Peripheral Blood Mononuclear Cell Subsets Research Articles

Increased CRHR1 expression on monocytes from patients with AA enables a pro-inflammatory response to corticotrophin-releasing hormone.

Stress may play a key role in alopecia areata (AA), though the exact interactions of stress with AA remain undefined. Corticotropin-releasing hormone (CRH), the proximal regulator of the stress axis, has been recognized as an immunomodulatory factor in tissues and peripheral blood mononuclear cells (PBMCs). We used multicolour flow cytometry to identify receptor CRHR1 expression on PBMC subsets in AA patients (n = 54) and controls (n = 66). We found that CRHR1 was primarily expressed by circulating monocytes. CRHR1 expression on monocytes was enhanced in AA compared with controls (3.17% vs. 1.44%, p = 0.002, chi-squared test). AA incidence was correlated to elevated CD14+ monocyte numbers (R = 0.092, p = 0.036) and markedly independently correlated with increased CRHR1 expression (R = 0.215, p = 0.027). High CRHR1 expression was significantly related to chronic AA (disease duration >1 year; p = 0.003, chi-squared test), and large lesion area (AA area >25%; p = 0.049, chi-squared test). We also observed enhanced percentages of active monocytes and reduced CD16+ CD3- NK cell numbers in AA patients' PBMCs (p = 0.010; 0.025, respectively). Invitro CRH treatment of PBMCs and human monocyte cell line THP-1 promoted CD86 upregulation. The findings imply that stress-related factors CRH and CRHR1 contribute to AA development and progression where higher CRHR1 expression is associated with chronic AA and larger lesions.

Human hypofunctional NCF1 variants promote pulmonary fibrosis in the bleomycin-induced mouse model and patients with systemic sclerosis via expansion of SPP1+ monocytes-derived macrophages

ObjectiveWe assessed the role of a systemic lupus erythematosus causal hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in systemic sclerosis (SSc).MethodsAssociation of NCF1-H90 with SSc was performed in...

CD4+ Effective Memory T Cell Markers GBP2 and LAG3 Are Risk Factors for PTB and COVID-19 Infection: A Study Integrating Single-Cell Expression Quantitative Trait Locus and Mendelian Randomization Analyses.

Observational studies indicate that variations in peripheral blood mononuclear cell (PBMC) subsets are associated with an increased risk of pulmonary tuberculosis (PTB) and coronavirus disease 2019 (COVID-19), but causal validation is lacking. Here, we combined single-cell expression quantitative trait locus (sc-eQTL) and two-sample mendelian randomization (MR) analyses to elucidate the causal relationship between PBMC subsets and the occurrence of PTB and COVID-19 and verified by RT-qPCR. We observed an increase in the CD4+ Effective Memory T Cell (CD4+ TEM) cluster in both PTB and COVID-19 patients according to the single-cell transcriptional landscape of PBMC. Through MR analysis using an inverse variance weighted (IVW) method, we found strong evidence of positive correlations between CD4+ TEM cell markers (GBP2, TRAV1-2, and ODF2L) and PTB, and between markers (LAG3 and SLFN5) and COVID-19, especially highlighted by lead eQTL-SNPs of GBP2 (rs2256752, p = 4.76321 × 10-15) and LAG3 (rs67706382, p = 6.16× 10-16). Similar results were observed in validation sets, and no pleiotropy was detected in sensitivity analyses including weighted median (WM), MR-Egger, MR-pleiotropy residual sum and outlier, and leave-one-out analyses (all p > 0.05). We visualized the colocalization of marker-eQTLs and markers of PTB and COVID-19 genome-wide association study (GWAS) associations. Based on CellChat analyses, monocytes communicated predominantly with CD4+ TEM cells positively expressing PTB markers (GBP2, TRAV1-2, and ODF2L) and COVID-19 markers (LAG3 and SLFN5) in both PTB and COVID-19. Our data suggest a causal effect between two key CD4+ TEM cell markers (GBP2 and LAG3) and the risk for PTB and COVID-19 infection. Our findings provide novel insights into the biological mechanism for PTB and COVID-19 infection, but future single-cell studies are necessary to further enhance understanding of this find.

