T-cell Activation Responses Research Articles

Comparative immune profiling in survivors of the 2023 Nipah outbreak in Kerala state, India.

Immune profiling of Nipah virus (NiV) infection survivors is essential for advancing our understanding of NiV pathogenesis, improving diagnostic and therapeutic strategies, and guiding public health efforts to prevent future outbreaks. There is currently limited data available on the immune response to NiV infection. We aimed to elucidate the specific immune mechanisms involved in protection against NiV infection by analyzing the immune profiles of survivors of the Nipah outbreak in Kerala, India 2023. Immune cell populations were quantified and compared between survivors (up to 4 months post onset day of illness) and healthy controls. Statistical analysis was performed to explore associations between immune profiles and clinical outcomes. Immune signatures common to all three cases were: a heretofore undescribed persistent lymphopenia including the CD4+ Treg compartment with the relative expansion of memory Tregs; trends indicative of global leukopenic modulation were observed in monocytes and granulocytes including an expansion of putatively immunosuppressive low-density granulocytes described recently in the context of severe COVID-19; altered mucosal homing with respect to integrin beta-7 (ITGB7) expressing subsets; increased mobilization of activated T-cells (CD4+ and CD8+) and plasmablasts in the early phase of infection. Comparative analysis based on clinical presentation and outcome yielded lower initial viremia, increased activated T-cell responses, expanded plasmablasts, and restoration of ITGB7 expressing CD8+ T-cells as possible protective signatures. This longitudinal study delineates putative protective signatures associated with milder NiV disease. It emphasizes the need for the development of immunotherapeutic interventions such as monoclonal antibodies to blunt early viremia and ameliorate pathogenesis.

A Simple and Rapid Protocol for the Isolation of Murine Bone Marrow Suitable for the Differentiation of Dendritic Cells.

The generation of bone-marrow-derived dendritic cells is a widely used approach in immunological research to study antigen processing and presentation, as well as T-cell activation responses. However, the initial step of isolating the bone marrow can be time-consuming, especially when larger numbers of precursor cells are required. Here, we assessed whether an accelerated bone marrow isolation method using centrifugation is suitable for the differentiation of FMS-like tyrosine kinase 3 ligand-driven dendritic cells. Compared to the conventional flushing method, the centrifugation-based isolation method resulted in a similar bone marrow cell yield on Day 0, increased cell numbers by Day 8, similar proportions of dendritic cell subsets, and consequently a higher number of type 1 conventional dendritic cells (cDC1) from the culture. Although the primary purpose of this method of optimization was to improve experimental efficiency and increase the output of cDC1s, the protocol is also compatible with the differentiation of other dendritic cell subsets such as cDC2 and plasmacytoid dendritic cells, with an improved output cell count and a consistent phenotype.

