Teff Cells Research Articles

Autoimmune and Neoplastic Outcomes After the mRNA Vaccination: The Role of T Regulatory Cell Responses

A plethora of autoimmune disease incidences occured after COVID-19 mRNA injections were rolled out. Aggressive cancer cases have occurred in the bodies of recipients at sites where the mRNA was injected and at distant metastatic sites. The mRNA vaccines cause thymic involution (shrinking) and dysregulation of the T regulatory (Treg) and T effector (Teff) homeostatic cell balance. Activated immune cells deliver spike protein to the thymic epithelial cells, damaging them. The Treg/Teff balance may determine the fate of autoimmunity and/or cancer and is different for patients with cancer versus those without any cancerous tissues. Repeated mRNA injections lead to empirical evidence of impaired immune functions (elevated IgG4, PD-L1), associated with increased autoimmunity and cancer risks, and decreased resistance to infections. In the with-cancer recipients, the mRNA vaccine may be associated with either autoimmunity or further progression of the cancer(s) depending on the immunotherapy treatment the patient receives. When an antigen stimulates the immune system, specific T regulatory (Treg) and T effector (Teff) subpopulations develop from naïve T cells. An imbalance between Treg and Teff cells can lead to either cancer or autoimmunity. Treg cells are beneficial in that they protect from autoimmunity. However, they suppress the immune response to tumors. In this review, we analyze Treg responses after SARS-CoV-2 mRNA vaccination and find distinct pathological responses under differing conditions. Injection with modified mRNA can lead to a delayed but highly active immune response, resulting in overactivation of the inflammasome. The mRNA “vaccine” induces a very strong IgG antibody response, while suppressing CD8+ T cell activation. Exosomes distribute the recombinant, synthetic spike protein and the mRNA encoding it throughout the organism. In cancer patients, disease progression depends on the starting point of the cancer patient at the time of injection and the type of cancer treatment underway. Migration of circulating dendritic cells and Treg cells back to the thymus, while they are carrying spike protein, damages the medullary thymic epithelial cells and accelerates thymic involution, a direct cause of inflammaging and immunosenescence. In summary, the Treg responses to mRNA injections and subsequent mRNA-encoded SARS-CoV-2 spike protein expression may disrupt immune capacities resulting in accelerated development of autoimmune disease and cancer. The processes discussed here are consistent with both epidemiological findings and case reports.

Exploring CD26-/lo subpopulations of lymphocytes in asthma phenotype and severity: A novel CD4+ T cell subset expressing archetypical granulocyte proteins.

Asthma pathology may induce changes in naïve/memory lymphocyte proportions assessable through the evaluation of surface CD26 (dipeptidyl peptidase 4/DPP4) levels. Our aim was to investigate the association of asthma phenotype/severity with the relative frequency of CD26-/lo, CD26int and CD26hi subsets within different lymphocyte populations. The proportion of CD26-/lo, CD26int and CD26hi subsets within CD4+ effector T cells (Teff), total CD4- lymphocytes, γδ-T cells, NK cells and NKT cells was measured in peripheral blood samples from healthy (N = 30) and asthma (N = 119) donors with different phenotypes/severities by flow cytometry. We performed K-means clustering analysis and further characterised the CD4+CD26-/lo Teff cell subset by LC-MS/MS and immunofluorescence. Cluster analysis including clinical and flow cytometry data resulted in four groups, two of them with opposite inflammatory profiles (neutrophilic vs. eosinophilic). Neutrophilic asthma presented reduced CD4-CD26hi cells, which negatively correlated with systemic inflammation. Eosinophilic asthma displayed a general expansion of CD26-/lo subsets. Specifically, CD4+CD26-/lo Teff expansion was confirmed in asthma, especially in atopic patients. Proteomic characterisation of this subset with a TEM/TEMRA phenotype revealed upregulated levels of innate (e.g. MPO and RNASE2) and cytoskeleton/extracellular matrix (e.g. MMP9 and ACTN1) proteins. Immunofluorescence assays confirmed the presence of atypical proteins for CD4+ T cells, and an enrichment in 'flower-like' nuclei and MMP9/RNASE2 levels in CD4+CD26-/lo Teff compared to CD4+ T lymphocytes. There is an association between CD26 levels in different lymphocyte subsets and asthma phenotype/severity. CD4+CD26-/loTEMRA cells expressing innate proteins specific to eosinophils/neutrophils could be determinant in sustaining long-term inflammation in adult allergic asthma.