Longitudinal analysis of peripheral immune cells in patients with multiple sclerosis treated with anti-CD20 therapy.

Anti-CD20 therapy is a highly effective treatment for multiple sclerosis (MS). In this study, we investigated MS-related changes in peripheral blood mononuclear cell (PBMC) subsets compared to healthy controls and longitudinal changes related to the treatment. Multicolor spectral flow cytometry analysis was performed on 78 samples to characterize disease- and treatment-related PBMC clusters. Blood samples from MS patients were collected at baseline and up to 8 months post-treatment, with three collection points after treatment initiation. Unsupervised clustering tools and manual gating were applied to identify subclusters of interest and quantify changes. B cells were depleted from the periphery after anti-CD20 treatment as expected, and we observed an isolated acute, transitory drop in the proportion of natural killer (NK) and NKTcells among the main populations of PBMC (P = 0.03, P = 0.004). Major affected PBMC subpopulations were cytotoxic immune cells (NK, NKT, and CD8+ Tcells), and we observed a higher proportion of cytotoxic cells with reduced brain-homing ability and a higher regulatory function as a long-term anti-CD20-related effect. Additionally, anti-CD20 therapy altered distributions of memory CD8+ Tcells and reduced exhaustion markers in both CD4+ and CD8+ Tcells. The findings of this study elucidate phenotypic clusters of NK and CD8+ Tcells, which have previously been underexplored in the context of anti-CD20 therapy. Phenotypic modifications towards a more regulatory and controlled phenotype suggest that these subpopulations may play a critical and previously unrecognized role in mediating the therapeutic efficacy of anti-CD20 treatments.

Immune cell profiling of the ICAM1 p.K56M heart failure with preserved ejection fraction risk variant.

Intercellular adhesion molecule-1 (ICAM-1) facilitates inflammation via leucocyte recruitment and has been implicated in heart failure with preserved ejection fraction (HFpEF). Approximately 35% of African American individuals carry a copy of an ICAM1 missense variant (rs5491; p.K56M), which is associated with an increased risk of HFpEF. The pathways by which rs5491 increases HFpEF risk are not well defined. We evaluated the circulating immune cell profile of rs5491. Among African American individuals in the Multi-Ethnic Study of Atherosclerosis, we evaluated the associations of rs5491 with 29 circulating peripheral blood mononuclear cell subsets. The top immune cells were then related to echocardiographic measures of structure and function. Among 502 individuals with immune cell profiling (mean age 63years, 51% female), 191 individuals (38%) had at least one copy of rs5491. Each additional rs5491 allele was significantly associated with higher proportions of Tc17 CD8+ cytotoxic T cells (β=1.34, SE=0.45, P=9.5×10-5) and Tc2 CD8+ cytotoxic T cells (β=1.19, SE=0.44, P=0.00012). There were no other associations noted between rs5491 and the remaining immune cells. A higher proportion of Tc17 cells was significantly associated with a higher left ventricular ejection fraction, E/e' average and right ventricular systolic pressure (RVSP), while a higher proportion of Tc2 cells was significantly associated with a higher RVSP. The ICAM1 p.K56M variant (rs5491) carries a distinct and inflammatory T-cell subset profile. These cytotoxic T cells are in turn associated with alterations in cardiac function and adverse haemodynamics later in life, thus providing insight into pathways by which rs5491 may increase the risk of HFpEF.

Single-cell atlas of peripheral blood by CyTOF revealed peripheral blood immune cells metabolic alterations and neutrophil changes in intracranial aneurysm rupture.