(Invited) Lab-in-a-Tube Design Enables a Rapid Diagnosis of Infectious Disease

Disease diagnosis increasingly relies on molecular tests that are often limited by infrastructure constraints. For example, tuberculosis (TB) remains a leading cause of death from infectious disease, but an estimated 4.2 million (39.6%) new TB cases were not diagnosed in 2021 and only 57% were confirmed by gold-standard sputum culture or PCR-based tests, partially due to limited accessibility. New point-of-care tests are thus urgently needed to address coverage disparities for chronic, malignant, and infectious diseases.We have developed two smartphone-based assays that can address two diagnostic shortfalls: an on-chip interferon-gamma release assay (IGRA) that detects a T-cell activation response induced by pathogen antigens in fingerstick blood samples, and a lab-in-tube assay that employs CRISPR to sensitively detect pathogen DNA in sputum. Both approaches translate current diagnostic approaches to point-of-care devices that require minimal equipment, user expertise, and time.Results from our on-chip IGRA also correlated with clinical IGRA results when we employed two proteins associated with TNF-alpha receptor signaling (4-1BB and OX-40) to analyze T-cell activation, indicating this approach could detect infections in individuals with compromised interferon-gamma responses; as observed in HIV patients, who are at increased risk for TB. On-chip IGRA results from a SARS-CoV-2-specific assay also corresponded with both infection and vaccination history, suggesting this data could be employed as a high-throughput, high-capacity test to evaluate the effect of vaccination or previous infection to confer a protective immune response, evaluate its change over time, and predict the response to new variant strains to guide healthcare decisions. Mitogen-induced results could also guide immunotherapy decisions, as reduced IGRA values in patients receiving immune checkpoint inhibitors predict poor treatment response, reduced progression-free survival, and increased risk for interstitial pneumonia, while their change after adoptive T-cell therapy can be an independent predictor for overall survival. Results from our lab-in-tube TB DNA assay also had robust diagnostic performance when compared to gold-standard clinical assays but lacked their more extensive personnel, workflow, and infrastructure requirements and were performed in a closed-tube system that protected user from exposure to infectious material. This lab-in-tube approach also permits parallel multiplex analyses to be performed in a single tube, and thus can be employed to detect mutations associated with drug-resistance to guide treatment decisions. For example, we found that an assay with an optimized guide RNA accurately detected a single-nucleotide polymorphism (rpoB S450L) responsible for the majority of rifampin-resistant TB cases.Both these self-contained handheld devices can employ lyophilized reagents to limit cold-chain concerns, can be readily adapted to diagnose other disease conditions by substituting other disease-specific antibodies or guide RNAs, use rechargeable batteries to avoid infrastructure limitations, and can directly transmit results to central sites to improve disease control efforts.

Laboratory Evaluation of SARS-CoV-2 Specific T-Cell Immunity by ELISpot Interferon-γ Based Immunoassay

Cellular and humoral immune responses are fundamental for SARS-CoV-2 elimination, infection resolution, and protection against reinfection. Data on long-term SARS-CoV-2-specific T-cell responses are limited. We conducted this study to evaluate the presence of SARS-CoV-2 specific T lymphocytes using the ELISpot interferon-γ-based kit for outpatients with or without a history of SARS-CoV-2 infection. SARS-CoV-2 activated T-cell response was defined from peripheral blood mononuclear cells (PBMCs) of nine (n=9) outpatients- 6 women and 3 men, aged 32 to 73 years old, all from Varna city, Bulgaria. They were tested between November and December 2021. Eight of them – 88.9% were in contact with the COVID-19 virus from 1 to 20 months ago. The number of spots obtained provides a measure of the abundance of SARS-CoV-2 – sensitized effector T cells in the peripheral blood. A reactive T-cellular immune response was ob¬served 12 months after SARS-CoV-2 viral infection. In addition, IgG humoral immunity was analyzed and compared with the specific T-cell response in some participants. Compared with humoral immunity-re¬lated studies, those focusing on SARS-CoV-2-specific cellular immunity using the ElISpot immunoassay are relatively falling behind standardization. The SARS-CoV-2 specific ELISpot Interferon-γ immunoassay, described in our study, conducted with a small group of patients, can serve as an effective immunoassay to measure host T-cell responses and help further detailed understanding of pathogenetic mechanisms and persistence of cellular and humoral immunity against SARS-CoV-2.

Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1- Dysfunctional State.

