Upregulation Of CD86 Research Articles

Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands.

Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.

Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor.

Solid cancers Myeloid cells are prevalent in solid cancers, but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME), which hinders the effectiveness of cancer immunotherapies. Myeloid cells' natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers, including tumor infiltration, tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1, which is often upregulated by solid cancers to evade immune responses. Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1, while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells, we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally, we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy's effectiveness in vivo. Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1β. Moreover, CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors, infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Taken together, these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers.

Comprehensive Analysis of Immune Signatures in Primary Biliary Cholangitis and Autoimmune Hepatitis.

Primary Biliary Cholangitis (PBC) and Autoimmune Hepatitis (AIH) are autoimmune diseases that target hepatocytes and bile duct cells, respectively. Despite their shared autoimmune nature, the differences in immunologic characteristics between them remain largely unexplored. This study seeks to elucidate the unique immunological profiles of PBC and AIH, and to identify key differences. We comprehensively analyzed various T-cell subsets and their receptor expression in a cohort of 45 patients, including 27 PBC and 18 AIH cases. Both diseases exhibited T cell exhaustion and senescence along with a surge in inflammatory cytokines. Significantly increased CD38+HLA-DR+CD8+T cell populations were observed in both diseases. AIH was characterized by an upregulation of CD8+TEMRA, CD4+TEM, and CD4+TEMRA cells, and a concurrent reduction in Treg cells. In contrast, PBC displayed a pronounced presence of Tfh cells and a contraction of CD4-CD8-T cell populations. Correlation analysis revealed that NKP46+NK frequency was closely tied to ALT and AST levels, and TIGIT expression on T cells was associated with GLB level in AIH. In PBC, there is a significant correlation between Tfh cells and ALP levels. Moreover, the identified immune landscapes in both diseases strongly related to disease severity. Through logistic regression analysis, γδ T, TIGIT+Vδ2 T, and Tfh1 cell frequencies emerged as distinct markers capable of differentiating PBC from AIH. In conclusion, our analyses reveal that PBC and AIH share similarities and differences regarding to immune profiles. And γδ T, TIGIT+Vδ2 T, and Tfh1 cell frequencies are potential noninvasive immunological markers that can differentiate PBC from AIH.

Tetracyclines enhance antitumor T-cell immunity via the Zap70 signaling pathway

BackgroundCancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we...

Abstract 5302: CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors

Abstract INTRODUCTION: CD137 is a TNF receptor superfamily (TNFRSF9) costimulatory receptor expressed by T cells and NK cells. Clustering-mediated CD137 signaling enhances immune cell survival, proliferation, cytokine production and memory formation. CD40 (previously TNFRSF5) is a related superfamily member expressed primarily on B cells and antigen-presenting cells (APCs) which upon clustering is also a key modulator of T cell responses. Mesothelin (MSLN) is highly upregulated on tumor cells in ovarian and pancreatic cancer and select other solid tumour histologies. To avoid systemic toxicity, CB699 is designed to be immunologically active in the presence of any two of its three binding targets, thus enhancing both immune-immune and tumour-immune crosstalk. We present here the preclinical pharmacology of CB699. EXPERIMENTAL PROCEDURES: Binding of CB699 to CD40, CD137 or MSLN was measured by surface plasmon resonance and by flow cytometry. CD137 and/or CD40 agonism, immune cell activation and cytokine secretion were evaluated in vitro using reporter gene assays and primary cell coculture assays in the presence/absence of MSLN-expressing tumor cells. The ability of CB699 to enhance tumor cell killing was characterized in 3D spheroid cocultures. CB699 toxicology and toxicokinetics was assessed in a dose range-finding study in cynomolgus macaques. CB699 was also assessed in vivo with a human CD34+ cell engrafted mouse model. RESULTS: CB699 binds to primary and immortalized cells that express CD40, CD137 or MSLN, with low nanomolar affinity. CB699 also binds to cells from primary disaggregated human solid tumors. CB699 induces CD137 reporter cell activity in cocultures containing cells expressing either CD40 or MSLN. Additionally, CB699 induces CD40 reporter cell activity in cocultures containing cells expressing either CD137 or MSLN. CB699 induces IL-2 secretion and upregulation of CD86 on B cells in primary cocultures containing healthy human PBMCs and MSLN expressing cancer cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB699 and was coupled with immune cell expansion. CB699 showed significant pharmacology in vivo with immune cell engrafted mice. Finally, CB699 was well tolerated in a dose range finding non-human primate toxicology study. CONCLUSIONS: CB699 is a novel MSLN-binding CD137 and CD40 dual agonist that selectively enhances immune cell activity by enhancing both T cell and APC signaling axes. CB699 is positioned to enter clinical development. Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Holly Spencer, Minjung Song, Timothy Wong, Phillip D. Bartlett, Kieran Williams, Lukas Jasaitis, Philip Bland-Ward. CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors [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 5302.

