Effector Memory CD8 Research Articles

Analysis and validation of biomarkers of immune cell-related genes in postmenopausal osteoporosis: An observational study.

Postmenopausal osteoporosis (PMOP) is a common metabolic inflammatory disease. In conditions of estrogen deficiency, chronic activation of the immune system leads to a hypo-inflammatory phenotype and alterations in its cytokine and immune cell profile, although immune cells play an important role in the pathology of osteoporosis, studies on this have been rare. Therefore, it is important to investigate the role of immune cell-related genes in PMOP. PMOP-related datasets were downloaded from the Gene Expression Omnibus database. Immune cells scores between high bone mineral density (BMD) and low BMD samples were assessed based on the single sample gene set enrichment analysis method. Subsequently, weighted gene co-expression network analysis was performed to identify modules highly associated with immune cells and obtain module genes. Differential analysis between high BMD and low BMD was also performed to obtain differentially expressed genes. Module genes are intersected with differentially expressed genes to obtain candidate genes, and functional enrichment analysis was performed. Machine learning methods were used to filter out the signature genes. The receiver operating characteristic (ROC) curves of the signature genes and the nomogram were plotted to determine whether the signature genes can be used as a molecular marker. Gene set enrichment analysis was also performed to explore the potential mechanism of the signature genes. Finally, RNA expression of signature genes was validated in blood samples from PMOP patients and normal control by real-time quantitative polymerase chain reaction. Our study of PMOP patients identified differences in immune cells (activated dendritic cell, CD56 bright natural killer cell, Central memory CD4 T cell, Effector memory CD4 T cell, Mast cell, Natural killer T cell, T follicular helper cell, Type 1 T-helper cell, and Type 17 T-helper cell) between high and low BMD patients. We obtained a total of 73 candidate genes based on modular genes and differential genes, and obtained 5 signature genes by least absolute shrinkage and selection operator and random forest model screening. ROC, principal component analysis, and t-distributed stochastic neighbor embedding down scaling analysis revealed that the 5 signature genes had good discriminatory ability between high and low BMD samples. A logistic regression model was constructed based on 5 signature genes, and both ROC and column line plots indicated that the model accuracy and applicability were good. Five signature genes were found to be associated with proteasome, mitochondria, and lysosome by gene set enrichment analysis. The real-time quantitative polymerase chain reaction results showed that the expression of the signature genes was significantly different between the 2 groups. HIST1H2AG, PYGM, NCKAP1, POMP, and LYPLA1 might play key roles in PMOP and be served as the biomarkers of PMOP.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction

ABSTRACT Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

T cell specific Eomes Deletion Does Not Protect Against High Fat Diet Induced Large Artery Stiffening

We have found that T cells contribute to age-related large artery stiffness. The T-box transcription factor, Eomes is an important regulator of CD8+ (Cytotoxic) T cell differentiation from a naïve to a memory phenotype. We previously reported that these effector memory CD8+ T cells accumulate in the aorta of old mice. We aimed to investigate whether inhibiting CD8+ T cell memory differentiation in mice, specifically through T cell-specific Eomes deletion, would protect these animals from high-fat diet (HFD)-induced large artery stiffness. Mice with a CD8 driven Cre Recombinase were bred with mice with loxp sites surrounding the Eomes gene resulting in wild type (CD8 Eomes+/+) and T cell specific Eomes deleted (CD8 Eomesf/f) mice. Mice were randomly divided into a normal chow (NC) and HFD. Resulting in 8 mice of each genotype and diet. The NC group was given a normal diet containing 4% fat and the HFD group was given a high-fat diet containing 60% fat. At 4-8 months of age, doppler pulse wave velocity was used to assess large artery stiffness. Following euthanasia, aortic T cell phenotype was assessed using flow-cytometry. Group differences were assessed by two-way ANOVA and Tukey’s post-hoc test. Data shown as mean ± SD. Regardless of genotype, HFD had a significant impact on pulse wave velocity (PWV), indicating elevated aortic stiffness. In the HFD group, both CD8 Eomesf/f and CD8 Eomes+/+ mice exhibited higher PWV values (330.08±72.33 cm/s;312.5±53.74 cm/s) compared to their counterparts in the NC group (256.4±63.67 cm/s;259.2±66.36 cm/s, p=0.0172). To determine whether HFD-induced increases in aortic stiffness are accompanied by changes in aortic T cell phenotype, flow cytometric analysis was performed on the aorta. First, we observed, regardless of mouse genotype, HFD had a significant impact on CD4+ (Helper T) naïve cells, resulting in a blunted percentage of these cells. This dietary effect was observed in both CD8 Eomesf/f and CD8 Eomes+/+ mice (NCD CD8 Eomesf/f: 70.92±5.912%; NCD CD8 Eomes+/+ mice: 56.46±20.62%; HFD CD8 Eomesf/f: 47.5±7.865%; HFD CD8 Eomes+/+ mice: 50.58±13.907%; p=0.0206). In contrast, the percentages of CD8+ naïve cells did not display a significant change in response to the high-fat diet (p=0.6303). HFD significantly influenced CD4+ memory cells, resulting in an increase in percentages, regardless of mouse genotype (NCD CD8 Eomesf/f: 29.09± 5.909%; NCD CD8 Eomes+/+ mice: 44.27±19.17%; HFD CD8 Eomesf/f: 52.47±7.875%; HFD CD8 Eomes+/+ mice: 49.44±13.9%; p=0.02). However, HFD did not produce any significant alterations in CD8+ memory cell percentages (p=0.6). These results suggest that contrary to our hypothesis, T cell specific Eomes deletion is not suffcient to protect against diet induced large artery stiffness. However, this may be due to greater proportions of CD4+ memory cells in the aorta with high fat feeding. Funding: AHA 940023, NIH R01AG060395, K01AG061271. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