Chemokine receptors are required for effector T-cell trafficking to GVHD tissues but not to bone marrow

AbstractIn allogeneic hematopoietic stem cell transplantation (allo-SCT), alloreactive donor T cells mediate the graft-versus-leukemia effect but also attack nonhematopoietic tissues, causing graft-versus-host disease (GVHD). Reducing alloreactive T-cell trafficking to GVHD target tissues while allowing their access to bone marrow (BM) and spleen, major sites of malignant hematopoiesis, is a rational strategy for reducing the GVHD risk when using alloreactive T cells as a therapeutic. Here, we show that effector T-cell (Teff) entry into BM and spleen in unmanipulated mice and in mice that received transplantation without alloreactive T cells is augmented by pertussis toxin (PTX)-sensitive chemokine receptor signaling. However, unexpectedly, in the presence of a GVH response, chemokines no longer draw T cells into BM and spleen but remain critical for their recruitment to GVHD target tissues. Consistent with this, PTX-treated Teff cells were as efficacious as untreated T cells in killing leukemia cells in BM and spleen in mice with a concurrent GVHD response. These results suggest a strategy to improve the safety of alloreactive T-cell therapeutics in treating leukemias in the context of an allo-SCT.

Alternative activation of mast cells by CD4+ T helper cells.

Effector CD4+ T lymphocytes (Teff) infiltrate sites of inflammation and orchestrate the immune response by instructing local leukocytes. Mast cells (MCs) are tissue sentinel cells strategically located near blood vessels and T cell rich areas. MC/Teff cells interactions shape Teff cell responses but in turn, Teff cell action on MC is still poorly understood. Here, we analyzed the human MC/Teff cells interplay through both the application of RNAseq and functional assays. We showed that activated Teff cells induce a specific transcriptomic program in MCs including production of both inflammatory cytokines and chemokines, prostaglandin, and a FcεRI-dependent degranulation facilitation thereby driving them toward an inflammatory phenotype. Moreover, Teff cells induce in MCs the capacity to interact with CD4+ T cell through a wide-range of dedicated soluble and membrane ligands and to play the role of antigen presenting cells (APCs).

Grooved Microneedle Patch Augments Adoptive T Cell Therapy Against Solid Tumors via Diverting Regulatory T Cells.

The efficacy of adoptive T cell therapy (ACT) for the treatment of solid tumors remains challenging. In addition to the poor infiltration of effector T (Teff) cells limited by the physical barrier surrounding the solid tumor, another major obstacle is the extensive infiltration of regulatory T (Treg) cells, a major immunosuppressive immune cell subset, in the tumor microenvironment. Here, this work develops a grooved microneedle patch for augmenting ACT, aiming to simultaneously overcome physical and immunosuppressive barriers. The microneedles are engineered through an ice-templated method to generate the grooved structure for sufficient T-cell loading. In addition, with the surface modification of chemokine CCL22, the MNs could not only directly deliver tumor-specific T cells into solid tumors through physical penetration, but also specifically divert Treg cells from the tumor microenvironment to the surface of the microneedles via a cytokine concentration gradient, leading to an increase in the ratio of Teff cells/Treg cells in a mouse melanoma model. Consequently, this local delivery strategy of both T cell receptor T cells and chimeric antigen receptor T cells via the CCL22-modified grooved microneedles as a local niche could significantly enhance the antitumor efficacy and reduce the on-target off-tumor toxicity of ACT.

Characterizing immune microenvironment cell composition of chronic rhinosinusitis with nasal polyps based on mass cytometry technology

