Activation Marker CD69 Research Articles

Elucidating the Role of Tolerogenic Dendritic Cells in Immune Tolerance and Disease Progression in Multiple Myeloma

Introduction: Previous research has demonstrated that programmed death-ligand 1 (PD-L1) in multiple myeloma (MM) cells not only suppresses anti-tumor immune responses but also enhances malignant potential through the PD-L1-PD-1 interaction [Ishibashi M, et al. Cancer Immunol Res. 2016]. However, it has not yet been shown that monotherapy and combination therapies utilizing PD-1/PD-L1 antibodies are sufficiently effective for MM. One reason for this is the absence of myeloma-specific cytotoxic T lymphocytes induction in the MM tumor microenvironment. In certain cancers, tumor-infiltrating dendritic cells (DCs) undergo conversion into tolerogenic DCs, which are crucial for inducing and maintaining T-cell tolerance, although this remains unclear in MM. Thus, this study aimed to investigate the function and induction mechanisms of tolerogenic DCs in the MM microenvironment. Materials and Methods: (1) Flow cytometry (FCM) was employed to assess the two conventional DC (cDC; lineage negative, HLA-DR +CD11c high) subsets, cDC1 (CD141 + cDC) and cDC2 (CD1c + cDC), in bone marrow (BM) samples obtained from MM patients (n=17) and healthy controls (n=13). (2) CD14 + monocytes were cultured with IL-4 and GM-CSF to generate monocyte-derived DCs (moDCs). Simultaneously, moDCs were co-cultured with MM cell lines using a trans-well system. The characteristics and functions of moDCs were analyzed using real-time PCR, RNA-sequencing (RNA-seq), western blotting, ELISA, and FCM after lipopolysaccharide stimulation. Results: The frequency of cDC1 subsets was significantly lower in MM patients compared to controls, while the major cDC2 subset did not differ. Similarly, when moDCs were co-cultured with MM cells, moDC numbers were markedly reduced. These co-cultured moDCs exhibited downregulated expression of activation markers CD80, CD86, and CD83, along with decreased phosphorylation of ERK and S6K. Conversely, these moDCs upregulated the expression of immunosuppressive factors IL-10, ARG1, and NOS2 through increased transcription factor C/EBPβ. Moreover, these moDCs inhibited T-cell proliferation, activation, and interferon-γ production in comparison to control moDCs, indicating the presence of immature and tolerogenic phenotypes. However, these phenotypes were not observed in moDCs co-cultured with soluble factors secreted from MM and stromal cells. Treatment with AZD3965 improved the phenotype of moDCs co-cultured with MM cells, reversing their suppressive effect on T-cell proliferation. AZD3965 targets monocarboxylate transporter 1 (MCT1) in MM cells, thereby suppressing extracellular lactate accumulation and acidic pH in the MM microenvironment. Furthermore, RNA-seq analysis comparing moDCs co-cultured with and without MM cells revealed downregulation of gene sets associated with activated DCs in moDCs co-cultured with MM. Notably, moDCs co-cultured with MM showed high enrichment of gene sets related to lysosome biogenesis and NOD-like receptor signaling. This suggests that inflammasome activation in these moDCs leads to caspase-1 activation, resulting in increased levels of mature IL-1β and IL-18 in their cell culture supernatants. Additionally, co-culturing CD38-positive MM cells with CD203a/CD73-positive HS-5 stromal cells and NAD + resulted in the generation of adenosine in the cell culture supernatant. Adenosine promotes the immaturation of mature DCs; however, inflammasome activation does not occur in these DCs. Conclusions: Patients with MM exhibit a reduced frequency of cDC1 subsets, which are crucial for cross-presentation to CD8 + T cells. moDCs in acid pH conditions derived from MM cells display an immature phenotype and impaired function due to decreased DC activation signals. Furthermore, inflammasome activation promotes the secretion of IL-1β and IL-18, contributing to pro-tumorigenic inflammation in the MM microenvironment. Moreover, adenosine generated via the CD38/CD203a/CD73 adenosinergic pathway promotes the immaturation and dysfunction of moDCs. Thus, the characteristics of tolerogenic DCs may vary depending on the MM microenvironment. These findings highlight the induction of tolerogenic DCs by the metabolic microenvironment in MM, leading to immune tolerance and disease progression.

Impact of Psychological Distress on Immune Phenotype in CLL/SLL Patients Managed By Active Observation