An IL-12 mRNA-LNP adjuvant durably enhances the CD8+ T cell response to mRNA vaccination

Abstract The design of vaccines that can reliably induce protective CD8+ T cell activity has been considered a challenge for the vaccine field, but the use of viral vectors or mRNA encapsulated into lipid nanoparticles (LNPs) has overcome some of these issues. Because the cytokine interleukin-12 (IL-12) supports CD8+ T cell expansion and acquisition of effector functions, studies were performed to assess its contribution to the ability of mRNA vaccines to promote CD8+ T cell responses. mRNA-LNPs do not stimulate macrophage production of IL-12 in vitro nor does in vivo vaccination, and endogenous IL-12 is not required for the CD8+ T cell response to mRNA vaccination. However, the addition of IL-12 mRNA-LNPs into vaccination with LNPs that contain mRNA for the model antigen ovalbumin (OVA) results in enhanced OVA-specific CD8+ T cell expansion, improved acquisition of effector function, diversification of the CD8+ T cell effector pool through generation of a KLRG1hi effector subset and expanded effector and central memory CD8+ T cell populations. In both preventative vaccination against Listeria monocytogenes-OVA and therapeutic vaccination against B16 F0-OVA melanoma, addition of IL-12 mRNA-LNPs enhanced protection. Thus, modification of current mRNA vaccine formulations to induce IL-12 production provides a strategy to modify the CD8+ T cell response, enhance CD8+ T cell mediated protection and illustrates the utility of cytokine mRNA to tailor vaccine-induced immunity.

Abstract 1028: Cytotoxic CD4+ T Cells: Unraveling The Residual Risk In Lipid Lowering For Type 2 Diabetes Patients

Type 2 Diabetes (T2D) significantly increases the risk of cardiovascular diseases (CVDs), marked by severe plaque pathology resistant to standard cholesterol-lowering treatments. While the roles of the innate immune system and macrophages in CVDs are well-established, T cell alterations, particularly involving cytotoxic CD4 T cells (CD4 CTLs), remain unexplored in human atherosclerotic plaques. In our study, we aimed to identify the phenotype and function of CD4 CTLs in blood & plaque tissue, in the context of T2D. Using unbiased single-cell RNA sequencing (scRNA-seq) analysis, we examined over 70,000 immune cells from 22 plaques of patients undergoing carotid endarterectomy (T2D: n=12, Non-Diabetic (ND): n=10), & peripheral blood mononuclear cells (PBMCs) from the CHORD study (NCT04369664) (T2D: n=12, ND: n=4). scRNA-seq analysis revealed a diverse population of CD4 T cells, like naïve, central memory (CM), effector memory (EM), tissue resident memory (Trm), T regulatory (Tregs), & two clusters of cytotoxic (CTL) T cells expressing canonical cytotoxicity-related genes. Cluster 5 expressed GZMB, GNLY, PRF1 , whereas Cluster 10 expressed GZMA, GZMK . Cell-cell communication analysis demonstrated that both CTL clusters received signaling from dendritic cells and macrophages, including TREM2+ macrophages. CTL T cells reciprocally communicated with clusters of EM and EMRA CD8 T cells, underscoring their central role in plaque cell-cell communication. Notably, Cluster 5, one of these two CTL clusters, was expanded in T2D plaque samples (p=0.032) and expressed genes involved in chemotaxis, IL-2 signaling, & transcription factors (e.g., RUNX3, TBX21 ) involved in T cell activation. The equivalent of CD4 CTL phenotype was found to be expanded in T2D individuals compared to controls, & these elevated levels persisted in T2D patients following intensive lipid-lowering therapy, indicating resistance to the treatment regimen. In summary, our study emphasizes the crucial role of CD4 CTLs in the immune dynamics of atherosclerotic plaques. The observed resistance of CTLs to lipid-lowering interventions underscores the necessity for targeted therapeutic strategies to address the distinctive immune changes in T2D-associated cardiovascular diseases.