Objective: To analyze the cellular composition characteristics of the nasal tissue immune microenvironment in patients with control, chronic rhinosinusitis without nasal polyps (CRSsNP), non-eosinophilic chronic rhinosinusitis with nasal polyps (neCRSwNP), and eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) using mass cytometry flow technology. Methods: Thirteen CRS patients who underwent endoscopic nasal surgery at the Department of Otorhinolaryngology Head and Neck Surgery of Peking Union Medical College Hospital from March to December 2022 were recruited, including 8 males and 5 females, aged 22.3 to 58.3 years. Three control mucosae were obtained from normal ethmoid or sphenoid sinuses of patients with benign tumors of the temporal fossa or non-functional pituitary adenomas who underwent endoscopic surgery, excluding allergic rhinitis and sinusitis. Sixteen clinical tissue samples (3 of control, 3 of CRSsNP, 4 of neCRSwNP, and 6 of eCRSwNP) were prepared into single-cell suspensions. Mass cytometry flow detection was performed using a combination of 42 molecular markers to analyze the differences in cell subpopulations among the groups. Data were analyzed using GraphPad Prism 9. Results: Based on the mass cytometry flow results, cells from control, CRSsNP, neCRSwNP, and eCRSwNP were divided into seven main cell subgroups, with detailed subgrouping of T/NK cells and myeloid cells. In T/NK cells, compared with the control group, the number of NK CD56bright cells increased in the CRSsNP group, while NK CD56dim cells decreased; compared with the CRSsNP group, the eCRSwNP group showed a decrease in NKT cells and CD4+Tem cells; compared with the CRSsNP group, the eCRSwNP group showed a significant increase in CD25 expression within Treg cells; compared with the CRSsNP group, the eCRSwNP group showed a significant decrease in Tbet expression in CD8+Teff cells and CD8+TRM cells; in eCRSwNP, the expression of CD103 in CD8+TRM cells was significantly lower than in CRSsNP. In myeloid cells, compared with the other three groups, the eCRSwNP group showed a significant increase in macrophages and a significant decrease in cDC1 and monocytes; compared with the control group and CRSsNP, the eCRSwNP group also showed a significant decrease in resting state macrophages; compared with the CRSsNP group, the eCRSwNP group showed a significant decrease in the level of CX3CR1 within cDC2 and monocytes; the expression levels of NLRP3 in cDC2 and macrophages in the eCRSwNP group were significantly higher than in the other three groups; compared with the control group, the expression levels of Gata3 in cDC2 and macrophages in the eCRSwNP group were also significantly increased; additionally, the expression of CCR2 within monocytes in the eCRSwNP group was lower than in the CRSsNP group. In ILC, compared with the control group, the expression of CCR6 decreased in the eCRSwNP group. Conclusions: Compared with the control group, CRSsNP, and neCRSwNP, eCRSwNP shows an increase in macrophage number, a decrease in cDC1 and resting state macrophages, and depletion of protective cells CD103+CD8+TRM. Additionally, the expression levels of CCR2 and CX3CR1 in monocytes of eCRSwNP are decreased.

Integrated analysis of single-cell data on role of NKG7high CD8+ Teff cells in driving immune-related adverse events.

e24114 Background: Immune-related adverse events (irAEs) frequently occur during immunotherapy and can impact various organ systems, which dampen the clinical outcomes of immune checkpoint inhibitors (ICIs). However, the precise immune hallmarks associated with irAEs remain further explored. Methods: We collected public (Bukhari et al., Zhu et al., Su et al., and Ma et al.) and our own scRNA-seq data across patients with different irAE types or without irAEs. By comparing T cell compositions between irAE and no-irAE patients, the subcluster correlated with the occurrence of irAE was identified. Subsequently, the integrated analyses were performed to explore its mechanisms in driving irAEs in both peripheral blood and local tissues. Results: Compared to no-irAE patients, a subcluster of effector T (Teff) cells was observed to increase notably in the peripheral blood of irAE patients during immunotherapy. This specific subcluster, named "NKG7high CD8+ Teff cells," was characterized by the significant expression of effector genes such as GZMH, CCL5, and especially NKG7. In contrast to other CD8+ T cells, the NKG7high CD8+ Teff cells exhibited stronger cytotoxicity functions, increased exosome secretion, and enhanced MIF-CD74 interaction with monocytes, which suggests the mechanistic link between its increased infiltration and the development of irAEs. Further investigation showed these characteristics were more remarkable in NKG7high CD8+ Teff from irAE patients than no-irAE patients. Subsequently, the increase of NKG7high CD8+ Teff during immunotherapy was validated in our own scRNA from immune thyroiditis patients, which was not observed in no-irAE patients. Across various irAE types, NKG7high CD8+ Teff subsets, along with their NKG7 expression, demonstrated heightened infiltration and expression in immune-related arthritis and myocarditis samples. After investigating the role of NKG7high CD8+ Teff in peripheral blood, we focused on its distribution and roles in local irAE tissue. NKG7high CD8+ Teff cells accumulated in immune-related thyroiditis tissues, and recruited macrophages through the IFNG and CSF1 signaling pathways, thereby inducing an immune overreaction. Meanwhile, in immune-related myocarditis mice heart tissues, NKG7high CD8+ Teff cells could interact with macrophages via the SPP1 signaling pathway and cause endothelial cell damage through the FASL-FAS pathway. Conclusions: This comprehensive study revealed the intricate mechanisms of NKG7high CD8+ Teff in inducing irAEs, providing valuable insights into their roles and potential implications in irAE treatment.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Fasting and Glucose Metabolism Differentially Impact Peripheral Inflammation in Human Type 2 Diabetes.