T cells recognize several types of antigens in tumors, including aberrantly expressed, nonmutated proteins, which are therefore shared with normal tissue and referred to as self/shared-antigens (SSA), and mutated proteins or oncogenic viral proteins, which are referred to as tumor-specific antigens (TSA). Immunotherapies such as immune checkpoint blockade (ICB) can activate T-cell responses against TSA, leading to tumor control, and also against SSA, causing immune-related adverse events (irAE). To improve anti-TSA immunity while limiting anti-SSA autoreactivity, we need to understand how tumor-specific CD8+ T cells (TST) and SSA-specific CD8+ T (SST) cells differentiate in response to cognate antigens during tumorigenesis. Therefore, we developed a genetic cancer mouse model in which we can track TST and SST differentiation longitudinally as liver cancers develop. We found that both TST and SST lost effector function over time, but while TST persisted long term and had a dysfunctional/exhausted phenotype (including expression of PD1, CD39, and TOX), SST exited cell cycle prematurely and disappeared from liver lesions. However, SST persisted in spleens in a dysfunctional TCF1+PD-1- state: unable to produce effector cytokines or proliferate in response to ICB targeting PD-1 or PD-L1. Thus, our studies identify a dysfunctional T-cell state occupied by T cells reactive to SSA: a TCF1+PD-1- state lacking in effector function, demonstrating that the type/specificity of tumor antigen may determine tumor-reactive T-cell differentiation.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory Tcells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated Tcell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Mycobacterium tuberculosis impairs human memory CD4+ T cell recognition of M2 but not M1-like macrophages

Direct recognition of Mycobacterium tuberculosis (Mtb)-infected cells is required for protection by CD4+ Tcells. While impaired Tcell recognition of Mtb-infected macrophages was demonstrated in mice, data are lacking for humans. Using Tcells and monocyte-derived macrophages (MDMs) from individuals with latent Mtb infection (LTBI), we quantified the frequency of memory CD4+ Tcell activation in response to autologous MDMs infected with virulent Mtb. We observed robust Tcell activation in response to Mtb infection of M1-like macrophages differentiated using GM-CSF, while M2-like macrophages differentiated using M-CSF were poorly recognized. However, non-infected GM-CSF and M-CSF MDMs loaded with exogenous antigens elicited similar CD4+ Tcell activation. IL-10 was preferentially secreted by infected M-CSF MDMs, and neutralization improved Tcell activation. These results suggest that preferential infection of macrophages with an M2-like phenotype limits Tcell-mediated protection against Mtb. Vaccine development should focus on Tcell recognition of Mtb-infected macrophages.

T Cell-intrinsic Immunomodulatory Effects of TAK-981 (Subasumstat), a SUMO-activating Enzyme Inhibitor, in Chronic Lymphocytic Leukemia.

Novel targeted agents used in therapy of lymphoid malignancies are recognized to have complex immune-mediated effects. Sumoylation, a posttranslational modification of target proteins by small ubiquitin-like modifiers (SUMO), regulates a variety of cellular processes indispensable in immune cell activation. Despite this, the role of sumoylation in T-cell biology in context of cancer is not known. TAK-981 (subasumstat) is a small-molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein. Using T cells derived from patients with chronic lymphocytic leukemia (CLL), we demonstrate that targeting SAE activates type I IFN response. This is accompanied by largely intact T-cell activation in response to T-cell receptor engagement, with increased expression of CD69 and CD38. Furthermore, TAK-981 decreases regulatory T cell (Treg) differentiation and enhances secretion of IFNγ by CD4+ and CD8+ T cells. These findings were recapitulated in mouse models, suggesting an evolutionarily conserved mechanism of T-cell activation regulated by SUMO modification. Relevant to the consideration of TAK-981 as an effective agent for immunotherapy in hematologic malignancies, we demonstrate that the downstream impact of TAK-981 administration is enhancement of the cytotoxic function of CD8+ T cells, thus uncovering immune implications of targeting sumoylation in lymphoid neoplasia.

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor Tcell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ Tcells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ Tcells. Murine tumor models revealed that tumor-specific CD8+ Tcells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ Tcell activation in response to cancer is different from that of canonical CD8+ Tcell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ Tcell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

Strong T-cell activation in response to COVID-19 vaccination in multiple sclerosis patients receiving B-cell depleting therapies.