Introduction: Asymptomatic patients with a new diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) are usually managed by active observation; however, many patients report elevated levels of distress and worry during the ‘watchful waiting’ phase. Psychological distress may have clinical implications given that natural killer [NK] and T cells are not only sensitive to distress, but also critical in controlling disease progression. Thus, the purpose of this study was to examine associations of psychological functioning and quality of life (QOL) with biomarker expression and functional activity of immune cells from peripheral blood of CLL/SLL patients being managed with surveillance. Methods: We conducted a multisite, prospective study in patients with indolent B-cell neoplasms who were managed with surveillance. Participants completed measures of perceived stress (Perceived Stress Scale), anxiety and depressive symptoms (Hospital Anxiety and Depression Scale) and quality of life (FACT-G) at study entry and then every 6 months for up to 2 years or disease progression (whichever occurred first). Blood samples were collected at study entry and every 6 months for immune phenotyping using multiparametric flow cytometry (up to 13-color). Biomarkers of activation, viability, proliferation, exhaustion, and receptor expression were assessed on subsets of T, NK, and myeloid cells, and mean fluorescence intensity or percentage of cells staining were determined. Results: Between April 2016 and November 2020, 233 patients were enrolled at two academic institutions in Philadelphia, including 111 with a diagnosis of CLL/SLL. Of these, 76 patients also had viable samples for immune phenotyping. Participants were predominantly male (60.5%) with a mean age of 63.7 years (SD=9.1 years). Approximately 5% self-reported their race as Black or African American and 4% were of Hispanic ethnicity. At enrollment, the median time since diagnosis was 10.6 months (median follow-up time = 23.6 months). Multivariable regression analyses revealed that increased depressive symptoms were associated with a significant shift from lower percentages of non-classical monocytes (NCM) to higher percentages of classical monocytes (CM; see Figure 1). Similar significant population shifts from reduced NCM toward increased CM were also observed with increased levels of stress and anxiety. These results suggest that patients reporting increased stress, anxiety, and depressive symptoms have fewer of the “patrolling” NCM and increased “sensing” CM in peripheral blood. We also assessed expression of numerous innate immune receptors, including DNAM-1, which can stimulate responses by NK and T cells toward tumors. We observed lower expression of DNAM-1 on CD4 + helper T cells and CD8 + cytotoxic T cells in patients with increased depressive symptoms (p< 0.05). Similarly, patients reporting lower QOL also expressed lower levels of DNAM-1 on CD4 + and CD8 + T cells, as well as cytotoxic CD56 dim NK cells (p<0.05). As DNAM-1 is important for antitumor responses, our data suggest that patients with higher depressive symptoms have T and NK cells with reduced capacity to respond to CLL/SLL tumor cells. NKp30, another activating receptor on NK and CD8+ T cells promoting cytotoxicity, was also expressed at lower levels on CD56 dim NK cells and T cells in the peripheral blood of patients reporting depressive symptoms. Patients who reported higher levels of depressive symptoms had lower levels of expression of the activation marker CD69 on CD4+ and CD8+ T cells. Finally, longitudinal analyses revealed that higher levels of depressive symptoms predicted lower levels of PD-L1 on CD56 dim NK cells over time ( p = 0.04). Conclusion: In CLL/SLL patients, psychological functioning is associated with differential patterns of monocyte distribution. Greater depressive symptoms and lower quality of life predicted lower expression of innate immune receptors, such as DNAM-1 on CD4 + helper T cells and CD8 + cytotoxic T cells. Future directions will investigate whether these measures are associated with disease progression or the occurrence of second primary cancers, given the importance of DNAM-1 and NKp30 in immune surveillance for cancers and reactivation of latent viruses. These findings may have direct translational relevance and enable the early identification of patients at-risk for poor outcomes.

Durable natural killer cell response after three doses of SARS-CoV-2 inactivated vaccine in HIV-infected individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine can induce a potent cellular and humoral immune response to protect against SARS-CoV-2 infection. However, it was unknown whether SARS-CoV-2 vaccination can induce effective natural killer (NK) cell response in people living with human immunodeficiency virus (PLWH) and healthy individuals. Forty-seven PLWH and thirty healthy controls (HCs) inoculated with SARS-CoV-2 inactivated vaccine were enrolled from Beijing Youan Hospital in this study. The effect of SARS-CoV-2 vaccine on NK cell frequency, phenotype, and function in PLWH and HCs was evaluated by flow cytometry, and the response of NK cells to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation was also evaluated. SARS-CoV-2 vaccine inoculation elicited activation and degranulation of NK cells in PLWH, which peaked at 2 weeks and then decreased to a minimum at 12 weeks after the third dose of vaccine. However, in vitro stimulation of the corresponding peripheral blood monocular cells from PLWH with SARS-2-OS protein did not upregulate the expression of the aforementioned markers. Additionally, the frequencies of NK cells expressing the activation markers CD25 and CD69 in PLWH were significantly lower than those in HCs at 0, 4 and 12weeks, but the percentage of CD16 + NK cells in PLWH was significantly higher than that in HCs at 2, 4 and 12weeks after the third dose of vaccine. Interestingly, the frequency of CD16 + NK cells was significantly negatively correlated with the proportion of CD107a + NK cells in PLWH at each time point after the third dose. Similarly, this phenomenon was also observed in HCs at 0, 2, and 4 weeks after the third dose. Finally, regardless of whether NK cells were stimulated with SARS-2-OS or not, we did not observe any differences in the expression of NK cell degranulation markers between PLWH and HCs. s:SARS-CoV-2 vaccine elicited activation and degranulation of NK cells, indicating that the inoculation of SARS-CoV-2 vaccine enhances NK cell immune response.

Sex and Age Impact CD4+ T Cell Susceptibility to HIV In Vitro through Cell Activation Dynamics.

Cellular composition and the responsiveness of the immune system evolve upon aging and are influenced by biological sex. CD4+ T cells from women living with HIV exhibit a decreased viral replication ex vivo compared to men's. We, thus, hypothesized that these findings could be recapitulated in vitro and infected primary CD4+ T cells with HIV-based vectors pseudotyped with VSV-G or HIV envelopes. We used cells isolated from twenty donors to interrogate the effect of sex and age on permissiveness over a six-day activation kinetics. Our data identified an increased permissiveness to HIV between 24 and 72 h post-stimulation. Sex- and age-based analyses at these time points showed an increased susceptibility to HIV of the cells isolated from males and from donors over 50 years of age, respectively. A parallel assessment of surface markers' expression revealed higher frequencies of activation marker CD69 and of immune checkpoint inhibitors (PD-1 and CTLA-4) in the cells from highly permissive donors. Furthermore, positive correlations were identified between the expression kinetics of CD69, PD-1 and CTLA-4 and HIV expression kinetics. The cell population heterogeneity was assessed using a single-cell RNA-Seq analysis and no cell subtype enrichment was identified according to sex. Finally, transcriptomic analyses further highlighted the role of activation in those differences with enriched activation and cell cycle gene sets in male and older female cells. Altogether, this study brought further evidence about the individual features affecting HIV replication at the cellular level and should be considered in latency reactivation studies for an HIV cure.