Cytokines produced by peripheral T-helper 1/17 cells disproportionately contribute to the inflammation (i.e., metaflammation) that fuels type 2 diabetes (T2D) pathogenesis. Shifts in the nutrient milieu could influence inflammation through changes in T-cell metabolism. We aimed to determine whether changes in glucose utilization alter cytokine profiles in T2D. Peripheral blood mononuclear cells (PBMCs), CD4+ T-cells, and CD4+CD25- T-effector (Teff) cells were isolated from age-matched humans classified by glycemic control and BMI. Cytokines secreted by CD3/CD28-stimulated PBMCs and Teff were measured in supernatants with multiplex cytokine assays and a FLEXMAP-3D. Metabolic activity of stimulated CD4+ T-cells was measured by a Seahorse XFe96 analyzer. In this study, we demonstrated that T-cell stimulated PBMCs from non-fasted people with T2D produced higher amounts of cytokines compared to fasting. Although dysglycemia characterizes T2D, cytokine production by PBMCs or CD4+ T-cells in T2D was unaltered by hyperglycemic media. Moreover, pharmacological suppression of mitochondrial glucose oxidation did not change T-cell metabolism in T2D, yet enhanced cytokine competency. In conclusion, fasting and glucose metabolism differentially impact peripheral inflammation in human T2D, suggesting that glucose, along with fatty acid metabolites per our previous work, partner to regulate metaflammation. These data expose a major disconnect in the use of glycemic control drugs to target T2D-associated metaflammation.

Innate phase production of IFN-γ by memory and effector T cells expressing early activation marker CD69 during infection with Cryptococcus deneoformans in the lungs.

Cryptococcus deneoformans is a yeast-type fungus that causes fatal meningoencephalitis in immunocompromised patients and evades phagocytic cell elimination through an escape mechanism. Memory T (Tm) cells play a central role in preventing the reactivation of this fungal pathogen. Among these cells, tissue-resident memory T (TRM) cells quickly respond to locally invaded pathogens. This study analyzes the kinetics of effector T (Teff) cells and Tm cells in the lungs after cryptococcal infection. Emphasis is placed on the kinetics and cytokine expression of TRM cells in the early phase of infection. CD4+ Tm cells exhibited a rapid increase by day 3, peaked at day 7, and then either maintained their levels or exhibited a slight decrease until day 56. In contrast, CD8+ Tm cells reached their peak on day 3 and thereafter decreased up to day 56 post-infection. These Tm cells were predominantly composed of CD69+ TRM cells and CD69+ CD103+ TRM cells. Disruption of the CARD9 gene resulted in reduced accumulation of these TRM cells and diminished interferon (IFN) -γ expression in TRM cells. TRM cells were derived from T cells with T cell receptors non-specific to ovalbumin in OT-II mice during cryptococcal infection. In addition, TRM cells exhibited varied behavior in different tissues. These results underscore the importance of T cells, which produce IFN-γ in the lungs during the early stage of infection, in providing early protection against cryptococcal infection through CARD9 signaling.

Engineered IL2 for improved treatment of bladder cancer

Abstract Bladder cancer (BC) remains a formidable challenge despite its immunogenic nature and responsiveness to FDA-approved immunotherapy. Interleukin-2 (IL2), a potent cytokine with potential for cancer treatment, faces obstacles such as the simultaneous activation of immunosuppressive regulatory T cells (Tregs), high toxicity, and a short half-life. CD122-directed IL2 therapy (IL2c) has shown success in mouse BC models. To address clinical challenges, we engineered two IL2/anti-IL2 antibody (F10) fusion proteins: F10 IC (native IL2), potentially biased toward effector cell activation, and F10 H9T (H9T, an engineered IL2), potentially favoring memory T cells. In vitro F10 H9T induced stem-like T cells (TCF-1+) and central memory T cells (CD44+CD62L+), while IL2c promoted Teff cell (CD44+CD62L-) differentiation. In vivo, using an orthotopic mouse bladder cancer model (n=10/group), F10 IC was superior in activating anti-tumor Teff and NK cells over F10 H9T and IL2c. Unexpectedly, F10 IC also induced TCF-1+ T cells, suggesting a potentially valuable dual effect, but further validation is required. We provide a roadmap for designing and translating immunomodulatory cytokine/antibody fusion proteins to overcome challenges associated with IL2-based cancer therapies.

Surface CD6 regulates T cell adhesion and migration through direct ligand interaction and stabilization of VLA4

Abstract The CD6-ALCAM pathway is an important regulator of T cell activity and trafficking. However, its precise role in modulating T-cell adhesion and migration during inflammation is unclear. To investigate its role in adhesion and transendothelial migration, we used itolizumab, an anti-CD6 mAb that can induce cleavage of CD6 from the T cell surface when in the presence of monocytes. PBMCs were stimulated overnight or left unstimulated prior to treatment with itolizumab to generate CD6low T cells. Cells were then tested in standard adhesion and migration assays. The CD6 low T cells had ~ 50% decreased adherence to HUVEC cells compared to the control T cells (CD6high); similar results were observed by blocking ALCAM on HUVEC. Modulating CD6 on T cells or blocking ALCAM on the HUVEC also translated to a ~3-fold decrease in migration of Teff cells. Loss of CD6 was associated with reduced protein levels of VLA4, but not of other integrins, under both stimulated (~70%) and non-stimulated conditions (~40%). Antibody blockade of CD6 without cleavage did not affect VLA4 levels, but still decreased migration of unstimulated T cells though less so than in stimulated T cells, where VLA4 reduction is required to modulate migration. Our data demonstrate that CD6 contributes to cell adhesion/migration both through direct ligand interactions with ALCAM, and through the stabilization of cell membrane VLA4 and that these functions can be abrogated by modulation of cell surface levels of CD6.