Previous studies have found that the peripheral immune environment is closely related to the occurrence and development of intracranial aneurysms. However, it remains unclear how the metabolism of peripheral blood mononuclear cells (PBMCs) and the composition of polymorphonuclear leukocytes (PMNs) changes in the process of intracranial aneurysm rupture. This study utilized cytometry by time of flight technology to conduct single-cell profiling analysis of PBMCs and PMNs from 72 patients with IAs. By comparing the expression differences of key metabolic enzymes in PBMCs between patients with ruptured intracranial aneurysms (RIAs) and unruptured intracranial aneurysms, we found that most PBMCs subsets from RIA group showed upregulation of rate-limiting enzymes related to the glycolytic pathway. By comparing the composition of PMNs, it was found that the proinflammatory CD101+HLA DR+ subsets were increased in the RIA group, accompanied by a decrease in the anti-inflammatory polymorphonuclear myeloid-derived suppressor cells. In conclusion, this study showed the changes in the peripheral immune profile of RIAs, which is helpful for our understanding of the mechanisms underlying peripheral changes and provides a direction for future related research.

Single-cell RNA-Seq analysis reveals cell subsets and gene signatures associated with rheumatoid arthritis disease activity.

Rheumatoid arthritis (RA) management leans toward achieving remission or low disease activity. In this study, we conducted single-cell RNA sequencing (scRNA-Seq) of peripheral blood mononuclear cells (PBMCs) from 36 individuals (18 patients with RA and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFN-induced transmembrane 3-overexpressing (IFITM3-overexpressing) IFN-activated monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate-high disease activity (DAS28-CRP ≥ 3.2) and a decrease in nonclassical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of proinflammatory genes in the γδ T cell subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and nonclassical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of proinflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, and G0S2 and downregulation of genes including HLA-DQB1, HLA-DRB5, and TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity.

Abstract 5334: Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research

Abstract Background: The preclinical evaluation of novel immune therapies requires humanized immune system (HIS) mouse models. In previous studies we have demonstrated that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T and NK cells or CD34+ hematopoietic stem cells (HSC) can be used to establish a HIS model. With the development of next-generation NOG mice, lineage-specific differentiation of immune cell sub-populations of interest can be supported. Transplantation of cell line-derived (CDX) or patient-derived (PDX) tumor xenografts on HIS mice provides a full model for human tumors for the investigation of checkpoint inhibitors (CPI), as well as novel cell therapies and immune cell engagers. Methods: HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient mice. For humanization, NOG mice and next-generation NOG strains were used: NOG, NOG-EXL, hIL-2 NOG, hIL-6 NOG and FcResolv™ NOG mice were compared to each other for lineage-specific differentiation using single donors or a mixed HSC donor pool. Engraftment of immune cells was monitored by FACS analysis of blood every four weeks. In humanized NOG mice, CDX and PDX from different entities were s.c. transplanted and used to evaluate CPI. Results: Humanized hIL-2 NOG mice showed significantly decreased survival after HSC transplantation in comparison to the other mouse strains and had to be sacrificed within the first 6-8 weeks after HSC transplantation. In the other mouse strains, transplanted HSCs engrafted and differentiated mainly into B and T cells. NOG-EXL mice displayed the highest engraftment, with up to 80% of human cells in the blood, including a higher portion of myeloid cells. In humanized hIL-6 NOG mice a higher portion of monocytes could be determined. FcResolv™ NOG mice lack murine Fc gamma receptors in order to reduce confounding interactions between residual murine immune cells and antibody-based therapies, and they engraft and differentiate human HSCs similarly to the parent NOG strain. In humanized NOG mice, selected CDX and PDX tumors successfully engrafted without significant differences in tumor growth compared to non-humanized mice. CPI treatments induced tumor growth delay in selected models. Conclusions: Next-generation NOG mouse strains are characterized by a lineage-specific differentiation of immune cells depending on integrated human cytokines. Furthermore, we established a human tumor-immune-cell model for NOG mice using different entities of CDX or PDX in combination with CPI. Our human tumor-immune cell models allow preclinical translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Louise Baskin, Jens Hoffmann. Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research [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 5334.