Mapping the cellular landscape of the maternal-fetal interface in women with preterm birth and chronic chorioamnionitis

Abstract Preterm birth (PTB) affects over 15 million infants yearly. In up to 40% of late PTBs, placental examination reveals chronic chorioamnionitis (CCA), maternal mononuclear cell infiltration in the chorioamniotic membranes; yet, the cellular landscape remains fully unknown. Herein, we first used imaging mass cytometry (IMC) to map the phenotype and spatial distribution of immune cells at the maternal-fetal interface of women with PTB and CCA and non-CCA controls (n = 4 each). Cases exhibited a 5-fold increase in T cells, including effector memory CD4+ (p=0.02) and proliferative CD8+ T cells (p=0.04), and a 7-fold rise in CD163+CD206+ macrophages (p=0.05) compared to controls. NK cells were also present in both groups. Neighborhood analysis revealed that CD8+ T cells are in proximity to macrophages and CD4+Tbet+ T cells, and distanced from NK cells. Next, we performed spatial transcriptomics (STx) in each study group (n = 8 PTB with CCA, n = 7 PTB no CCA). STx highlighted diversity in several clusters of T cells, macrophages, and other immune cells found to be transcriptionally active in CCA. Gene ontology analysis indicated enrichment for CD8+ T cell activation, as well as IL-12 and IFNγ signaling in macrophages in CCA. Ongoing efforts are focused on validation and functional testing of such immune cells in order to unveil the complex interactions at the maternal-fetal interface in CCA, a placenta pathology associated with adverse outcomes in the mother and offspring.

Comparing the efficacy of combined versus single immune cell adaptive therapy targeting colorectal cancer.

Colorectal cancer (CRC) is the most frequent cancer with limited therapeutic achievements. Recently, adoptive cellular immunotherapy has been developed as an antitumor therapy. However, its efficacy has not been tested in CRC. This study investigated the ability of an immune cell cocktail of dendritic cells (DCs), T cells, and natural killer (NK) cells to overcome immunological hurdles and improve the therapeutic efficacy of cell therapy for CRC. CRC lysate-pulsed monocyte-derived DCs (Mo-DCs), CRC antigen-specifically expanded T cells (CTL), and in vitro-expanded NK cells were cultured from patient peripheral blood mononuclear cells (PBMC). The ability of the combined immune cells to kill autologous tumor cells was investigated by co-culturing the combined immune cells with patient-derived tumor cells. The Mo-DCs produced expressed T cell co-stimulating molecules like CD80, CD86, human leukocyte antigen (HLA)-DR and HLA-ABC, at high levels and were capable of activating naive T cells. The expanded T cells were predominantly CD8 T cells with high levels of CD8 effector memory cells and low levels of regulatory T cells. The NK cells expressed high levels of activating receptors and were capable of killing other cancer cell lines (K562 and HT29). The immune cell cocktail demonstrated a higher ability to kill autologous tumor cells than single types. An in vivo preclinical study confirmed the safety of the combined immune cell adaptive therapy showing no therapy-related death or general toxicity symptoms. The results suggested that combined immune cell adaptive therapy could overcome the limited efficacy of cell immunotherapy.

Aberrant CD8+T cells drive reproductive dysfunction in female mice with elevated IFN-γ levels.

Interferon-gamma (IFN-γ) is pivotal in orchestrating immune responses during healthy pregnancy. However, its dysregulation, often due to autoimmunity, infections, or chronic inflammatory conditions, is implicated in adverse reproductive outcomes such as pregnancy failure or infertility. Additionally, the underlying immunological mechanisms remain elusive. Here, we explore the impact of systemic IFN-γ elevation on cytotoxic T cell responses in female reproduction utilizing a systemic lupus-prone mouse model with impaired IFN-γ degradation. Our findings reveal that heightened IFN-γ levels triggered the infiltration of CD8+T cells in the pituitary gland and female reproductive tract (FRT), resulting in prolactin deficiency and subsequent infertility. Furthermore, we demonstrate that chronic IFN-γ elevation increases effector memory CD8+T cells in the murine ovary and uterus. These insights broaden our understanding of the role of elevated IFN-γ in female reproductive dysfunction and suggest CD8+T cells as potential immunotherapeutic targets in female reproductive disorders associated with chronic systemic IFN-γ elevation.