Modulating alloreactive immune responses by controlled dual delivery of Fas and IL-2R agonists using a lipocoacervate platform

Abstract Allogeneic islet transplantation is an effective treatment for type 1 diabetes (T1D). Graft rejection is controlled by immunosuppressive drugs that have adverse effects. An imbalance between Teffs and Tregs is the primary cause of T1D and graft rejection. Activated Teff cells express Fas and are sensitive to FasL-mediated apoptosis. Treg cells are relatively refractive to apoptosis and expand in response to IL-2. Thus, a combination of Fas and IL-2R agonists has the potential to modulate alloreactive responses for sustained graft survival. Given the off-target effects of systemic delivery of biologics, we established a lipocoacervate formulation consisting of a biodegradable polycation, PEAD, and polyanion heparin to form coacervate for controlled delivery of a novel Fas agonist, SA-FasL, and IL-2. Release kinetics and activities of proteins were assessed in vitro. The immunomodulatory efficacy of protein-loaded lipocoacervate was assessed in vivo. IL-2 and SA-FasL proteins showed a steady release over 30 days in vitro. IL-2 released on day 14 had minimal activity loss, whereas FasL showed ~20% activity loss on day 9. Treatment with SA-FasL/IL-2-loaded lipocoacervate modulated in vivo alloreactive T cell responses, resulting in an increased Treg/Teff cell ratio. In conclusion, lipocoacervate is an effective platform for controlled and sustained release of SA-FasL and IL-2 biologics to modulate allo and autoreactive T cells with significant therapeutic potential for T1D.

Beneficial mechanisms of dimethyl fumarate in autoimmune uveitis: insights from single-cell RNA sequencing

BackgroundDimethyl fumarate (DMF) is a fumaric acid ester that exhibits immunoregulatory and anti-inflammatory properties. However, the function of DMF in autoimmune uveitis (AU) is incompletely understood, and studies comprehensively exploring the impact of DMF on immune cells are still lacking.MethodsTo explore the function of DMF in uveitis and its underlying mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) on the cervical draining lymph node (CDLN) cells of normal, experimental autoimmune uveitis (EAU), and DMF-treated EAU mice. Additionally, we integrated scRNA-seq data of the retina and CDLNs to identify the potential impact of DMF on ocular immune cell infiltration. Flow cytometry was conducted to verify the potential target molecules of DMF.ResultsOur study showed that DMF treatment effectively ameliorated EAU symptoms. The proportional and transcriptional alterations in each immune cell type during EAU were reversed by DMF treatment. Bioinformatics analysis in our study indicated that the enhanced expression of Pim1 and Cxcr4 in EAU was reversed by DMF treatment. Further experiments demonstrated that DMF restored the balance between effector T (Teff) /regulatory T (Treg) cells through inhibiting the pathway of PIM1-protein kinase B (AKT)-Forkhead box O1 (FOXO1). By incorporating the scRNA-seq data of the retina from EAU mice into analysis, our study identified that T cells highly expressing Pim1 and Cxcr4 were enriched in the retina. DMF repressed the ocular infiltration of Teff cells, and this effect might depend on its inhibition of PIM1 and CXCR4 expression. Additionally, our study indicated that DMF might reduce the proportion of plasma cells by inhibiting PIM1 expression in B cells.ConclusionsDMF effectively attenuated EAU symptoms. During EAU, DMF reversed the Teff/Treg cell imbalance and suppressed the ocular infiltration of Teff cells by inhibiting PIM1 and CXCR4 expression. Thus, DMF may act as a new drug option for the treatment of AU.

Abstract 580: APP as a novel immune checkpoint: Expression in T cells and its clinical implications for immuno-oncology drug refractory