Bovine NK subsets in the afferent lymph and lymph nodes have distinct expression of naïve and activation-associated cell surface expressed molecules, and are differentially stimulated by BCG vaccination

Bovine natural killer (bNK) cells are heterogeneous cell populations defined by constitutive expression of the natural cytotoxicity receptor, NKp46 (CD335). Two major subsets of bNK cells, classified by differential expression of CD2, display divergent functions in innate immunity, and are hypothesised to contribute to adaptive immunity following vaccination. Here we characterised phenotypic variation of bNK cells within afferent lymph and lymph node (LN) tissues and between CD2+ and CD2- bNK subsets, and report phenotypic changes induced by BCG vaccination. CD2- bNK cells, which dominate in the afferent lymph and LN, displayed lower expression of the activation marker CD25 within the LN, with CD25+ cells being less than half as frequent as in afferent lymph. Furthermore, we found bNK cells had a lower expression of CD45RB, associated in cattle with naïve cell status, within LN compared to afferent lymph. Following BCG vaccination, bNK cells in afferent lymph draining the vaccination site showed increased CD2-CD25+ frequencies and increased expression of CD25 on CD2+ bNK cells, although the frequency of these cells remained unchanged. In summary, we provide an overview of the phenotype of bNK cells within bovine lymphatic tissues, and provide an indication of how subsets may diverge following BCG vaccination.

Regulatory T cells inhibit autoantigen-specific CD4+ T cell responses in lupus-prone NZB/W F1 mice.

Progressive loss of regulatory T cell (Treg)-mediated control over autoreactive effector T cells contributes to the development of systemic lupus erythematosus (SLE). Accordingly, we hypothesized that Treg may also have the capacity to suppress the activation of autoreactive CD4+ T cells that are considered to drive autoimmunity. To investigate whether Treg are involved in the control of autoreactive CD4+ T cells, we depleted CD25+ Treg cells either in vivo or in vitro, or combined both approaches before antigen-specific stimulation with the SLE-associated autoantigen SmD1(83-119) in the NZB/W F1 mouse model either after immunization against SmD1(83-119) or during spontaneous disease development. Frequencies of autoantigen-specific CD4+ T cells were determined by flow cytometry using the activation marker CD154. Both in vitro and in vivo depletion of CD25+ Treg, respectively, increased the frequencies of detectable autoantigen-specific CD4+ T cells by approximately 50%. Notably, the combined in vivo and in vitro depletion of CD25+ Treg led almost to a doubling in their frequencies. Frequencies of autoantigen-specific CD4+ T cells were found to be lower in immunized haploidentical non-autoimmune strains and increased frequencies were detectable in unmanipulated NZB/W F1 mice with active disease. In vitro re-addition of CD25+ Treg after Treg depletion restored suppression of autoantigen-specific CD4+ T cell activation. These results suggest that the activation and expansion of autoantigen-specific CD4+ T cells are partly controlled by Treg in murine lupus. Depletion of Treg therefore can be a useful approach to increase the detectability of autoantigen-specific CD4+ T cells allowing their detailed characterization including lineage determination and epitope mapping and their sufficient ex vivo isolation for cell culture.

IMMU-14. SINGLE CELL RNASEQ OF PBMCS FROM NEWLY-DIAGNOSED GLIOBLASTOMA PATIENTS

Abstract INTRODUCTION A multitude of recently published papers have found immunosuppression caused by glioblastoma (GBM) is not limited to the tumor microenvironment and impacts the peripheral immune system. Our group has recently reported an expansion of granulocyte macrophage precursors, a subset of hematopoietic stem and progenitor populations (HSPCs), in glioma-bearing mice relative to healthy, age-matched controls. Thus we sought to determine if GBM causes cell fate dysfunction in CD34+ HSPCs in patients. METHODS CD34+ cells were magnetically isolated from peripheral blood of newly-diagnosed GBM patients and healthy age-matched donors and processed for single-cell RNA sequencing. CD34+ cells were magnetically isolated from peripheral blood of newly-diagnosed GBM patients and healthy age-matched donors and processed for single-cell RNA sequencing using the 10X Genomics platform. CellRanger was used for primary analysis with default parameters. After quality control removal of doublets, dead, and low quality cells automated cell type annotation was performed using the Azimuth Hubmap Consortium human bone marrow reference. The annotated single cell object was then read into R and Seurat was used for differential expression analysis and visualization. RESULTS AND CONCLUSIONS There we no significant expansions of stem and progenitor populations. GBM patient CD34+ samples had upregulation of VWF, a marker of myeloid-bias cell subset. Within the mature immune cell compartments, we observed a significant expansion of NK cells in GBM patients including 1,997 genes differentially expressed in the NK cell cluster between cohorts. This includes downregulation of SLAMF6, which triggers cytolytic activity in NK cells, in GBM patients. Upregulation of early NK cell activation markers CD69 and JUN were seen in GBM patient samples. These results strongly suggest NK are likely activated in the context of GBM, but may not be functional.