Immunocompromised individuals, including multiple sclerosis (MS) patients on certain immunotherapy treatments, are considered susceptible to complications from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and specific vaccination regimens have been recommended for suitable protection. MS patients receiving anti-CD20 therapy (aCD20-MS) are considered especially vulnerable due to acquired B-cell depletion and impaired antibody production in response to virus infection and COVID-19 vaccination. Here, the humoral and cellular responses are analyzed in a group of aCD20-MS patients (n=43) compared to a healthy control cohort (n=34) during the first 6 months after a 2-dose cycle mRNA-based COVID-19 vaccination. Both IgG antibodies recognizing receptor binding domain (RBD) from CoV-2 spike protein and their blocking activity against RBD-hACE2 binding were significantly reduced in aCD20-MS patients, with a seroconversion rate of only 23.8%. Interestingly, even under conditions of severe B-cell depletion and failed seroconversion, a significantly higher polyfunctional IFNγ+ and IL-2+ T-cell response and strong T-cell proliferation capacity were detected compared to controls. Moreover, no difference in T-cell response was observed between forms of disease (relapsing remitting- vs progressive-MS), anti-CD20 therapy (Rituximab vs Ocrelizumab) and type of mRNA-based vaccine received (mRNA-1273 vs BNT162b2). These results suggest the generation of a partial adaptive immune response to COVID-19 vaccination in B-cell depleted MS individuals driven by a functionally competent T-cell arm. Investigation into the role of the cellular immune response is important to identifying the level of protection against SARS-CoV-2 in aCD20-MS patients and could have potential implications for future vaccine design and application.

High-fat diet blunts T-cell responsiveness in Nile tilapia

The reduced stress resistance and increased disease risk associated with high-fat diet (HFD) in animals have attracted increasing attention. However, the effects of HFD on adaptive immunity in early vertebrates, especially non-tetrapods, remain unknown. In this study, using Nile tilapia (Oreochromis niloticus) as a model, we investigated the effects of HFD on the primordial T-cell response in fish. Tilapia fed with an HFD for 8 weeks showed impaired lymphocyte homeostasis in the spleen, as indicated by the decreased number of both T and B lymphocytes and increased transcription of proinflammatory cytokines interferon-γ and interleukin-6. Moreover, lymphocytes isolated from HFD-fed fish or cultured in lipid-supplemented medium exhibited diminished T-cell activation in response to CD3ε monoclonal antibody stimulation. Moreover, HFD-fed tilapia infected by Aeromonas hydrophila showed decreased T-cell expansion, increased T-cell apoptosis, reduced granzyme B expression, and impaired infection elimination. Additionally, HFD attenuated adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) activity in tilapia lymphocytes, which in turn upregulated fatty acid synthesis but downregulated fatty acid β-oxidation. Altogether, our results suggest that HFD impairs lymphocyte homeostasis and T cell-mediated adaptive immune response in tilapia, which may be associated with the abnormal lipid metabolism in lymphocytes. These findings thus provide a novel perspective for understanding the impact of HFD on the adaptive immune response of early vertebrates.

Immune-regulating strategy against rheumatoid arthritis by inducing tolerogenic dendritic cells with modified zinc peroxide nanoparticles

In hypoxic dendritic cells (DCs), a low level of Zn2+ can induce the activation of immunogenic DCs (igDCs), thereby triggering an active T-cell response to propel the immune progression of rheumatoid arthritis (RA). This finding indicates the crucial roles of zinc and oxygen homeostasis in DCs during the pathogenesis of RA. However, very few studies have focused on the modulation of zinc and oxygen homeostasis in DCs during RA treatment. Proposed herein is a DC-targeting immune-regulating strategy to induce igDCs into tolerogenic DCs (tDCs) and inhibit subsequent T-cell activation, referred to as ZnO2/Catalase@liposome-Mannose nanoparticles (ZnCM NPs). ZnCM NPs displayed targeted intracellular delivery of Zn2+ and O2 towards igDCs in a pH-responsive manner. After inactivating OTUB1 deubiquitination, the ZnCM NPs promoted CCL5 degradation via NF-κB signalling, thereby inducing the igDC-tDC transition to further inhibit CD4+ T-cell homeostasis. In collagen-induced arthritis (CIA) mice, this nanoimmunoplatform showed significant accumulation in the spleen, where immature DCs (imDCs) differentiated into igDCs. Splenic tDCs were induced to alleviate ankle swelling, improve walking posture and safely inhibit ankle/spleen inflammation. Our work pioneers the combination of DC-targeting nanoplatforms with RA treatments and highlights the significance of zinc and oxygen homeostasis for the immunoregulation of RA by inducing tDCs with modified ZnO2 NPs, which provides novel insight into ion homeostasis regulation for the treatment of immune diseases with a larger variety of distinct metal or nonmetal ions.Graphical