Intratumor injection of BCG Ag85A high-affinity peptides enhanced anti-tumor efficacy in PPD-positive melanoma

As one of the scheduled immunization vaccines worldwide, virtually all individuals have been vaccinated with BCG vaccine. In order to verify the hypothesis that delivering BCG high-affinity peptides to tumor areas could activate the existing BCG memory T cells to attack tumor, we firstly predicted the HLA-A*0201 high-affinity peptides of BCG Ag85A protein (KLIANNTRV, GLPVEYLQV), and then, A375 melanoma cells and HLA-A*0201 PBMCs (from PPD-positive adults) were added to co-incubated with the predicted peptides in vitro. We found that the predicted BCG high-affinity peptides could be directly loaded onto the surface of tumor cells, enhancing the tumor-killing efficacy of PBMCs from PPD-positive volunteer. Then, we constructed PPD-positive mice model bearing B16F10 subcutaneous tumors and found that intratumor injection of BCG Ag85A high-affinity peptides (SGGANSPAL, YHPQQFVYAGAMSGLLD) enhanced the anti-tumor efficacy in PPD-positive melanoma mice. Along with the better anti-tumor efficacy, the expression of PDL1 on tumor cell surface was also increased, and stronger antitumor effects occurred when further combined with anti-PD1 antibody. For microenvironment analysis, the proportion of effector memory T cells was increased and the better treatment efficacy may be attributed to the elevated effector memory CD4 + T cells within the tumor. In conclusion, using the existing immune response of BCG vaccine by delivering high-affinity peptides of BCG to tumor area is a safe and promising therapy for cancer.

Causal relationship between the immune cells and ankylosing spondylitis: univariable, bidirectional, and multivariable Mendelian randomization.

Ankylosing spondylitis (AS) is an autoimmune disease that affects millions of individuals. Immune cells have been recognized as having a crucial role in the pathogenesis of AS. However, their relationship has not been fully explored. We chose to employ Mendelian randomization (MR) to investigate the potential correlation between immune cells and AS. We sourced the data on immune cells from the latest genome-wide association studies (GWASs). We obtained data on AS from the FinnGen consortium. Our comprehensive univariable MR analysis covered 731 immune cells to explore its potential causal relationship with AS. The primary analysis method was inverse-variance weighted (IVW). Additionally, we used Cochran's Q test and the MR-Egger intercept test to assess the presence of pleiotropy and heterogeneity. We examined whether our results could be influenced by individual single-nucleotide polymorphisms (SNPs) using the leave-one-out test. We conducted a bidirectional MR to investigate the reverse relationship. We also applied multivariable MR to decrease the potential influence between the immune cells. Overall, our univariable MR analysis revealed eight immune cells associated with AS. Among these, four immune cells contributed to an increased risk of AS, while four immune cells were identified as protective factors for AS. However, the Bonferroni test confirmed only one risk factor and one protective factor with a significance level of p < 6.84E-05. CD8 on effector memory CD8+ T cell could increase the risk of AS (p: 1.2302E-05, OR: 2.9871, 95%CI: 1.8289-4.8786). HLA DR on CD33dim HLA DR+ CD11b+ could decrease the risk of AS (p: 1.2301E-06, OR: 0.5446, 95%CI: 0.4260-0.6962). We also identified a bidirectional relationship between CD4 on CD39+ activated CD4 regulatory T cells and AS utilizing the bidirectional MR. To address potential confounding among immune cells, we employed multivariable MR analysis, which revealed that only one immune cell had an independent effect on AS. HLA DR on CD33dim HLA DR+ CD11b+ could decrease the risk of AS (p: 2.113E-06, OR: 0.0.5423, 95%CI: 0.4210-0.6983). Our findings were consistently stable and reliable. Our findings indicated a potential link between immune cells and AS, which could provide a new idea for future research. Nevertheless, the specific underlying mechanisms require further exploration.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Size Tuning of Mesoporous Silica Adjuvant for One-Shot Vaccination with Long-Term Anti-Tumor Effect.