Detection of Mycobacterium tuberculosis DNA in CD34+ peripheral blood mononuclear cells of adults with tuberculosis infection and disease

ObjectivesTo investigate whether Mycobacterium tuberculosis (Mtb) DNA is detected in peripheral blood mononuclear cells (PBMC) of subjects with tuberculosis (TB) or TB infection (TBI) living in a low-burden country. MethodsWe prospectively enrolled 57 patients with TB, 41 subjects with TBI, and 39 controls in Rome, Italy. PBMC were isolated, cluster of differentiation (CD)34+ and CD34− cells were immunomagnetic separated, DNA was extracted, and digital polymerase chain reaction for IS6110 and rpoB sequences was used to detect Mtb DNA in PBMC subsets and unfractionated PBMC. ResultsWe detected Mtb DNA at a low copy number in CD34+ cells in 4o f 30 (13%) patients with TB, 2 of 24 (8%) subjects with TBI, and 1 of 24 (4%) controls. Mtb DNA was detected in unfractionated PBMC in 3 of 51 (6%) patients with TB, 2 of 38 (5%) subjects with TBI, and 2 of 36 (6%) controls. In CD34− cells, only 1 of 31 (3%) subjects with TBI tested positive for Mtb DNA. ConclusionsMtb DNA was detected at low frequencies and levels in the PBMC of subjects with TBI and donors with TB living in a low-burden country. In particular, Mtb DNA was detected more frequently in CD34+ cells, supporting the hypothesis that these cells may represent a Mtb niche. This finding informs biological understanding of Mtb pathogenesis and may support the development of a microbial blood biomarker for Mtb infection.

Association of peripheral B cells and delirium: combined single-cell sequencing and Mendelian randomization analysis.

Delirium seriously affects the prognosis of patients and greatly reduces the ability to work and live. Peripheral inflammatory events may contribute to the development of delirium, the mechanism of which is still unclear. There is a lack of effective diagnostic and treatments for delirium in clinical practice. The study aims to investigate alterations in peripheral immune cell subsets under inflammatory stress and to explore causal associations with delirium. Single-cell transcriptional sequencing data of human peripheral blood mononuclear cells (PBMC) before and after lipopolysaccharide (LPS) intervention were processed by the Seurat package in R software. PBMC subsets and cellular markers were defined after downscaling and clustering by the Harmony algorithm to identify characteristic subsets in the context of inflammatory stress. Subsequently, a two-sample Mendelian randomization (MR) study was used to explore the causal associations of these inflammation-related PBMC subsets and their molecular phenotypes with delirium. Based on publicly available genetic data, the study incorporated 70 PBMC-associated immune traits, including 8 types of circulating immune cells, 33 B cell subsets and molecular phenotypes, 13 T cell subsets, and 16 B cell-associated cytokines. The results were also validated for robustness, heterogeneity, and horizontal pleiotropy. Under LPS-induced inflammatory stress, B cells, T cells, monocytes, and dendritic cells in human PBMC showed significant activation and quantitative changes. Of these, only lymphocyte and B cell counts were causally associated with delirium risk. This risk link is also seen in the TNF pathway. Further studies of B cells and their subsets revealed that this association may be related to unswitched memory B cells and CD27 expressed on memory B cells. Annotation of the screened SNPs revealed significant polymorphisms in CD27 and CD40 annotated by rs25680 and rs9883798, respectively. The functions of the key annotated genes may be related to the regulation of immune responses, cell differentiation, proliferation, and intercellular interactions. The present study revealed the potential possibility that B cell, memory B cell subset, and TNF-related molecules may be involved in the development of delirium due to peripheral inflammation, which can provide clues for further investigation of delirium prevention and treatment strategies.

Single-cell RNA sequencing reveals immune cell dysfunction in the peripheral blood of patients with highly aggressive gastric cancer.