T-cell responses to SARS-CoV-2 Omicron spike epitopes with mutations after the third booster dose of an inactivated vaccine.

The rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Omicron variant contains more than 30 mutations that mediate escape from antibody responses elicited by prior infection or current vaccines. Fortunately, T‐cell responses are highly conserved in most individuals, but the impacts of mutations are not clear. Here, we showed that the T‐cell responses of individuals who underwent booster vaccination with CoronaVac were largely protective against the SARS‐CoV‐2 Omicron spike protein. To specifically estimate the impact of Omicron mutations on vaccinated participants, 16 peptides derived from the spike protein of the ancestral virus or Omicron strain with mutations were used to stimulate peripheral blood mononuclear cells (PBMCs) from the volunteers. Compared with the administration of two doses of vaccine, booster vaccination substantially enhanced T‐cell activation in response to both the ancestral and Omicron epitopes, although the enhancement was slightly weakened by the Omicron mutations. Then, the peptides derived from these spike proteins were used separately to stimulate PBMCs. Interestingly, compared with the ancestral peptides, only the peptides with the G339D or N440K mutation were detected to significantly destabilize the T‐cell response. Although more participants need to be evaluated to confirm this conclusion, our study nonetheless estimates the impacts of mutations on T‐cell responses to the SARS‐CoV‐2 Omicron variant.

Strong T-cell activation in response to SARS-CoV-2 vaccination in patients with multiple sclerosis on B-cell depleting therapies (P1-1.Virtual)

Strong T-cell activation in response to SARS-CoV-2 vaccination in patients with multiple sclerosis on B-cell depleting therapies (P1-1.Virtual)

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector Tcells that exert antitumor function. Immunotherapy unleashes Tcell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 Tcell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 Tcells. Thus, glutaminase inhibition negatively impacts CD8 Tcells activated by anti-PD1 immunotherapy.

Modulation of Dendritic Cells by Microbiota Extracellular Vesicles Influences the Cytokine Profile and Exosome Cargo.

Gut bacteria release extracellular vesicles (BEVs) as an intercellular communication mechanism that primes the host innate immune system. BEVs from E. coli activate dendritic cells (DCs) and subsequent T-cell responses in a strain-specific manner. The specific immunomodulatory effects were, in part, mediated by differential regulation of miRNAs. This study aimed to deepen understanding of the mechanisms of BEVs to drive specific immune responses by analyzing their impact on DC-secreted cytokines and exosomes. DCs were challenged with BEVs from probiotic and commensal E. coli strains. The ability of DC-secreted factors to activate T-cell responses was assessed by cytokine quantification in indirect DCs/naïve CD4+ T-cells co-cultures on Transwell supports. DC-exosomes were characterized in terms of costimulatory molecules and miRNAs cargo. In the absence of direct cellular contacts, DC-secreted factors triggered secretion of effector cytokines by T-cells with the same trend as direct DC/T-cell co-cultures. The main differences between the strains influenced the production of Th1- and Treg-specific cytokines. Exosomes released by BEV-activated DCs were enriched in surface proteins involved in antigen presentation and T-cell activation, but differed in the content of immune-related miRNA, depending on the origin of the BEVs. These differences were consistent with the derived immune responses.