Despite recent clinical successes in cancer immunotherapy, it remains difficult to initiate a long-term anti-tumor effect. Therefore, repeated administrations of immune-activating agents are generally required in most cases. Herein, we propose an adjuvant particle size tuning strategy to initiate a long-term anti-tumor effect by one-shot vaccination. This strategy is based on the size-dependent immunostimulation mechanism of mesoporous silica particles. Hollow mesoporous silica (HMS) nanoparticles enhance the antigen uptake with dendritic cells around the immunization site in vivo. In contrast, hierarchically porous silica (HPS) microparticles prolong cancer antigen retention and release in vivo. The size tuning of the mesoporous silica adjuvant prepared by combining both nanoparticles and microparticles demonstrates the immunological properties of both components and has a long-term anti-tumor effect after one-shot vaccination. One-shot vaccination with HMS-HPS-ovalbumin (OVA)-Poly IC (PIC, a TLR3 agonist) increases CD4+ T cell, CD8+ T cell, and CD86+ cell populations in draining lymph nodes even 4 months after vaccination, as well as effector memory CD8+ T cell and tumor-specific tetramer+CD8+ T cell populations in splenocytes. The increases in the numbers of effector memory CD8+ T cells and tumor-specific tetramer+CD8+ T cells indicate that the one-shot vaccination with HMS-HPS-OVA-PIC achieved the longest survival time after a challenge with E.G7-OVA cells among all groups. The size tuning of the mesoporous silica adjuvant shows promise for one-shot vaccination that mimics multiple clinical vaccinations in future cancer immunoadjuvant development. This study may have important implications in the long-term vaccine design of one-shot vaccinations.

IgG4-related disease and B-cell malignancy due to an IKZF1 gain-of-function variant

BackgroundMonoallelic loss-of-function IKZF1 (IKAROS) variants cause B-cell deficiency or combined immunodeficiency, whereas monoallelic gain-of-function IKZF1 variants have recently been reported to cause hypergammaglobulinemia, abnormal plasma cell differentiation, autoimmune and allergic manifestations, and infections. MethodsWe studied seven relatives with autoimmune/inflammatory diseases and lymphoproliferative diseases. We analysed biopsy results and performed whole-exome sequencing and immunological studies. ResultsDisease onset occurred at a mean age of 25.2 years (range: 10-64, years). Six patients suffered from autoimmune/inflammatory diseases, four had confirmed immunoglobulin G4-related disease (IgG4-RD), and five developed B-cell malignancies: lymphoma in four and multiple myeloma in the remaining patient. Patients without immunosuppression were not particularly prone to infectious diseases. Three patients suffered from life-threatening COVID-19 pneumonia, one of whom had autoantibodies neutralizing interferon (IFN)-α. The recently described IKZF1 gain-of-function p.R183H variant was found in the five affected relatives tested and in a six-year-old asymptomatic girl. Immunological analysis revealed hypergammaglobulinemia and high frequencies of certain lymphocyte subsets (exhausted B cells, effector memory CD4 T cells, TEMRA and CD28-CD57+ CD4+ and CD8+ T cells, Th2 and Tfh2 cells) attesting to immune dysregulation. Partial clinical responses to rituximab and corticosteroids were observed, and treatment with lenalidomide, which promotes IKAROS degradation, was initiated in three patients. ConclusionHeterozygosity for gain-of-function IKZF1 variants underlies autoimmunity/inflammatory diseases, IgG4-RD and B-cell malignancies, the onset of which may occur in adulthood. Clinical and immunological data are similar to those for patients with unexplained IgG4-RD. Patients may therefore benefit from treatments inhibiting pathways displaying IKAROS-mediated overactivity.

Alterations in peripheral T- and B-cell subsets in patients with systemic sclerosis.

To determine the alteration of peripheral T and B cell subsets in patients with systemic sclerosis (SSc) and to evaluate their correlation with the progression of SSc. We recruited 47 SSc patients and 45 healthy controls (HCs) in this study. Demographic and clinical data were then collected. Flow cytometry was used to detect the proportions of 44 different T and B cell subsets in circulating blood. The proportion of total B cells (p = .043) decreased in SSc patients, together with similar frequencies of total T cells, CD4+ T cells, and CD8+ T cells in both groups. Several subsets of T and B cells differed significantly between these two groups. Follicular helper T cells-1 (Tfh1) (p < .001), helper T cells-1 (Th1) (p = .001), regulatory T cells (Treg) (p = .004), effector memory CD8+ T cells (p = .041), and cytotoxic T cells-17 (Tc17) (p = .01) were decreased in SSc patients. Follicular helper T cells-2 (Tfh2) (p = .001) and, helper T cells-2 (Th2) (p = .001) levels increased in the SSc group. Regulatory B cells (Breg) (p = .015) were lower in the SSc group, together with marginal zone (MZ) B cells (p < .001), memory B cells (p = .001), and non-switched B cells (p = .005). The modified Rodnan skin score (mRSS) correlated with helper T cells-17 (Th17) (r = -.410, p = .004), Tfh1 (r = -.321, p = .028), peripheral helper T cells (Tph) (r = -.364, p = .012) and plasma cells (r = -.312, p = .033). The alterations in T and B cells implied immune dysfunction, which may play an essential role in systemic sclerosis.