Highly aggressive gastric cancer (HAGC) is a gastric cancer characterized by bone marrow metastasis and disseminated intravascular coagulation (DIC). Information about the disease is limited. Here we employed single-cell RNA sequencing to investigate peripheral blood mononuclear cells (PBMCs), aiming to unravel the immune response of patients toward HAGC. PBMCs from seven HAGC patients, six normal advanced gastric cancer (NAGC) patients, and five healthy individuals were analysed by single-cell RNA sequencing. The expression of genes of interest was validated by bulk RNA-sequencing and ELISA. We found a massive expansion of neutrophils in PBMCs of HAGC. These neutrophils are activated, but immature. Besides, mononuclear phagocytes exhibited an M2-like signature and T cells were suppressed and reduced in number. Analysis of cell-cell crosstalk revealed that several signalling pathways involved in neutrophil to T-cell suppression including APP-CD74, MIF-(CD74+CXCR2), and MIF-(CD74+CD44) pathways were increased in HAGC. NETosis-associated genes S100A8 and S100A9 as well as VEGF, PDGF, FGF, and NOTCH signalling that contribute to DIC development were upregulated in HAGC too. This study reveals significant changes in the distribution and interactions of the PBMC subsets and provides valuable insight into the immune response in patients with HAGC. S100A8 and S100A9 are highly expressed in HAGC neutrophils, suggesting their potential to be used as novel diagnostic and therapeutic targets for HAGC.

Metabolic alterations of peripheral blood immune cells and heterogeneity of neutrophil in intracranial aneurysms patients.

Intracranial aneurysms (IAs) represent a severe cerebrovascular disease that can potentially lead to subarachnoid haemorrhage. Previous studies have demonstrated the involvement of peripheral immune cells in the formation and progression of IAs. Nevertheless, the impact of metabolic alterations in peripheral immune cells and changes in neutrophil heterogeneity on the occurrence and progression of IAs remains uncertain. Single-cell Cytometry by Time-of-Flight (CyTOF) technology was employed to profile the single-cell atlas of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) in 72 patients with IAs. In a matched cohort, metabolic shifts in PBMC subsets of IA patients were investigated by contrasting the expression levels of key metabolic enzymes with their respective counterparts in the healthy control group. Simultaneously, compositional differences in peripheral blood PMNs subsets between the two groups were analysed to explore the impact of altered heterogeneity in neutrophils on the initiation and progression of IAs. Furthermore, integrating immune features based on CyTOF analysis and clinical characteristics, we constructed an aneurysm occurrence model and an aneurysm growth model using the random forest method in conjunction with LASSO regression. Different subsets exhibited distinct metabolic characteristics. Overall, PBMCs from patients elevated CD98 expression and increased proliferation. Conversely, CD36 was up-regulated in T cells, B cells and monocytes from the controls but down-regulated in NK and NKT cells. The comparison also revealed differences in the metabolism and function of specific subsets between the two groups. In terms of PMNs, the neutrophil landscape within patients group revealed a pronounced shift towards heightened complexity. Various neutrophil subsets from the IA group generally exhibited lower expression levels of anti-inflammatory functional molecules (IL-4 and IL-10). By integrating clinical and immune features, the constructed aneurysm occurrence model could precisely identify patients with IAs with high prediction accuracy (AUC=0.987). Furthermore, the aneurysm growth model also exhibited superiority over ELAPSS scores in predicting aneurysm growth (lower prediction errors and out-of-bag errors). These findings enhanced our understanding of peripheral immune cell participation in aneurysm formation and growth from the perspectives of immune metabolism and neutrophil heterogeneity. Moreover, the predictive model based on CyTOF features holds the potential to aid in diagnosing and monitoring the progression of human IAs.