TMIC-69. TARGETING TUMOR-ASSOCIATED MACROPHAGES IN THE IMMUNE-MICROENVIRONMENT OF MENINGIOMAS

Abstract Meningiomas represent the most common primary brain malignancies in adults with a subset of tumors exhibiting aggressive clinical behavior. Immunotherapy might present a new treatment strategy but is highly dependent on the immunological composition of the tumor microenvironment. Our previous data have shown that tumor-associated macrophages (TAMs) make up the main immune cell population in meningiomas with a significant negative impact on patient outcome. In this study, we investigated whether TAMs from meningioma tissue could be reprogrammed to an immunologically active and tumoricidal phenotype. For this purpose, CD11b+ sorted macrophages derived from > 40 patients were treated with small molecule inhibitors targeting the colony-stimulating factor-1 receptor (CSF-1R). In a first analysis, the direct treatment response of CD11b+ patient-derived macrophages was investigated by various techniques including flow cytometry and bulk RNA-sequencing of treated TAMs and further analysis of the macrophage-conditioned media after treatment. In addition, we studied the influence of CSF-1R-targeted macrophage treatment on the phenotype and functional activity of T cells to assess a potential indirect treatment response in the tumor microenvironment. Our data revealed that CSF-1R-targeted treatment of CD11b+ TAMs induced significant changes in the protein and gene expression of macrophage polarization markers towards a more immunologically active state and a significantly higher metabolic nitric oxide production as another sign of immunological activation. Subsequent analysis of indirect effects on T cells showed not only a significantly increased expression of the T cell activation marker CD69+, but also a significantly increased tumor cell killing by autologous T cells after macrophage-targeted treatment. Together these data suggest both a direct and indirect CSF-1R-targeted macrophage treatment response in the local tumor microenvironment and give first promising results on the efficacy of macrophage-targeted immunotherapy in human meningiomas.

Lipid Handling Induces Ferroptosis and Conveys Sensitivity to Anti-BCMA Immunotherapy in Multiple Myeloma

Lipid metabolic changes in cancer are attracting increased attention. BAFF/APRIL axis is a multifaceted immune regulator of adipose tissue function and an emerging target for anti-BCMA therapy in multiple myeloma (MM). However, lipid analyses are scarce in MM onset and response to treatment. First, we measured metabolites and proteins in sera obtained from matched bone marrow and peripheral blood of 17 newly diagnosed and 9 relapsed/refractory MM patients, by LC-MS/MS. String analysis identified crucial pathways including fibrinogen regulation, inflammatory response, structural integrity of lipoproteins and lipid transport, with a significant increase of apoliproteins APO-A1 (key component of HDL particle and cofactor for lecithin-cholesterol acyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters), A2,C2 (which hydrolyzes triglycerides to provide free fatty acids for cells), C3, components of HDL and LDL, confirming a deranged lipid handling in RR-MM patients. Second, we studied the lipid metabolic changes upon anti-BCMA exposure with Elranatamab (EL), an investigational, off-the-shelf, humanized BCMA-CD3-directed bispecific antibody engineered to elicit potent T-cell-mediated anti-MM activity. Combining in vitro assays, FACS, qRT-PCR, immunofluorescence, Western Blot and RNA-seq we evaluated the response to EL treatment in 3 human myeloma cell lines U266, NCI-H929 and OPM2 (3 cell lines chosen for their different basal bioenergetics profile). MM cells were co-incubated with various concentrations of EL in presence of CD3+T cells isolated from healthy donors in 96- well U-bottom plates for 4 h and 24 h at an E:T ratio of 1:5 and 1:10, then stained with anti-CD138-PE antibody . CD138 + cells were detected by flow cytometry and counted as surviving MM cells. BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) and BODIPY 493/503 for Neutral Lipid Droplets Staining for FACS and immunofluorescence were used to quantify lipid peroxidation and cytoplasmatic LDs on surviving CD138 + cells. Despite the BCMA target was equally expressed on U266, NCI-H929 and OPM2, the sensitivity to EL was different, inducing cell cycle arrest in the G2/M phase in U266 and NCI-H929 cell lines but not in OPM2, in E:T ratio independent-manner. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. Oil-Red-O staining revealed an increased lipid content after EL exposure in all tested MM cell lines, with OPM2 the richest in basal lipids content. To dissect the contribution of lipophagy versus lipolysis we performed an autophagy flux analysis by western blot and RNA-seq essay, showing that lipolysis was the emerging response to EL treatment. In EL-sensitive U266 we found an increase of lipid peroxides, hallmark of ferroptosis, a form of cell death due to missed control of membrane lipid peroxidation, a phenomenon negligible in EL-resistant OPM2. Lipidomics demonstrated higher levels of monounsaturated fatty acids (MUFA) than polyunsaturated fatty acids (PUFA) in OPM2, associated to an increased expression of Stearoyl CoA desaturase 1 (SCD1), the enzyme that catalyzes the rate-limiting step in the production of MUFA. While the PUFA oxidation is pro-ferroptotic, MUFA activation promotes a ferroptosis-resistant cell state. Indeed, a ferroptosis-primed resistant phenotype in EL-sensitive U266 was associated to downregulation of lipid peroxides scavengers GPX4 and SLC7A11 and up-regulation of pro-ferroptotic acyl-CoA synthetase long-chain family member 4 (ACSL4). Exogenous MUFA (via supplementation with palmitic acid 125 µM conjugated to BSA 1:6) alleviated ferroptosis induction in U266 making them resistant to EL, a phenotype associated to overexpression of CD36, the main free fatty acids (FFA) transporter. Conversely, in OPM2, inducing iron-dependent lipid peroxidation through RSL3 (3 µM) treatment rescued EL resistance, resulting in a time-dependent increase in cytosolic lipid peroxidation beginning at 6 hours, and overt cell death via ferroptosis at 18 hours. Taken together, our findings suggest that MUFA/PUFA ratio drives ferroptosis and mediates EL sensitivity, addressing lipid handling as a novel means to tailor immunotherapy in RRMM.