Abstract Amyloid precursor protein (APP) is a type I transmembrane protein expressed in various cell types and tissues. We previously confirmed that APP expressed in T cells is a binding partner protein of a novel immune checkpoint contactin 4 (CNTN4), and APP is a central molecule for conveying the immunosuppressive signal of CNTN4 to T cells. We further profiled an expression of APP in tumor-infiltrating T cells from tumors of NSCLC patients who received anti-PD-1 as a neoadjuvant treatment combined with chemotherapy using single-cell RNA analysis. As a result, the proportion of APP-expressing CD4+ Teff or CD8+ Teff cells was higher in stable disease (SD) patients than in partial response (PR) patients. In contrast, we observed higher PDCD1-expressing CD4+ Tnaïve and CD4+ Teff cell proportions in PR patients than in SD patients. When we performed a Differential Expression Gene analysis between the PR and SD patients within Teff cells, we observed that the PR patients’ CD8+ Teff cells manifested a higher IFNG and PDCD1, while the SD patients’ CD8+ Teff cells predominantly exhibited elevated APP levels. Subsequent pathway analysis also suggested a negative relationship between immune-related pathways and APP expression in CD8+ Teff cells. Such findings imply that the predominant presence of APP in T cells is associated with I/O refractory. From a biological view, we demonstrated increased APP expression in T cells by stimulation with an anti-CD3 antibody or antigen-presenting cell. We also identified that upon stimulation with an anti-CD3 antibody, transcription factors for T-cell activation bind to the proximal promoter of APP and increase its expression. Furthermore, it was confirmed that APP in T cells not only increases its expression upon anti-CD3 antibody stimulation but also translocates from the cytoplasm to the membrane through the Golgi apparatus. When anti-CD3 antibody stimulation of T cells persists, the membrane expression of APP increases for 14 to 24 h, then gradually decreases when the stimulation disappears and becomes the same as before stimulation at 48 h. T cells differentiate into various subsets and play an essential role in adaptive immunological responses to foreign substances. In particular, CD4+ (helper) T cells differentiate into different T-cell subsets, such as Th1, Th2, Th17, Th19, Th22, Tfh, and Treg, to govern the overall immune response by producing cytokines that can recruit other cells of the immune system. So, we investigated whether APP is expressed on T cell subsets including naïve, effector, memory, and exhausted T-cells. We identified that APP is expressed in various CD4+ and CD8+ T cell subsets upon stimulation with anti-CD3 antibody. Taken together, our results suggest that APP is expressed on multiple functional T cells and that increased expression of APP on T cells in TIL may increase resistance to immunotherapy. Citation Format: Mi Young Cha, Bu-Nam Jeon, Gihyeon Kim, Areum Jeong, Sang-Moo Park, Kyung Mi Park, Hansoo Park. APP as a novel immune checkpoint: Expression in T cells and its clinical implications for immuno-oncology drug refractory [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 580.

Abstract 7176: Optimization of T cell co-stimulatory agonists: A semi-mechanistic PKPD model integrating drug properties and tumor-immune interactions

Abstract Background: Despite the success of some immune checkpoint inhibitors in the treatment of cancer, T-cell co-stimulation has faced limited clinical success due to the intricacies around the optimal engagement of agonistic antibodies to co-stimulatory receptors, and the absence of biomarkers for patient selection. Understanding agonist-binding characteristics, pharmacokinetics, T cell subpopulation activation and differentiation kinetics, T cell life span in the tumor microenvironment, and the impact of T cell/tumor interactions on tumor growth kinetics is crucial for the positive clinical outcomes of T cell agonists. Currently there is not a suitable model to describe these complex interactions. Methods: We investigated the pharmacokinetic (PK), pharmacodynamics (PD), and anti-tumor efficacy of an anti-TNFR2 agonist, HFB3-1, and an anti-OX40 agonist, HFB10-1, in syngeneic tumor models. Flow cytometry was employed to profile post-treatment tumor infiltrating lymphocytes (TILs) in dissociated tumor tissues. In vitro binding affinity was determined using Biolayer Interferometry. A semi-mechanistic model, integrating PK, tumor growth, and immune interaction networks (interactions among Teff, Treg, and tumor cells), was developed in Berkeley Madonna (version 10.5.1). This was subsequently adapted to a human tumor growth model with T cell interaction networks to assess the impact of doses and dosing regimens on tumor growth inhibition. Results: A semi-mechanistic PKPD model was successfully developed incorporating drug properties including binding affinity, agonistic effect, ADCC activity, and pharmacokinetics, and tumor growth characteristics including tumor growth kinetics, TIL composition, interaction, proliferation, and turnover. This model was applied to investigate the effect of FcγR, dose, and dosing frequency on in vivo efficacy. Following incorporation of human PK and human tumor microenvironment properties into the model, tumor growth characteristics were simulated under varying human dosing regimens. Simulation results indicated that lower and less frequent doses optimize T cell co-stimulation for optimal anti-tumor activity. Conclusion: Our semi-mechanistic PKPD model elucidates the intricate interactions between T cell co-stimulatory agonists and the tumor-immune cells in the tumor microenvironment. The model advocates for pulsatile agonism as the optimal anti-tumor activity for T cell co-stimulation agents. This model serves as a valuable tool for guiding dose and dose regimen optimization for T cell co-stimulatory agonists in clinical development. Citation Format: Jinping Gan, William Hedrich, Yun-Yueh Lu, Wenhua Xu, Robert H. Andtbacka, Francisco Adrian, Liang Schweizer. Optimization of T cell co-stimulatory agonists: A semi-mechanistic PKPD model integrating drug properties and tumor-immune interactions [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 7176.