Transplacental migration of maternal natural killer and T cells assessed by ex vivo human placenta perfusion

IntroductionThe transplacental passage of cells between a mother and her fetus, known as microchimerism, is a less studied process during pregnancy. The frequency of maternal microchimeric cells in fetal tissues in physiological pregnancies and mechanisms responsible for transplacental cell trafficking are poorly understood. This study aimed to evaluate the placental trafficking of maternal peripheral blood mononuclear cells (PBMC) using human ex vivo placenta perfusion. MethodsTen placentas and maternal PBMC were obtained after healthy pregnancies. Flow cytometry was used to characterize PBMC subtypes. They showed a higher percentage of CD3+ T cells compared to CD56+ NK cells. The isolated PBMC were stained with a fluorescent dye and perfused through the maternal circuit of the placenta in an ex vivo perfusion system. Subsequent immunofluorescence staining for CD3+ T cells and CD56+ NK cells was performed on placental tissue sections, and the number of detectable PBMC in different tissue areas was counted using fluorescence microscopy. ResultsThe applied method allowed discrimination of perfused autologous maternal cells from cells resident in the placenta before perfusion. Further, it allows additional immunohistochemical labelling and distinction of immune cell subsets. Perfused PBMC were detected in all analyzed placentas, mostly in contact to the syncytiotrophoblast. CD3+ T cells were identified more frequently than CD56+ NK cells and some CD3+ T cells were found inside fetoplacental tissues and vasculature. The results indicate that also other PBMCs than T or NK cells adhere to or enter villous tissue, but they have not been specified in this analysis. DiscussionPrevious studies have detected maternal cells in the fetal circulation which we could mimick in our ex vivo placenta perfusion experiments with fluorescence labelled autologous maternal PBMC. The applied experimental settings did not allow comparison of transmigration abilities of PBMC subsets, but slight modifications of the model will permit further studies of cell transfer processes and microchimerism in pregnancy.

Abstract A005: Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models

Abstract Background The preclinical evaluation of novel immune therapies requires humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By xenotransplantation of cell-line-derived (CDX) or patient-derived (PDX) tumors on humanized mice, we successfully generated a full human tumor-immune-cell model for different disease entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies, and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient 1st and 2nd generation NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities were s.c. (i.e. lymphoma) and/or i.v. (leukemia) transplanted on those humanized mice. Tumor growth of orthotopic models was followed up by BLI measurement. Blood and tumor samples were analysed by for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. The 2nd generation NOG mice are showing an increased total engraftment and lineage specific differentiation of cells. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Check point inhibitor treatments (Ipi, Nivo, or Pembro) induced tumor growth delay in selected models. BLI measurement is a suitable method to follow up systemic disease models and treatment effects over time. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on different immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been successfully used for preclinical evaluation of novel check point inhibitors, cell therapies and immune cell engagers. Our human tumor-immune-cell models allow preclinical translational research on tumor immune biology as well as evaluation of new therapies, drug combinations, as well as biomarker identification and validation in subcutaneous and orthotopic models. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Joshua Alcaniz, Glenn Smits, Wolfgang Walther, Jens Hoffmann. Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A005.

A-098 Preliminary Tests of Cell Viability, Proteins and mRNA after Freezing PBMC for the Study of Chronic and Acute Lymphoproliferative Leukemias

Abstract The peripheral blood mononuclear cells (PBMC) cryopreserved are widely used in human immunology. However, PBMC, like other cell types, are sensitive to the freezing process. The impact of cryopreservation on T and B cell subsets of PBMC remains a challenge. For human PBMC, some reports have not observed significant changes between fresh and frozen cells in terms of proportions, phenotype and functionality, as well as antigen-specific responses or response to mitogens. Others, however, report that cryopreservation alters the proportions of human PBMC subsets in various immune responses. With the interest of studying chronic and acute lymphoproliferative leukemias (CLL and ALL), the authors’ objective was to test the viability of PBMC cells obtained from patients with CLL and ALL before and after freezing, following a simplified protocol. Methods On blood samples collected in EDTA, readings and cell counts were performed. Samples with a suggestive diagnosis of CLL and ALL were selected for the separation of PBMC whose analyzes were performed before and after freezing. The freezing of the PBMC was carried out in several stages and the whole process is carried out with care and in a gradual and simplified way of freezing until reaching the liquid nitrogen phase. Cells were kept frozen in freezing medium (RPMI plus 3% DMSO and 20% fetal bovine serum) for 1 week. Afterwards, staining with Trypan blue and counting in a Neubauer chamber were carried out. Student’s t test for independent variables and a significance level of 5% were considered. Results Table 1. Conclusion The description of this technique allows its use for analysis of gene expression profile, being able to contribute with the description of the molecular signature of leukemias.