Mapping the landscape of chromatin dynamics during na\xefve CD4+\u2009T-cell activation

T-cell activation induces context-specific gene expression programs that promote energy generation and biosynthesis, progression through the cell cycle and ultimately cell differentiation. The aim of this study was to apply the omni ATAC-seq method to characterize the landscape of chromatin changes induced by T-cell activation in mature naïve CD4+ T-cells. Using a well-established ex vivo protocol of canonical T-cell receptor signaling, we generated genome-wide chromatin maps of naïve T-cells from pediatric donors in quiescent or recently activated states. We identified thousands of individual chromatin accessibility peaks that are associated with T-cell activation, the majority of which were annotated intronic and intergenic enhancer regions. A core set of 3268 gene promoters underwent chromatin remodeling and concomitant changes in gene expression in response to activation, and were enriched in multiple pathways controlling cell cycle regulation, metabolism, inflammatory response genes and cell survival. Leukemia inhibitory factor (LIF) was among those factors that gained the highest accessibility and expression, in addition to IL2-STAT5 dependent chromatin remodeling in the T-cell activation response. Using publicly available data we found the chromatin response was far more dynamic at 24-h compared with 72-h post-activation. In total 546 associations were reproduced at both time-points with similar strength of evidence and directionality of effect. At the pathways level, the IL2-STAT5, KRAS signalling and UV response pathways were replicable at both time-points, although differentially modulated from 24 to 72 h post-activation.

Abstract 1677: Characterization of the pharmacodynamic activity of AGEN1181, an Fc-enhanced CTLA-4 antibody, alone and in combination with the PD-1 antibody balstilimab

Abstract AGEN1181 is a novel, Fc-engineered monoclonal antibody that targets cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), with enhanced FcyR-dependent functionality designed to improve T cell priming and activation, deplete intratumoral regulatory T cells (Treg), and improve memory T cell response. AGEN1181 is currently undergoing Phase I clinical evaluation, both as a single agent and in combination with the anti-PD-1 antibody balstilimab, in patients with advanced solid tumors (NCT03860272). Encouraging signals of clinical benefit have emerged as part of this ongoing study, including complete and durable responses in patients that would traditionally be considered unresponsive to conventional CTLA-4/PD-1 therapy. Here we report on pharmacodynamic analyses associated with AGEN1181 exposure in patients on study in this trial. As monotherapy, AGEN1181 was administered on a once every 3 or once every 6 week cycle (doses escalated from 0.1 - 4 mg/kg). Alternatively, AGEN1181 was dosed once every six weeks in combination with balstilimab (3 mg/kg once every 2 weeks). Tumor mutational burden (TMB) and gene expression were assessed from pre-treatment and on-treatment biopsies by whole exome sequencing (WES) and RNASeq, respectively. Multiplex immunohistochemistry was used to assess Treg depletion and CD8 T-cell activation in response to AGEN1181. TCR repertoire was analyzed in PBMCs collected on-treatment with AGEN1181. FcγRIIIA genotyping was performed by real-time PCR. Immunophenotyping of peripheral blood mononuclear cells collected pre- and post-treatment were analyzed by flow cytometry. Consistent with its proposed mechanism of action, dose-dependent pharmacodynamic effects have been observed in patients while on-treatment with AGEN1181. Peripheral CD4+Ki67+, CD4+ICOS+ and CD4+HLA-DR+ T cells increased over baseline in response to monotherapy. This effect was further enhanced in subjects treated with AGEN1181 when given in combination with balstilimab. Responders expressed either the low affinity FcγRIIIA allele or were heterozygous - suggesting that AGEN1181 has the potential to expand clinical activity to patients with low affinity Fc binding, in contrast to conventional CTLA-4 inhibitors. Additionally, we show that AGEN1181 induced selective Treg depletion within the tumor microenvironment with concomitant increases in CD8 T cell infiltration. TCRSeq analysis demonstrated increased clonality and decreased downsampled diversity, providing further evidence that the Fc-enhanced activity of AGEN1181 can promote improved T cell priming and Treg depletion. In summary, the pharmacodynamic analyses presented here are consistent with the Fc-enhanced design of AGEN1181 and its potential to broaden the therapeutic reach of CTLA-4-based immunotherapy. Citation Format: Irina Shapiro, Min Lim, Simarjot Pabla, Steven J. O'Day, Anthony El-Khoueiry, Chethan Ramamurthy, Andrea J. Bullock, Michael S. Gordon, Jennifer Buell, Dhan Chand, Anna Wijatyk. Characterization of the pharmacodynamic activity of AGEN1181, an Fc-enhanced CTLA-4 antibody, alone and in combination with the PD-1 antibody balstilimab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1677.