Abstract 172: Targeting the poliovirus receptor to activate T cells and induce myeloid-derived suppressor cells to differentiate to M1-like macrophages via the IFNγ-pSTAT1-IRF8 axis in cancer therapy

Abstract T cell immunoglobulin and ITIM domain (TIGIT) is one of the most important immune checkpoints expressed on lymphocytes, and poliovirus receptor (PVR, also CD155) serves as the most crucial ligand for TIGIT, harboring an important function in cancer cells and influencing the tumor microenvironment (TME). While it's well-established that TIGIT blockade could reverse immunosuppression, the question of whether direct inhibition of PVR yields comparable results remains to be fully elucidated. In this study, we generated a mouse gene Pvr knock out (KO) Lewis lung cancer (LLC) cell line and found that the Pvr-KO tumors tended to develop a pro-inflammatory TME, as reflected by a downregulation of monocytic-myeloid derived suppressor cells (M-MDSCs) and an upregulation of CD8+ central memory T cells. Through bulk RNA-seq and multi-cytokines measurement, we quantified the transcription and protein levels of immunostimulatory cytokines and chemokines and found they were significantly upregulated in Pvr-KO TME, including GM-CSF, IFN-γ, TNF-α, IL-2/4/6/10, CCLs (including CCL2/3/4/5) and CXCLs (including CXCL1/2/5). We further employed single-cell RNA sequencing (scRNA-seq) on 20,026 intra-tumoral CD45+ immune cells. Using a conserved MDSCs gene set and the 'AddModuleScore' function of Seurat package, we identified the clusters of MDSCs and found the differentiation ability of M-MDSCs in Pvr-KO TME was enhanced. Through pseudo-temporal ordering, we found the M-MDSCs in the Pvr-KO TME have a trend to differentiate towards M1-like macrophages, which could play an essential positive role in anti-tumor immunity. We further focused on the scRNA-seq of T cell subsets and found the enhanced function of cytotoxic CD8+ T cells, leading to increased production of IFN-γ. In the intercellular communications analysis, a significant upregulation of the IFNG-IFNR signal between T cells: MDSCs in the Pvr-KO group was observed. Moreover, we found that Irf8, a key factor in myeloid cell differentiation, was significantly upregulated in the M-MDSCs of Pvr-KO tumor. We then employed CUT & Tag sequencing and confirmed that IFN-γ induces phosphate Stat1, which then binds on the promoter of Irf8. These findings suggest that the IFN-γ-Stat1-Irf8 axis plays a crucial role in MDSCs differentiation and may be a potential target for therapeutic intervention in cancer. Next, an anti-PVR nanobody was synthesized and its efficacy was evaluated in the context of LLC and 4T1 tumor grafts. When in combination with anti-PD-1 therapy, anti-PVR exhibited a remarkable capacity to reduce tumor size in both tumor models with no notable indications of toxicity observed. Our findings provide new insights into the role of PVR in modulating the TME and offer potential strategies for enhancing the effectiveness of immunotherapy. Citation Format: Mingyang Feng, Sirui Tan, Qizhi Ma, Benxia Zhang, Yue Chen, Qiu Li, Yongsheng Wang. Targeting the poliovirus receptor to activate T cells and induce myeloid-derived suppressor cells to differentiate to M1-like macrophages via the IFNγ-pSTAT1-IRF8 axis in cancer therapy [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 172.

Abstract 6744: Activating anti-MDS immunity with polarized dendritic cell vaccines