Pathological proliferation: a potential mechanism for poor CD4+ T cell recovery in people living with HIV.

People living with HIV (PLWH) fail to achieve normalization of CD4+ T cell counts and function, especially in immunological non-responders (INRs). The frequencies of Ki67+CD4+ T cells were inversely associated with CD4+ T cell counts in HIV infected patients. Early ART did not normalize CD4+ T cell proliferation. However, the features of the abnormal proliferation CD4+ T cell in INRs are far from known. PLWH were divided into INRs (n= 16) and immunological responders (IRs, n= 53) groups. Mass cytometry was applied to peripheral blood T cells to profile the immune cells and liquid chip technique was used to measure plasma levels of cytokines and chemokines. Correlation analyses were conducted to evaluate associations between the degree of CD4+ T cell proliferation and immune function. The percentage of Ki67+ CD4+ T cells were significant higher in INRs, and we defined these cells with significant higher level of Ki67, as over-proliferating cells. No significant difference of markers' expression (HLA-DR, CD38, CD57, PD-1, PD-L1, CD107a, perforin) was found between INRs and IRs. Compared with naïve CD4+ T cells in INRs, Ki67+ CD4+ T cells exhibited lower levels of CD57 and CD38. Whereas Ki67+ T cells exhibited higher levels of CD38 and CD57 and activation compared with differentiated mature central memory CD4+ T cells and effector memory CD4+ T cells. Ki67+ cells did not show higher levels of senescence and activation compared to certain Ki67- CD4+ central memory T cells in IRs. Furthermore, Ki67+ CD4+ Tcm cells exhibited positive correlations with pro-inflammatory cytokines. We proposed and validated the hypothesis of "pathological proliferation" in INRs: excessive proliferation of CD4+ T cells in INRs may be accompanied by aberrant activation, senescence and loss of immune function. Eventually, such over-proliferating but poor-quality cells in INRs result in incomplete recovery of both CD4+ T cell counts and function. An intervention that enhancing the proliferative capacity or functional ability or both of CD4+ T cell in INRs might therefore be beneficial.

High-Dimensional Mass Cytometry Reveals Emphysema-associated Changes in the Pulmonary Immune System.

Rationale: Chronic inflammation plays an important role in alveolar tissue damage in emphysema, but the underlying immune alterations and cellular interactions are incompletely understood. Objectives: To explore disease-specific pulmonary immune cell alterations and cellular interactions in emphysema. Methods: We used single-cell mass cytometry (CyTOF) to compare the immune compartment in alveolar tissue from 15 patients with severe emphysema and 5 control subjects. Imaging mass cytometry (IMC) was applied to identify altered cell-cell interactions in alveolar tissue from patients with emphysema (n = 12) compared with control subjects (n = 8). Measurements and Main Results: We observed higher percentages of central memory CD4 T cells in combination with lower proportions of effector memory CD4 T cells in emphysema. In addition, proportions of cytotoxic central memory CD8 T cells and CD127+CD27+CD69- T cells were higher in emphysema, the latter potentially reflecting an influx of circulating lymphocytes into the lungs. Central memory CD8 T cells, isolated from alveolar tissue from patients with emphysema, exhibited an IFN-γ response upon anti-CD3 and anti-CD28 activation. Proportions of CD1c+ dendritic cells, expressing migratory and costimulatory markers, were higher in emphysema. Importantly, IMC enabled us to visualize increased spatial colocalization of CD1c+ dendritic cells and CD8 T cells in emphysema in situ. Conclusions: Using CyTOF, we characterized the alterations of the immune cell signature in alveolar tissue from patients with chronic obstructive pulmonary disease stage III or IV emphysema versus control lung tissue. These data contribute to a better understanding of the pathogenesis of emphysema and highlight the feasibility of interrogating the immune cell signature using CyTOF and IMC in human lung tissue. Clinical trial registered with www.clinicaltrials.gov (NCT04918706).