Flightless I Homolog Reverses Enzalutamide Resistance through PD-L1-Mediated Immune Evasion in Prostate Cancer.

Tumor cells can evade immune surveillance and immune killing during the emergence of endocrine therapy resistance in prostate cancer, but the mechanisms underlying this phenomenon are still unclear. Flightless I homolog (FLII) is a coregulator for transcription factors in several malignancies. Here, we have demonstrated that endocrine therapy resistance can induce an immunosuppressive prostate tumor microenvironment and immune evasion through FLII downregulation, which leads to activation of the YBX1/PD-L1 signaling pathway. FLII expression negatively correlated with expression of PD-L1 in tumors. Mechanism studies demonstrated that FLII physically interacted with YBX1 to inhibit nuclear localization of YBX1 and thereby suppress transcription of PDL1 in enzalutamide-resistant tumors. Restoration of FLII expression reversed enzalutamide resistance through activation of T-cell responses in the tumor microenvironment through inhibition of the YBX1/PD-L1 pathway. We also found that reversal of endocrine therapy resistance and immune evasion was mediated by proliferation of effector CD8+ T cells and inhibition of tumor infiltration by regulatory T cells and myeloid-derived suppressor cells. Taken together, our results demonstrate a functional and biological interaction between endocrine therapy resistance and immune evasion mediated through the FLII/YBX1/PD-L1 cascade. Combination therapy with FLII expression and endocrine therapy may benefit patients with prostate cancer by preventing tumor immune evasion.

Tuning Antibody Presentation to Enhance T-Cell Activation for Downstream Cytotoxicity

Cytotoxic effector cells are an integral component of the immune response against pathogens and diseases such as cancer and thus of great interest to researchers who wish to enhance the native immune response. Although researchers routinely use particles to stimulate cytotoxic T cells, few studies have comprehensively investigated: (1) beyond initial activation responses (i.e., proliferation and CD25/CD69 expression) to downstream cancer-killing effects and (2) how to drive cytotoxic T-cell responses by adjusting biomolecular and physical properties of particles. In this study, we designed particles displaying an anti-CD3 antibody to activate cytotoxic T cells and study their downstream cytotoxic effects. We evaluated the effect of antibody immobilization, particle size, molecular surface density of an anti-CD3 antibody, and the inclusion of an anti-CD28 antibody on cytolytic granule release by T cells. We found that immobilizing the anti-CD3 antibody onto smaller nanoparticles elicited increased T-cell activation products for an equivalent delivery of the anti-CD3 antibody. We further established that the mechanism behind increased cancer cell death was associated with the proximity of T cells to cancer cells. Functionalizing particles additionally with the anti-CD28 antibody at an optimized antibody density caused increased T-cell proliferation and T-cell binding but we observed no effective increase in cytotoxicity. Meaningfully, our results are discussed within the context of commercially available and widely used anti-CD3/28 Dynabeads. These results showed that T-cell activation and cytotoxicity can be optimized with a molecular presentation on smaller particles and thus, offer exciting new possibilities to engineer T-cell activation responses for effective outcomes.