Development of Chimeric Antigen Receptor (CAR) T Cells Targeting MET in Lymphomas and Solid Tumors

Currently, multiple versions of CAR T cells targeting the cell surface protein CD19 are approved by the FDA to treat refractory B-cell malignancies. However, durable remissions range from 60% in B-cell leukemias to 40% in B-cell lymphomas. Major reasons for resistance to CD19-CAR treatment relate to heterogeneous or loss of CD19 expression. In addition, CD19-CAR leads to B-cell depletion which increases the risk for infection. These limiting factors necessitate research into alternative targets for CAR therapy against B-cell lymphomas. While HGF/MET signaling has been extensively studied in solid tumors, studies have also shown important roles of HGF/MET in hematologic malignancies. Approximately 30-70% of diffuse large B-cell lymphomas (DLBCL) express MET with moderate to strong intensity; as do other lymphomas such as classic Hodgkin lymphoma, Burkitt lymphoma, primary effusion lymphoma, and multiple myeloma. We hypothesize that these MET-positive B-cell lymphomas can potentially be targeted with chimeric antigen receptor (CAR) T-cells against MET receptor. We first looked at the DepMap database and found that a significant subset of aggressive and large B-cell lymphoma cell lines express MET such as Raji, Karpas-422, OCI-LY3, and OCI-LY132. Of note, some of them, like OCI-LY3 and OCI-LY132, lack detectable CD19. Flow cytometry using anti-MET antibody confirmed MET expression in these cell lines. Further analysis of RNAseq data from 500 CD19-CAR T-naïve primary patient samples (GSE125966) revealed MET expression in 62% (294/474) of CD19-positive and 61% (15/26) of CD19-low to negative DLBCL . We then constructed a second-generation anti-MET-CAR by fusing the scFv fragment of an anti-MET monoclonal antibody with the human CD28 hinge/transmembrane/cytoplasmic domain and CD3z cytoplasmic domain. Stable MET-CAR Jurkat T cells were incubated with various target lymphoma cells for 16-24 hours, and the percentage of Jurkat T cells expressing the activation marker CD69 was determined by flow cytometry. MET-CAR Jurkat showed increased CD69 expression in MET-positive lymphoma cell lines such as Karpas-422 and OCI-LY3. Karpas-422 demonstrated dose dependent activation of MET-CAR Jurkat cells, with higher CD69 expression in response to increasing amounts of MET antigen. OCI-LY3, a lymphoma cell line negative for CD19 but positive for MET, led to only basal level of CD69 expression in CD19-CAR Jurkat but elevated CD69 level in MET-CAR Jurkat. After showing MET-CAR Jurkat T cell activation by lymphoma cell lines, we tested cytotoxicity using primary human CD3+ T-cells against A549, a lung cancer cell line with known high level of MET expression, as a positive control for MET-CAR mediated cytotoxicity. Activated primary human T cells were electroporated with MET-CAR mRNA. Twelve hours post electroporation, the reprogrammed T cells were mixed with A549 cells at a 5:1 effector: target ratio (E:T). CellCyte proliferation analysis showed reduced A549 cell proliferation in the presence of MET-CAR T cells, in comparison to mock electroporated CD3+ T cells. We further demonstrated that the same mRNA MET-CAR T-cell was able to mediate substantial cytotoxicity against DLBCL cell lines such as Karpas-422 and OCI-LY3 ( Figure 1). At varying E:T ratio, MET-CAR T cell exhibited significant cytotoxicity against Karpas-422. Also, consistent with the CD69 expression data, while CD19-CAR T cell showed only low level of cytotoxicity against the CD19-/MET+ OCI-LY3 cell line, MET-CAR T cell was able to mediate substantial tumor cell lysis. In vivo testing against a xenograft model is currently underway. In summary, the preliminary data demonstrate that MET-CAR is capable of mediating cytotoxicity against MET-positive DLBCL. Some cell lines such as OCI-LY3 are negative for CD19 but positive for MET expression, and MET-CAR is capable of mediating cell lysis while CD19-CAR is ineffective. These results suggest that some large B-cell lymphomas negative for CD19, either intrinsic or secondary to loss of CD19 as a result of resistance mechanism, may show MET expression and will likely benefit from MET-targeted immunotherapy. Figure caption: Figure 1. Cytotoxicity of MET-CAR T cell against DLBCL cell lines. (A) MET-CAR against Karpas-422 at various E:T ratio. (B) Cytotoxicity of MET-CAR against OCI-LY3 which is positive for MET but negative for CD19.E:T ratio is 10:1.