Abstract 3858: Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS)

Abstract Introduction: NeoICB is a promising approach for pts with DDLPS and UPS. However, biomarkers to identify which pts benefit from this treatment and understanding the mechanisms of response and resistance to ICB remains a challenge. Methods: We conducted a randomized phase 2 trial of neoICB (anti-PD1 +/- CTLA4) in pts with resectable DDLPS (n=17) and UPS (n=10). Pts with UPS received concurrent neoICB and radiation. The primary endpoint of the trial was pathological response; 3 DDLPS and 9 UPS pts were responders (R) and 14 DDLPS and 1 UPS pts were non-responders (NR). Peripheral blood mononuclear cells (PBMCs) were collected at baseline, prior to cycle 2 of neoICB (C2D1), and at the time of surgery (SURG). We performed mass cytometry (CyTOF) to identify immune cells associated with response and their dynamic changes with neoICB. We designed a custom 41-parameter panel using CD45 live cell barcoding with 31 markers for immune profiling, of which 23 were selected to detect key cell lineage and 8 to detect signaling markers. Batch correction and high dimensional data analyses were performed using CyCombine and CyTOF workflow R packages. Longitudinal comparisons between immune cell populations were conducted using Mann-Whitney U Test. Results: Of 79 available PBMC samples from 27 pts, 63 passed quality control after thawing (≥30% viability) and data clean-up (at least 10,000 events). Data was down-sampled to 5000 events for analysis. Of 19 immune cell types identified, monocytes were the most common followed by CD4+ and CD8+ T cells. There was an early increase in CD38+ effector/effector memory (EM) T cells at C2D1 in pts with DDLPS (CD8+ p=0.005, CD4+ p&amp;lt;0.001) and a similar increase observed in pts with UPS (CD8+ C2D1 p=NS, SURG p=0.034; CD4+ C2D1 p=0.024, SURG p=0.06) and these cells had higher Ki67 expression than CD38- T cells (p&amp;lt;0.005). At baseline, R pts with DDLPS had higher fractions of CD38+ EM CD8+ T cells than NR pts (p=0.044). Analysis in the UPS cohort was limited by the low number of NR pts. Higher expression of TIGIT was observed on CD38+ EM CD8+ T cells compared to CD38- counterparts at all time points. Conclusion: Peripheral CD38+ EM CD8+ T cells are associated with response to neoICB in pts with DDLPS. Longitudinal peripheral immune profiling reveals an increase in CD38+ EM CD8+ and CD4+ T cells with neoICB in pts with DDLPS and UPS. These cells are proliferative and express higher levels of TIGIT, which could be a future target for therapeutic exploration. Importantly, changes in the peripheral immune populations may precede changes in the tumor microenvironment (TME) as preliminary analyses of the TME did not identify early (C2D1) changes. TME imaging mass cytometry analyses are currently underway to associate peripheral and intratumor changes in immune populations. Citation Format: Elise F. Nassif, Hsinyi Lu, Michael Stanford, Karen Millerchip, Diana Romero, Jared Malke, Brenna Matejka, Jeffrey E. Gershenwald, Susmita Kumari, Duncan H. Mak, Angelique J. Lin, Jared K. Burks, Wei-Lien Wang, Alexander J. Lazar, Neeta Somaiah, Cara Haymaker, Christina L. Roland, Emily Z. Keung. Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS) [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 3858.

ENPP2/Autotaxin: The potential drug target for alcoholic liver disease identified through Mendelian randomization analysis.

At present, there is still a lack of radical drug targets for intervention in alcoholic liver disease (ALD), and drug discovery through randomized controlled trials is a lengthy, risky, and expensive undertaking, so we aimed to identify effective drug targets based on human genetics. We used Mendelian randomization (MR) and Bayesian colocalization analysis to investigate 2639 genes encoding druggable proteins and examined the causal effects on ALD (PMID 34737426: 456348 European with 451 cases and 455 897 controls). In addition, we conducted the mediation analysis to explore the potential mechanism using the genome-wide association study (GWAS) data of blood biomarkers as mediators. We finally identified the drug target: ENPP2/Autotaxin and genetically proxied ENPP2/Autotaxin was causally associated with the risk of ALD (OR = 2.28, 95% CI: 1.64 to 3.16, p = 7.49E-7). In addition, we found that the effect of ENPP2/Autotaxin on ALD may be partly mediated by effector memory CD8+ T cell (the proportion of mediation effect: 8.49%). Our integrative analysis suggested that genetically determined levels of circulating ENPP2/Autotaxin have a causal effect on ALD risk and are a promising drug target.