Abstract Less than 50% of patients with higher-risk myelodysplastic syndromes (MDS) show clinical response to standard-of-care hypomethylating (HMA) therapy. Significant enthusiasm existed for immunotherapy/HMA combinations for these patients, but studies have failed to deliver on the initial promise. We have demonstrated that patients with MDS have a deficiency in antigen-presenting cells, called CD141Hi dendritic cells (DC). DC-deficient MDS patients have reduced response to HMAs, worse overall survival, and show less response to cancer vaccination. We hypothesized that restoration of this population using a novel DC vaccination approach - alpha-type-1 polarized dendritic cells (αDC1) - might compensate for this deficiency and restore immunity. To generate αDC1s, we collected monocytes from MDS patients and healthy donors (HD) and cultured them for 6 days in vitro in the presence of recombinant human GM-CSF and IL-4 followed by an additional 18-24 hours in the presence of αDC1 maturation factors (poly-I:C/IFNα/IFNγ/TNFα/IL-1β). Overall, we attempted to generate αDC1s from 15 MDS samples; 13/15 exhibited intermediate to very high-risk disease and 10/15 are currently receiving HMA therapy. We successfully cultured cells from 8/15 samples (53%). Of the 8 MDS samples, 5 were collected from patients receiving HMA therapy (62.5%). We were unable to successfully culture αDC1s from the other 5 patients receiving HMA therapy, and thus the effect of HMA therapy on aDC1 generation remains unclear. To test the function of MDS αDC1s, we focused on two signals necessary to activate immune responses: 1) the expression of co-stimulatory molecules and 2) the production of IL-12p70. Flow cytometric analysis on αDC1s and immature DCs from MDS samples revealed intra-patient variation in the fold-change surface expression of CD40/80/83/86 and HLA-DR proteins. Additionally, there was a trend towards decreased up-regulation of CD80 and CD86 in MDS specimens compared to HD. To assess production of IL-12p70, we stimulated immature DCs and αDC1s with CD40L and performed ELISA analysis on the media supernatant. We found that 3 out of 4 samples could produce IL-12p70 (range 27 - 5,480 pg/ml). To date, our results suggest that generation of MDS patient-derived αDC1 cells is feasible and additional work is being performed to fully characterize their ability to activate anti-MDS immune responses. Since αDC1s have an established safety profile in patients with solid tumors, we anticipate that we will be able to rapidly translate our results to the clinic. Citation Format: Daniel Alvarado, Jennifer Peresie, Adam Brinkman, Cheryl Allen, Weijian Jiang, Pawel Kalinski, Tiffany Tanaka, Elizabeth Griffiths, Michael Nemeth. Activating anti-MDS immunity with polarized dendritic cell vaccines [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 6744.

Abstract 6647: Established 3D tumor-spheroid co-culture models demonstrate macrophage plasticity and unveil potential supportive roles of macrophages in the T cell-tumor-macrophage crosstalk post radiation therapy

Abstract Ionizing radiation therapy (RT) has been characterized as an immunogenic event that in some tumors results in cell-death mediated activation of innate inflammatory and immunostimulatory mediators. These inflammatory mediators in turn modulate lymphocyte effector function in the tumor environment that can contribute to radiation-mediated control of tumors. We aim to model these interactions ex vivo, to identify the critical signals regulating immune function. We demonstrate that it is necessary to establish a three-dimensional model for crosstalk between tumor cells and immune cells to recapitulate myeloid and T cell responses to radiation. We have established 3D tumor spheroid co-culture models using murine MC38 colon carcinoma and Panc02-SIY pancreatic carcinoma cell lines with bone-marrow derived macrophages (BMDM). The spheroid cultures were exposed to 12 Gy RT and maintained in culture over a period of 5 days. Analyses were done using live-cell imaging and flow cytometry. Our findings demonstrate a decrease in hypoxia post RT, and exposure to RT increased the phagocytosis of dying cancer cells by macrophages. This was followed by a significant increase in CD80 and CD86 expression on the surface of macrophages in co-cultures on day 3 and day 4 post RT. These data matched our analyses of tumors treated in vivo with radiation and analyzed on day 4 post RT. In addition to RT-mediated upregulation of costimulatory molecules on macrophages, we also observed an increase in PD-L1 expression that may also impact T cell responses. To test T cell function in the 3D model, antigen-specific T cells were added to the Panc-02-SIY and BMDM spheroid co-cultures, which resulted in enhanced T cell proliferation in RT-treated groups. To assess the mechanism of macrophage activation in co-cultures, we evaluated HMGB1 as an innate adjuvant that may cause TLR4-mediated macrophage activation; however, the pattern of activation did not match that of RT. The involvement of the nucleic acid sensing cGAS-STING pathway was assessed by using BMDM from STING knockout (KO) mice in the 3D co-cultures. Similar upregulation of CD80 and CD86 were observed on STING KO macrophages in 3D co-cultures on day 4 post RT, indicating the observed phenomena to be independent of STING signaling. Ongoing studies aim to decrypt the innate downstream signaling pathways involved in RT-mediated polarization of macrophages so as to identify potential targeted therapies for tumor-associated macrophages, and also translate this to patient tumors. Citation Format: Aanchal Preet Kaur, Alejandro F. Alice, Megan R. Potuznik, Gwen Kramer, Jason Baird, David J. Friedman, Marka R. Crittenden, Michael J. Gough. Established 3D tumor-spheroid co-culture models demonstrate macrophage plasticity and unveil potential supportive roles of macrophages in the T cell-tumor-macrophage crosstalk post radiation therapy [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 6647.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [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 6330.