The BCMA-Targeted Fourth-Generation CAR-T Cells Secreting CST6 Against Multiple Myeloma and Suppresses Osteolytic Lesions

651 Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction: Multiple myeloma (MM) is characterized by osteolytic lesions, and our previous research has shown that cystatin E/M (CST6) effectively mitigates MM bone disease by inhibiting osteoclast differentiation. However, the therapeutic application of recombinant CST6 protein faces challenges due to its short serum half-life in mouse models, necessitating maintenance of constantly elevated CST6 concentrations for optimal efficacy. In this study, we explore the potential of utilizing fourth-generation B cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR)-T cell therapies to address this limitation. By leveraging the secretion capabilities of CST6, we engineered BCMA-CST6-CAR-T cells to precisely target nascent focal lesions. Our results demonstrate that these engineered CAR-T cells effectively target MM cells and concurrently suppress osteolytic lesions, presenting a promising approach for combating MM and its associated bone pathology. Methods: The BCMA-CST6-CAR vector comprises BCMA-scFv, a 4-1BB co-activation domain, and CD3ζ. To facilitate separate expression, a P2A self-cleaving peptide was introduced between CAR and CST6. CAR-T cells were co-cultured with MM1.S cells at a 5:1 ratio for 24 hours. To assess in vivo anti-tumor activity and anti-bone resorption of BCMA-CST6-CAR-T cells, we engrafted luciferase-expressing MM1.S cells into NSG mice. On day 7, mice with established MM received CAR-T cell infusions. At day 35, the mice were euthanized, and we analyzed serum CST6 concentrations using ELISA. Additionally, we collected mouse tibiae to perform μCT scans and TRAP staining to evaluate bone resorption. Results: The co-culturing of BCMA-CST6-CAR-T cells with MM1.S cells resulted in a remarkable 77.2% lysis of MM1.S cells after 24 hours, with no significant difference in killing observed between the BCMA-CAR-T and BCMA-CST6-CAR-T groups. However, T cell activation marker CD69 exhibited a significant increase in the BCMA-CST6-CAR-T group (70.9%) compared to the MOCK-CAR-T group (9.2%). In the MM-bearing mouse model, bioluminescence imaging revealed that BCMA-CST6-CAR-T cells exhibited excellent antitumor activity, leading to near-complete tumor clearance by day 21. Notably, both BCMA-CST6-CAR-T and BCMA-CAR-T cells showed similar effects in inhibiting tumor growth. The in vivo serum concentration of CST6 was significantly higher in the BCMA-CST6-CAR-T group (938.4 ng/ml) compared to the MOCK group (60.9 ng/ml) and BCMA-CAR-T group (64.2 ng/ml) (P < 0.001), highlighting the robust secretion of CST6 protein by BCMA-CST6-CAR-T cells. Moreover, μCT images of mouse tibiae demonstrated that BCMA-CST6-CAR-T cells effectively suppressed osteolytic lesions in MM-bearing mice. This finding was further corroborated by TRAP staining of mouse tibia sections, which showed a significant reduction in osteoclast numbers and the proportion of bone surface occupied by osteoclasts in the presence of BCMA-CST6-CAR-T cells. Conclusion: These results underscore the potent antitumor and anti-bone resorption effects of BCMA-CST6-CAR-T cells and their potential as a promising therapeutic approach for multiple myeloma.

The Expression of Activation Markers CD25 and CD69 Increases during Biologic Treatment of Psoriasis.

CD (cluster of differentiation) 69 and CD25 are considered early and late markers of the activation of lymphocytes, respectively. CD25 is a part of the IL-2 receptor and is present on the surface of immune and non-immune cells, with high amounts on activated lymphocytes and regulatory T cells. CD69 is expressed on various types of white blood cells, including newly activated lymphocytes, lymphocytes infiltrating tissues isolated from subjects with chronic auto-inflammatory diseases, several subtypes of memory T cells and regulatory T cells. Primarily, CD69 was considered to be an early marker of the activation of lymphocytes, but, right now, data derived from in vitro and in vivo studies have revealed the immunomodulatory role of this surface antigen. In 84 patients with psoriasis, of whom 28 were treated with different biologic drugs, as well as in 29 healthy control subjects, the expression of CD25 and CD69 on different subtypes of peripheral blood mononuclear cells (PBMCs) was studied with the use of flow cytometry. Significantly higher levels of CD3/CD69-, CD8/CD69- and CD19/CD69-positive PBMCs as well as within CD3+ cells were present in subjects suffering from psoriasis when compared to healthy controls. In patients with psoriasis who were treated with biologic drugs, the levels of CD3/CD69-, CD4/CD69- and CD19/CD69-positive PBMCs, and CD3/CD69 within CD3+ cells, CD4/CD69 within CD4+ cells, CD4/CD25 within CD4+ cells and CD19/CD69 within CD19+ cells were significantly higher than before therapy. Our results support a role for activation markers, especially CD69, in psoriasis. Further research is warranted to fully clarify their significance in this common dermatosis, especially during biologic treatment.

Differential Immune-Modulating Activities of Cell Walls and Secreted Metabolites from Probiotic Bacillus coagulans JBI-YZ6.3 under Normal versus Inflamed Culture Conditions.

Spore-forming probiotic bacteria, including Bacillus coagulans, are resilient and produce a variety of beneficial metabolites. We evaluated the immune-modulating effects of the novel probiotic strain Bacillus coagulans JBI-YZ6.3, where the germinated spores, metabolite fraction, and cell wall fraction were tested in parallel using human peripheral blood mononuclear cell cultures under both normal and lipopolysaccharide-induced inflamed culture conditions. The expression of CD25 and CD69 activation markers was evaluated via flow cytometry. Supernatants were tested for cytokines, interferons, chemokines, and growth factors using Luminex arrays. The germinated spores were highly immunogenic; both the cell wall and metabolite fractions contributed significantly. Under normal culture conditions, increased levels of immune activation were observed as increased expressions of CD25 and CD69 relative to natural killer cells, suggesting an increased ability to attack virus-infected target cells. On monocytes, a complex effect was observed, where the expression of CD25 increased under normal conditions but decreased under inflamed conditions. This, in combination with increased interleukin-10 (IL-10) and decreased monocyte chemoattractant protein-1 (MCP-1) production under inflamed conditions, points to anti-inflammatory effects. The production of the stem cell-related growth factor granulocyte colony-stimulating Factor (G-CSF) was enhanced. Further research is warranted to characterize the composition of the postbiotic metabolite fraction and document the characteristics of immunomodulating agents secreted by this probiotic strain.