Abstract 1749: Esophageal adenocarcinoma single-cell resolution ecotypes and subtypes mediate chemoradiation resistance

Abstract Esophageal adenocarcinoma (EAC) is an aggressive and lethal disease with a 5-year survival rate less than 20%. The current standard of care for locally advanced EAC patients is neoadjuvant chemoradiation (CRT) followed by surgery and the pathological response rate is low in clinic. An unresolved question is how the cancer cells and their tumor microenvironment (TME) mediate CRT resistance in EAC. To address this question, we performed single-cell RNA sequencing on 84 longitudinal samples from a cohort of patients (N=47) containing 20 good responders (GR) and 27 non-responders (NR). In total, we obtained 302,918 single cells which enabled us to delineate the phenotypic heterogeneity landscape in EAC at baseline, and identify reprogramming in both the immune and tumor cells during treatment that lead to CRT resistance. We identified 4 major cell type compartments in the EAC TME: lymphoid cells (T cells, NK cells, B cells, and plasma cells), myeloid cells (macrophages, neutrophils, dendritic cells, and mast cells), stromal cells (fibroblasts and endothelial cells) and epithelial cells. Our data shows that the TME enriched in lymphoid cells, including resident and effector memory CD8 T cells, follicular helper CD4 T cells, and B cells was associated with better CRT responses. Moreover, a significant myeloid cell infiltration was observed across most patients during CRT and the infiltrated macrophages from GR harbored a more pro-inflammation phenotype. In addition, we identified 4 major ecotypes of the TME cells and 4 major expression subtypes of the cancer cells. Notably, the interactions between TME ecotypes and cancer subtypes are significantly associated with CRT responsiveness. In summary, this study has provided valuable insights into the biology of EAC CRT response, and identified novel predictive biomarkers and actionable targets to enhance CRT efficacy. Citation Format: Rui Ye, Yawei Qiao, Shanshan Bai, Emi Sei, Min Hu, Jianzhong He, Nan Li, Yifan Wang, Pablo Lopez, Wei Zhang, Fei Ye, Brian Weston, Emmanuel Coronel, William Ross, Phillip Ge, Bhutani Manoop, Nicholas Navin, Steven Lin. Esophageal adenocarcinoma single-cell resolution ecotypes and subtypes mediate chemoradiation resistance [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 1749.

Abstract 1122: Inhibition of HDAC6 enhanced radiation therapy induced antitumor response in melanoma

Abstract Patients with advanced melanoma are often treated with radiation therapy (RT) in combination with immunotherapies. Immunogenic tumor cell death induced by radiation is known to activate innate and adaptive immunity. Macrophage activity in the irradiated tumor microenvironment (TME) has been implicated to play a critical role in post-RT innate immune responses. However, tumor-associated macrophages (TAMs) due to their phenotypic plasticity convert from initial antitumor M1 phenotype to protumor M2 macrophages resulting in relapse and metastasis of any surviving tumor cells. Therefore, strategies regulating tumor macrophages post-radiation can enhance the therapeutic effectiveness of radiation therapy. Here, we report targeting HDAC6 function with a novel selective inhibitor (SP-2-225) as a potential therapeutic candidate for combination with RT. When administered intraperitoneally at a dose of 25mg/kg, SP-2-225 resulted in significant tumor suppression in the immunocompetent SM1 murine melanoma model. Phenotyping of tumor immune infiltrate indicated a profound decrease in M2 macrophages resulting in an M1/M2 macrophage ratio and an increase in CD8 effector memory T-cells. Further, in-vitro co-culture studies using trans-well assays indicated that irradiated tumor cells attract M2 macrophages, and treatment with SP-2-225 abrogated the M2 macrophage migration but did not alter M1 macrophage migration. In addition, 24 hours post-RT, intratumor administration of SP-2-225 treated M1 macrophages as adoptive cell therapy resulted in diminished tumor growth. The combination of conditioned M1 macrophages with an irradiated tumor microenvironment resulted in an increased M1/M2 ratio and infiltration of effector memory and central memory CD8 T-cells underscoring the influence of transplanted macrophages on the local antitumor immunity. Therefore, our findings demonstrate the ability of HDAC6 inhibitors to regulate macrophage function and phenotype as a cell therapy for use in combination with radiation therapy. Citation Format: Xintang Li, Manasa Suresh, Hawa Coulibaly, David Quiceno-Torres, Nithya Gajendran, Karen Tan, Satish Noonepalle, Scott Grindrod, Anatoly Dritschilo, Alejandro Villagra. Inhibition of HDAC6 enhanced radiation therapy induced antitumor response in melanoma [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 1122.