Abstract 2666: Genetic ablation of B2M leads to resistance to PD-1/PD-L1 blockade in vivo

Abstract Immune checkpoint inhibitors (ICIs) has revolutionized the treatment landscape of various cancers by reinvigorating the exhausted T cells in patients. However, the therapeutic efficacy is largely confined due to the primary or acquired resistance to anti-PD-1 (L1) therapy in many patients. Recently, the mechanisms of resistance to ICIs has been extensively elucidated from different aspects and emerging sequencing data from clinical samples has pointed to IFN-γ signaling defects and antigen presentation loss in patients who are resistant to PD-1(L1) blockade. The loss-of-function mutations in JAK1 and JAK2 results in lack of response to IFN-γ signaling and incapacity to upregulate PD-L1 and MHC-I, which subsequently leads to noninflammatory TMEs and resistance to anti-PD-1/PD-L1. The loss-of-function of B2M is likely a common resistance mechanism that the intratumoral cytotoxic CD8+ T cells are failed to be boosted owing to antigen presentation loss. In order to obtain higher clinical benefits in various cancers, it’s appealing and urgent to develop mechanism-based strategies to overcome resistance to ICI-based immunotherapy. To this end, we developed a genetically acquired resistant tumor model with B2M mutations in syngenic MC38 tumor cells using CRISPR/Cas9 technology, which is ‘hot’ tumor to PD-1/PD-L1 blockade. The expression of B2M was detected by western blotting and flow cytometry. Mouse B2M-knockout MC38 tumors became resistant to PD-1/PD-L1 blockade in vivo. Notably, we found a remarkable increase of CD8+ T cell infiltration in B2m-KO MC38 tumors in comparison to parental MC38 tumors upon anti-PD-1 treatment, which further explains that antigen presentation loss results in inactivation of cytotoxic CD8+ T cells within the tumor microenvironments (TMEs), subsequently leading to resistance and poor survival to ICI therapy.In summary, our data consistently indicated the importance of MHC-I expression in T cell activation in the TMEs and the possibility to dig out the pathway involved in MHC-I expression independent of IFN-γ signaling by CRISPR-based screening to overcome MHC-I deficiency-induced resistance in the future. In addition, it’s possible to leverage the cytotoxic NK cells and CD4+ T cells in antitumor immunity against MHC-II+ tumors even with the loss of MHC-I molecules. For example, high dose of IL-2RB-biased IL-2 agonist preferentially binding to the dimeric IL-2R can stimulate Teff and NK cells independent of immune checkpoint expression. Likewise, cytokine modified anti-PD-1 fusion formulation like anti-PD-1-IL-2 mutein and anti-PD-1-IL-15 mutein are promising to overcome the resistance. Furthermore, it has been suggested that the resistance due to genetic mutations of MHC-I could be overcome by combining NF-B targeted therapies. Citation Format: Yanlei Zhang, Hechun Ma, Boang Han, Yi Li, Ping Yang, Zhen Li, Dongxiao Feng, Lei Ci, Ruilin Sun, Daniel X. He. Genetic ablation of B2M leads to resistance to PD-1/PD-L1 blockade in vivo [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 2666.

Abstract 661: An oral Cbl-b inhibitor with sustained T cell activation demonstrated robust anti-tumor efficacy along with enhanced infiltration and activation of functional T cells