The involvement of circulating CD69+ CD56bright natural killer cells in weight loss before bariatric surgery: A retrospective cohort study.

As the impact of the immune system on weight loss prior to bariatric surgery has never been proven, we elucidated the clinical utility of the immune system as an indicator of preoperative weight loss before bariatric surgery. We examined the relationships between preoperative weight loss and biochemical and clinical data at the initial visit in 34 obese patients. Patients were divided according to preoperative weight loss, and peripheral blood mononuclear cells were compared using flowcytometry. The Δpreoperative excess weight loss [Δpre-EWL: pre-EWL (%)/period of preoperative weight loss (days)] showed negative correlations with total and subcutaneous fat area (P = .02, r = -0.41, P = .02, r = -0.42 respectively). The Δpre-EWL and Δpreoperative total weight loss (Δpre-TWL) were negatively correlated with white blood cell count, lymphocyte count, and C-reactive protein (CRP) levels at the initial visit (Δpre-EWL; P = .02, r = -0.37, P = .01, r = -0.41, P = .008, r = -0.45, Δpre-TWL; P = .01, r = -0.40, P = .01, r = -0.42, P = .01, r = -0.42, respectively). Multivariate regression modeling showed that both Δpre-EWL and Δpre-TWL were significantly associated with lymphocyte count (Δpre-EWL; P = .01, Δpre-TWL; P = .01). A comparison between the high (Δ pre-EWL > 0.098) and low weight loss group (Δ pre-EWL < 0.098) demonstrated a significant difference in the expression of the activation marker CD69 on CD56bright Natural killer (NK) cells (P = .01), whereas there was no difference in the frequency of T cells, Natural killer T cells, or NK cells. Additionally, high CRP levels were associated with CD69 expression in CD56bright NK cells (P = .01, R = 0.57). Peripheral lymphocytes, especially CD69-positive CD56bright NK cells, are involved in preoperative weight loss after bariatric surgery, and systemic inflammation may inhibit weight loss before surgery.

Exploring TCR-like CAR-Engineered Lymphocyte Cytotoxicity against MAGE-A4.

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.

SAT411 The Influence Of Gender Affirming Hormone Therapy On Markers Of Platelet Activation In Transgender Women

Abstract Disclosure: L.M. van Zijverden: None. M.H. Schutte: None. M.C. Madsen: None. J.J. van Diemen: None. C.M. Wiepjes: None. A. Thijs: None. M. den Heijer: None. Background: Transgender women often use hormone therapy to obtain body characteristics that comply with their experienced gender. This is important to improve quality of life and mental wellbeing. However, results from several studies show that trans women who use gender affirming hormones have an increased risk of cardiovascular disease (CVD) compared to cisgender women and men. The exact mechanisms that could explain this risk are yet undetermined. In general, platelet activation plays an important role in the development of CVD. This process might be influenced by feminizing hormone therapy, since platelets express estrogen receptors on their cell surface. The aim of our research was to gain insight in the role of platelet activation in CVD development in trans women. We therefore investigated the effect of estradiol on two markers of platelet activation: CD62p and CD63. Methods: In this prospective cohort study 17 trans women between 19 and 48 years old were included. They had no history of prior hormone therapy or known platelet disorders, and did not use antiplatelet medication. Treatment consisted of estradiol and anti-androgens. Blood was drawn at baseline and at 2, 12 and 52 weeks after the start of estradiol. We measured the percentage of platelets that were positive for activation markers CD62p and CD63. We also measured the fluorescence intensity of these markers. This is a way to measure the relative number of markers per platelet. These two measurements were then combined into a new outcome measure, called binding index (BI). Geometric means of the binding indices at each time point were calculated. We also calculated the percentage changes in binding index over time. Results: For CD62p, BI increased from 2.4 (95%CI 1.5, 3.7) at baseline to 4.6 (95%CI 2.0, 10.5) at week 2, reflecting a difference of 96% (95%CI -9, 318). After week 2, BI decreased to 2.6 (95%CI 1.3, 5.3) at week 12 and 1.4 (95%CI 1.0, 1.9) at week 52. The difference between week 52 compared to baseline was -40% (95%CI -69, 14). CD63 BI increased from 10.7 (95%CI 7.1, 16.1) at baseline with 32% (95%CI -32, 158) to 14.1 (95%CI 8.8, 22.8) at week 2. At week 12 and 52, BI was 13.1 (95%CI 7.4, 23.2) and 9.3 (95%CI 5.9, 14.6), respectively. The difference between week 52 compared to week 0 was -13% (95%CI -51, 53). Conclusions: The observed trends show an increase in binding index of both platelet activation markers directly after hormone therapy initiation. This could partially explain the increased rates of cardiovascular disease in transgender women in the early stages of their hormone therapy. However, we observe a decrease of CD62p and CD63 towards baseline values at twelve weeks of hormone therapy. This decrease continues until week 52. Hence, our results do not provide an explanation for an increased rate of CVD in trans women on the longer term. Presentation: Saturday, June 17, 2023

Flow Cytometric Analysis of Lymphocyte Subsets of Covid-19 Patients from A Single Centre in Turkey

