was not robust. Despite its essential role in antigen presentation, enhancing proteasomal processing is an unexploited strategy for improving vaccines. We examined 20 peptides in silico and experimentally, which showed that a tyrosine substitution (Y6-pepVIII) maximizes proteasome cleavage and survival in a subcutaneous tumor model. In an intracranial glioma model, Y6-pepVIII showed a 62 and 31% improvement in median survival compared to control animals and pepVIII-vaccinated mice. Y6-pepVIII vaccination altered tumor-infiltrating lymphocyte subsets and expression of PD-1 on intratumoral T cells. Combination with anti–PD-1 therapy cured 45% of the Y6-pepVIII–vaccinated mice but was ineffective for pepVIII-treated mice. LC-MS/MS analysis of proteasome-digested pepVIII and Y6-pepVIII revealed that most fragments were similar but more abundant inY6-pepVIII digests and 77% resulted from proteasome-catalyzed peptide splicing (PCPS). We identified 10 peptides that bound human and murine MHC class I. Nine were PCPS products and only one peptide was colinear with EGFRvIII, indicating that PCPS fragments may be a component of MHC class I recognition. Despite not being colinear with EGFRvIII, two of three PCPS products tested were capable of increasing survival when administered independently as vaccines. We hypothesize that the immune response to a vaccine represents the collective contribution from multiple PCPS and linear products. Our work suggests a strategy to increase proteasomal processing of a vaccine that results in an augmented immune response and enhanced survival in mice.

Hsc70-4 aggravates PolyQ-mediated neurodegeneration by modulating NF-κB mediated immune response in Drosophila.

Huntington's disease occurs when the stretch of CAG repeats in exon 1 of the huntingtin (htt) gene crosses the permissible limit, causing the mutated protein (mHtt) to form insoluble aggregates or inclusion bodies. These aggregates are non-typically associated with various essential proteins in the cells, thus disrupting cellular homeostasis. The cells try to bring back normalcy by synthesizing evolutionary conserved cellular chaperones, and Hsp70 is one of the families of heat shock proteins that has a significant part in this, which comprises of heat-inducible and cognate forms. Here, we demonstrate that the heat shock cognate (Hsc70) isoform, Hsc70-4/HSPA8, has a distinct role in polyglutamate (PolyQ)-mediated pathogenicity, and its expression is enhanced in the polyQ conditions in Drosophila. Downregulation of hsc70-4 rescues PolyQ pathogenicity with a notable improvement in the ommatidia arrangement and near-normal restoration of optic neurons leading to improvement in phototaxis response. Reduced hsc70-4 also attenuates the augmented immune response by decreasing the expression of NF-κB and the antimicrobial peptides, along with that JNK overactivation is also restored. These lead to the rescue of the photoreceptor cells, indicating a decrease in the caspase activity, thus reverting the PolyQ pathogenicity. At the molecular level, we show the interaction between Hsc70-4, Polyglutamine aggregates, and NF-κB, which may be responsible for the dysregulation of signaling molecules in polyQ conditions. Thus, the present data provides a functional link between Hsc70-4 and NF-κB under polyQ conditions.