Abstract The E3 ubiquitin ligase Casitas B-lineage lymphoma b (Cbl-b) is a member of the highly conserved family of Cbl (casitas b-lineage lymphoma) proteins, which associates with multi-immune response regulations including Teff and NK activation, Treg differentiation. Cbl-b KO mice showed enhanced immune responses and resistance to tumor transplantation. Previously, we reported a novel Cbl-b inhibitor, ZM-8026, which robustly activated T cells and efficiently inhibited tumor growth as monotherapy or in combination with anti-PD-1. Here, we further demonstrated that Cbl-b inhibition restores the Teff cells' function in immune suppression conditions, such as in the presence of PGE2, A2AR agonist, and TGF-β. CD8+T proliferation and cytokines induction were maintained in the presence of MDSC. Furthermore, CD8+T function was reversed from an exhausting status induced by a continuous anti-CD3/CD28 activation for a long period. To investigate whether continuous target occupancy was necessary for sustained TCR activation, the dynamic impacts of Cbl-b inhibition on CD8+T cell activation were explored using a washout assay. The results showed that the high-level activated T cells lasted for 48 h after more than 4hs preincubation with 1 µM Cbl-b inhibitor followed by a washout, however, short and weak activation states were found with less than 4hs preincubation or low inhibitor concentration treatment (< 0.1 uM). This finding revealed sufficient inhibitor concentration and treatment time ensure high T-cell activation. Further, in vivo efficacy studies demonstrated less anti-tumor growth efficacy for intermittent dosing (Q2D, Q3D) compared with QD dosing. In vitro and in vivo pharmacokinetics studies exhibited favorable ADME profiles and bioavailability of our Cbl-b inhibitor for oral administration in mice, rats, and dogs. At least in two syngeneic models, our Cbl-b inhibitor exhibited efficient anti-tumor growth with more than 70% TGI (tumor growth inhibition). In a CT26 syngeneic model, complete tumor growth inhibition in 6 of 8 mice was found in the Cbl-b inhibitor combined with an anti-PD1 antibody. The complete regression mice were re-challenged with the same tumor cells, and no measurable tumors were found for up to 30 days, suggesting the treatment-induced immune memory. Tumor samples in mice were collected, RNAseq showed that activated functions of tumor-infiltrated T, NK, DC, and macrophage were dramatically increased in the Cbl-b inhibitor treatment group, and the T and NK activation signature genes such as Gzmb, Ifng were significantly upregulated by RT-PCR. In conclusion, our Cbl-b inhibitor exhibited a favorable PK profile in multi-preclinical animals. The sustained T cell activation in vitro was identified and robust anti-tumor growth in vivo was demonstrated with optimized dosing frequency. Citation Format: Feng Zhou, Guimei Yang, Yajing Liu, Liting Xue, Weijie Chen, Zhengtao Li, Xiaowu Liu, Jian Li, Renhong Tang. An oral Cbl-b inhibitor with sustained T cell activation demonstrated robust anti-tumor efficacy along with enhanced infiltration and activation of functional T cells [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 661.

Abstract P251: Circulating Immune Cell Phenotypes Are Associated With Inflammatory Biomarkers in the Framingham Heart Study Offspring Cohort

Age-related changes in circulating immune cells have been associated with inflammation and immunosenescence, increasing susceptibility to cardiovascular disease. This study investigated the association between circulating immune cell phenotypes and inflammatory biomarkers in the Framingham Heart Study Offspring cohort. Participants (N=865, 52% female, mean age 62) were immunophenotyped using peripheral blood mononuclear cells with 37 immune cell phenotypes and 6 composite ratio measures, including T cells, B cells, NK cells, and monocytes. The inflammatory biomarkers were profiled in plasma samples using the OLINK Proteomics inflammation panel. Pairwise cross-sectional associations between 43 circulating immune cell phenotypes and 68 protein biomarkers were investigated using linear mixed-effect models accommodating familial correlations and adjusting for covariates (sex, age, number of apolipoprotein E ( APOE) ε2 and ε4 alleles, and cytomegalovirus levels), with FDR ≤0.05 to declare significant associations. Age-stratified analyses were performed to explore effect modification. CD8 T naïve cells (CD8Tn) were negatively associated with several proteins ( Figure ). Significant associations were also observed for CD4 T cell subtypes including higher levels of CD4 T helper cells associated with lower Flt3L and TNFRSF9, higher levels of CD4 T central memory (Tcm) and CD4 T effector (Teff) cells associated with lower TNFB, and higher CD4 Teff associated with lower CD40 levels. Among associations identified in the full sample, we observed a large proportion of associations between CD4 T cell subtypes and CD8 Tn in the older age group (>60 years) but not the younger age group. Our results suggest that people tend to have more inflammation-associated immune responses during aging. Future studies are needed to improve understanding of the contribution of these inflammation-immune cell associations to cardiovascular disease.

A single-agent fusion of human IL-2 and anti-IL-2 antibody that selectively expands regulatory T cells

The occurrence of many autoimmune diseases takes root on the disrupted balance among Treg cells, Teff cells, etc. Low-dose interleukin-2 (IL-2) cytokine demonstrates promising clinical efficacy in the expansion of Treg cells and the treatment of autoimmune diseases. However, its clinical application is hindered by the small therapeutic index and short half-life. Previous studies have shown that non-covalent complex of human IL-2 and anti-IL-2 antibody biases cytokine activity towards Treg cells and extends IL-2’s half-life. The clinical translation of such complex is non-trivial. In this study, we discover an anti-human IL-2 antibody and engineer a covalently-linked single-agent fusion of human IL-2 and its antibody that selectively expands Treg cells and exhibits superior disease control activity in animal models of ulcerative colitis and systemic lupus erythematosus, with proper safety profile and good developability. These studies pave the road for its clinical development in diverse autoimmune diseases.