Background:&#x0D; Lymphocyte counts have been shown to negatively correlate with the severity and different outcomes in Covid-19. The aim of this single centre study is to analyse the distribution of lymphocyte subsets in response to SARS-CoV-2 infection and its relation to the severity, course and prognosis of the disease.&#x0D; Methods:&#x0D; Blood samples were obtained from 67 consecutive patients between April 2020 and July 2020. Data on other laboratory parameters, and clinical course were collected retrospectively from patient files and patints were defined to have as severe or non-severe (mild/moderate) disease. Leukocyte subsets to be studied were identified by using flow cytometric analysis. Patients were allocated into 3 groups based on the day of blood sample collection for the flow cytometric analysis: Days 0-7, Days 8-14 and Days &gt;14 as Group I, Group II and Group III, respectively. In 10 available of 67 patients an additional flow cytometric analysis was done 7-10 days after the initial sampling.&#x0D; Results:&#x0D; Lower total lymphocyte, CD3 positive, CD4 positive and B-cell counts were identified in severe infection compared to non-severe infection group which were also correlated with high serum CRP, D-dimer and ferritin levels. NK and monocyte counts were not different between the two groups. Activation markers CD38 and HLA-DR on CD4 and CD8 positive lymphocytes also were not different in either group. &#x0D; Conclusion:&#x0D; CD3 and CD4 lymphopenia were lower in accordance with previous studies and were associated with severe disease. The expectancy of high activation markers was not met. Future studies with detailed subgroup analyses at different time-points, and immune profiling after vaccination as well as during new infection in vaccinated patients will shed more light on our general knowledge of the immune response to COVID-19 and viruses in general.

Comparison of the effect of autoclaved and non-autoclaved live soil exposure on the mouse immune system

Background. Lack of exposure to the natural microbial diversity of the environment has been linked to dysregulation of the immune system and numerous noncommunicable diseases, such as allergies and autoimmune disorders. Our previous studies suggest that contact with soil material, rich in naturally occurring microbes, could have a beneficial immunoregulatory impact on the immune system in mice and humans. However, differences in the immunomodulatory properties of autoclaved, sterile soil material and non-autoclaved, live soil material have not been compared earlier.Results. In this study, we exposed C57BL/6 mice to autoclaved and live soil powders that had the same rich microbiota before autoclaving. We studied the effect of the soil powders on the mouse immune system by analyzing different immune cell populations, gene expression in the gut, mesenteric lymph nodes and lung, and serum cytokines. Both autoclaved and live soil exposure were associated with changes in the immune system. The exposure to autoclaved soil resulted in higher levels of Rorγt, Inos and Foxp3 expression in the colon. The exposure to live soil was associated with elevated IFN-γ concentration in the serum. In the mesenteric lymph node, exposure to live soil reduced Gata3 and Foxp3 expression, increased the percentage of CD8 + T cells and the expression of activation marker CD80 in XCR1+SIRPα− migratory conventional dendritic cell 1 subset.Conclusions. Our results indicate that exposure to the live and autoclaved soil powders is not toxic for mice. Exposure to live soil powder slightly skews the immune system towards type 1 direction which might be beneficial for inhibiting type 2-related inflammation. Further studies are warranted to quantify the impact of this exposure in experimental type 2 inflammation.

Abstract 003: Aberrant Host Immune Responses Specific To Microbial-derived Ligands Are Linked To Hypertension In BPH/2J Mice

It has been established that derangements in host-microbiota homeostasis leads to hypertension. However, the precise mechanisms remain largely unknown. Microbial-derived ligands released into the host circulation are sensed and eliminated by various host innate and adaptive immune mechanisms. Herein, we focused on such microbial-derived ligands and hypothesized that defect in host innate and adaptive immune response to counter microbial-derived ligands is linked to hypertension. To test this hypothesis, both in vitro and in vivo studies were conducted. First, bone marrow-derived macrophages from Blood Pressure Normal (BPN/3J, systolic blood pressure: 113.2±2.20 mm Hg) and Blood Pressure Hypertensive (BPH/2J, systolic blood pressure: 143.9±4.10 mm Hg) mice (male, 8 weeks) were challenged with various Toll-like receptor (TLR) ligands of microbial origin. Unstimulated macrophages from BPH mice displayed reduced levels of the co-stimulatory activation markers CD40, CD80 and CD86 (~2 fold) compared to macrophages from BPN/3J mice. Such differences, however, was normalized upon stimulation with ligands for TLR3 (poly-I:C), TLR4 (LPS), TLR5 (flagellin), TLR7/8 (R848) and TLR9 (CpG-DNA). Despite so, BPH-macrophages displayed blunted IL-6 and lipocalin-2 (~2 fold) levels in response to all the aforementioned TLR ligands when compared to macrophages from BPN mice. To confirm these findings in vivo , we challenged BPN and BPH mice with the microbial-derived ligand, flagellin. Interestingly, even though BPH mice had reduced cytokine responses to flagellin as monitored by levels of Lcn2 (BPN 320.9 ± 22.20 vs BPH 230.8 ± 12.40, p&lt;0.05) and CXCL1 (BPN 40.9 ± 4.6 vs BPH 23.8 ± 2.4, p&lt;0.05), they displayed higher levels of flagellin-specific IgG (BPN vs BPH O.D 450 , 0.51 ± 0.10 vs 0.93 ± 0.07, p&lt;0.05) and substantially lower levels of flagellin-specific IgA (BPN vs BPH O.D 450 , 0.44 ± 0.13 v s 0.13 ± 0.03, p&lt;0.05). Collectively, both in vitro and in vivo studies demonstrate that both innate and adaptive immune responses specifically against microbial ligands is substantially deranged in the hypertensive state.

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor.

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.