Abstract 5227: Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues

Abstract Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies, offering hope to patients with limited treatment options. However, extending this therapy to myeloid malignancies and solid tumors has proven challenging due to co-expression of targetable antigens on hematopoietic progenitors and healthy tissues. In this context, γδ T cells show promise, as they can directly identify and eliminate malignant cells via recognition of multiple tumor-associated stress antigens rarely expressed on normal tissues. We leveraged the tumor-sensing capabilities of γδ T cells with enhanced tumor localization by employing a non-signaling CAR (nsCAR) that excludes the CD3ζ domain, facilitating targeted tumor cell killing while sparing healthy tissues. We designed second-generation lentiviral constructs for anti-CD33-scfv for in vitro evaluation against acute myeloid leukemia (AML) lines HL-60, KG-1a, and MOLM-13 and the chronic myeloid leukemia (CML) line K-562 Additionally, we tested CD33/CD123 dual-targeting nsCAR constructs (ns-dCAR) to determine if the addition of CD123 targeting enhanced the therapeutic index. We also tested whether a secreted IL-15 (sIL-15) would enhance CAR-T cell fitness. We observed significant upregulation of CD69 in Jurkat cells transduced with signaling CARs in co-culture with KG-1a cells but not in those with nsCAR constructs. We also noticed a time-dependent reduction (43%) in the CD3ζ+ CD33CAR+ population over a 7-day extended coculture while the nsCAR+ Jurkat population remained stable, suggesting potential mitigation of activation-induced cell death (AICD). Expanded and activated γδ T cells from healthy donors were then transduced with nsCAR lentiviral vectors. The ns33CAR+ γδ T cells exhibited enhanced killing capability against HL-60 (up to 1.3x) and K-562 (up to 1.6x) compared to unmodified γδ T cells (UTD) in a 24-hour cytotoxicity assay. Incorporation of sIL-15 into the nsCD33 CAR construct also increased killing across all 4 cell lines (up to 1.8x for HL-60, up to 2.6x for KG-1a, up to 2.0x for MOLM-13 and up to 2.0x for K-562). Minimal cytotoxicity (<10%) was observed for nsCARs or UTDs against normal CD33+/CD123+ cells from healthy donor PBMC or CD34+ HPSCs. The ns-dCAR constructs did not improve in vitro AML killing compared to ns33CAR alone, although ns-dCARs generally exhibited lower transduction efficiency. In summary, our findings suggest that combining nsCAR constructs with γδ T cells may widen the therapeutic window to expand the reach of CAR-T therapy to cancers with limited target antigen expression on critical healthy tissues. Further optimization to improve integration and incorporation of membrane-bound IL-15 co-expression holds the potential to enhance both the efficacy and safety profiles of next-generation adoptive cell therapies against a broader spectrum of cancers. Citation Format: Lei Ding, Yanjie Li, Jiexue Li, Mariska Ter Haak, Lawrence Lamb. Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues [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 5227.

Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

Unveiling the immunomodulatory effect of the novel probiotic Akkermansia muciniphila and its protective effect in vitro

Akkermansia muciniphila, a bacterium found in the human microbiota, has gained interest due to its potential health benefits. Previous studies have linked its absence to inflammatory disorders, while also suggesting its role in maintaining a healthy gut barrier. However, there is limited information on its specific effects on the immune system. Therefore, the aim of this research was to analyze the in vitro response triggered by A. muciniphila employing RAW 264.7 macrophages. The study focused on investigating the production of cytokines and nitric oxide, along with evaluating the expression of inflammatory surface cellular markers. Additionally, we assessed its potential to protect against intestinal infections, using Salmonella enterica serovar Enteritidis as a model. Our findings reveal a modulation effect of A. muciniphila with pro-inflammatory features, including the release of pro-inflammatory cytokines and upregulation of CD40 and CD80 surface markers, in contrast with previous reported data. Importantly, A. muciniphila could protect against Salmonella infection by promoting macrophage activation, appearing as a promising probiotic candidate for the control of intestinal infections.

Effects of ageing and frailty on circulating monocyte and dendritic cell subsets

Ageing is associated with dysregulated immune responses, resulting in impaired resilience against infections and low-grade inflammation known as inflammageing. Frailty is a measurable condition in older adults characterized by decreased health and physical impairment. Dendritic cells (DCs) and monocytes play a crucial role in initiating and steering immune responses. To assess whether their frequencies and phenotypes in the blood are affected by ageing or frailty, we performed a flow cytometry study on monocyte and DC subsets in an immune ageing cohort. We included (n = 15 in each group) healthy young controls (HYC, median age 29 years), healthy older controls (HOC, 73 years) and Frail older controls (76 years). Monocyte subsets (classical, intermediate, non-classical) were identified by CD14 and CD16 expression, and DC subsets (conventional (c)DC1, cDC2, plasmacytoid (p)DC) by CD11c, CD1c, CD141 and CD303 expression. All subsets were checked for TLR2, TLR4, HLA-DR, CD86, PDL1, CCR7 and CD40 expression. We observed a lower proportion of pDCs in HOC compared to HYC. Additionally, we found higher expression of activation markers on classical and intermediate monocytes and on cDC2 in HOC compared to HYC. Frail participants had a higher expression of CD40 on classical and non-classical monocytes compared to the HOC group. We document a substantial effect of ageing on monocytes and DCs. Reduced pDCs in older people may underlie their impaired ability to counter viral infections, whereas enhanced expression of activation markers could indicate a state of inflammageing. Future studies could elucidate the functional consequences of CD40 upregulation with frailty.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

Causal relationship between the immune phenotype of monocytes and myasthenia gravis: A Mendelian randomization study

BackgroundMonocytes play an essential role in developing autoimmune diseases; however, their association with myasthenia gravis (MG) development is unclear. MethodsWe performed a two-sample Mendelian randomization analysis to assess the causal relationship between monocyte-associated traits and MG, reviewing summary statistics of genome-wide association studies (GWAS). ResultsUsing the inverse variance weighted method, the following were found to be causally associated with MG: HLA-DR on monocytes (OR, 1.363; 95% CI, 1.158–1.605; P = 2E-04), HLA-DR on CD14+ monocytes (OR, 1.324; 95% CI, 1.183–1.482; P = 1.08E-06), HLA-DR on CD14+CD16− monocytes (OR, 1.313; 95% CI, 1.177–1.465; P = 1.07E-06), CD40 on monocytes (OR, 1.135; 95% CI, 1.012–1.272; P < 0.05), CD40 on CD14+CD16− monocytes (OR, 1.142; 95% CI, 1.015–1.285; P < 0.05), CD40 on CD14+CD16+ monocytes (OR, 1.142; 95% CI, 1.021–1.278; P < 0.05), CD64 on CD14+CD16+ monocytes (OR, 1.286; 95% CI, 1.019–1.623; P < 0.05). ConclusionsThe present study suggests a causal relationship between the upregulation of CD40, HLA-DR, and CD64 on monocytes and the development of MG. Altered monocyte function may potentially be a risk factor for MG and a therapeutic target.

Artificial Targets: a versatile cell-free platform to characterize CAR T cell functionin vitro.

Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.

Upregulation of CD8+ regulatory T cells following liver-directed AAV gene therapy

Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.

Identification and analysis of alloreactive T lymphocytes from peripheral blood mononuclear cells.

Alloreactive T-cell responses against mismatched MHC or minor histocompatibility antigens may result in deleterious graft-versus-host disease (GVHD) and increased morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, these T-cell responses may be directed against residual tumor cells (the graft-versus-tumor effect, GVT), thus preventing relapse of the disease. Recent findings have shown that CD45RA+ naïve T cells, but not CD45RA- memory T cells are the major contributors to GVHD, thus leading to clinical trials where CD45RA+-depleted, memory-enriched T-cell products are adoptively transferred following allo-HSCT to prevent GVHD and enhance immune reconstitution. However, residual alloreactivity may still be present in the memory T-cell compartment, thus contributing to prevent disease relapse by GVT. Here, we describe a simple cell-based protocol to identify alloreactive naïve and memory T cells by co-culturing T-cell subsets and third-party antigen-presenting cells. The responding cells are identified following dilution of carboxyfluorescein succinimidyl ester (CFSE) and upregulation of the activation marker CD25. These CFSE-diluting cells can be further phenotyped by high-dimensional flow cytometry, or purified with a cell sorter for downstream genomic and functional assays.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.