Evaluation of quantitative biomarkers of aging in human PBMCs.

Functional decline with age contributes significantly to the burden of disease in developed countries. There is growing interest in the development of therapeutic interventions which slow or even reverse aging. Time and cost constraints prohibit the testing of a large number of interventions for health and lifespan extension in model organisms. Cell-based models of aging could enable high throughput testing of potential interventions. Despite extensive reports in the literature of cell properties that correlate with donor age, few are robustly observed across different laboratories. This casts doubt on the extent that aging signatures are captured in cultured cells. We tested molecular changes previously reported to correlate with donor age in peripheral blood mononuclear cells (PBMCs) and evaluated their suitability for inclusion in a panel of functional aging measures. The tested measures spanned several pathways implicated in aging including epigenetic changes, apoptosis, proteostasis, and intracellular communication. Surprisingly, only two markers correlated with donor age. DNA methylation age accurately predicted donor age confirming this is a robust aging biomarker. Additionally, the apoptotic marker CD95 correlated with donor age but only within subsets of PBMCs. To demonstrate cellular rejuvenation in response to a treatment will require integration of multiple read-outs of cell function. However, building a panel of measures to detect aging in cells is challenging and further research is needed to identify robust predictors of age in humans.

Supervised clustering of peripheral immune cells associated with clinical response to checkpoint inhibitor therapy in patients with advanced melanoma

Immune therapy with checkpoint inhibitors (CPIs) is a highly successful therapy in many cancers including metastatic melanoma. Still, many patients do not respond well to therapy and there are no blood-borne biomarkers available to assess the clinical outcome. To investigate cellular changes after CPI therapy, we carried out flow cytometry-based immune monitoring in a cohort of 90 metastatic melanoma patients before and after CPI therapy using the FlowSOM algorithm. To evaluate associations to the clinical outcome with therapy, we divided the patients based on progression-free survival. We found significant associations with CPI therapy in both peripheral blood mononuclear cell and T-cell subsets, but with the most pronounced effects in the latter. Particularly CD4+ effector memory T-cell subsets were associated with response with a positive correlation between CD27+HLA-DR+CD4+ effector memory T cells in a univariate (odds ratio: 1.07 [95% confidence interval 1.02-1.12]) and multivariate regression model (odds ratio: 1.08 [95% confidence interval 1.03-1.14]). We also found a trend towards stronger accumulation of CD57+CD8+ T cells in non-responding patients. Our results show significant associations between immune monitoring and clinical outcome of therapy that could be evaluated as biomarkers in a clinical setting.

Blood CD8+ Naïve T-Cells Identify MS Patients with High Probability of Optimal Cellular Response to SARS-CoV-2 Vaccine.

This single-center study included 68 multiple sclerosis (MS) patients who received the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination from one of several approved vaccine preparations in Spain. Blood samples were collected one to three months after the second dose of the vaccine had been administered. Cellular immune responses to the vaccine were assessed using QuantiFERON analysis, and peripheral blood mononuclear cell subsets were assayed using flow cytometry. Response associated with higher percentages of total lymphocytes, naïve CD4+ T-cells (p = 0.028), CD8+ T-cells (p = 0.013), and, mostly, naïve CD8+ T-cells (p = 0.0003). These results were confirmed by analyzing absolute numbers (p = 0.019; p = 0.002, and p = 0.0003, respectively). Naïve CD8 T-cell numbers higher than 17 cells/μL were closely associated with an optimal cellular response to SARS-CoV-2 vaccination (odds ratio: 24.0, confidence interval: 4.8-460.3; p = 0.0001). This finding clearly shows that independent of the treatment received, higher numbers of naïve CD8+ T-cells yield a strong cellular response to SARS-CoV-2 vaccines in MS patients. If this finding is validated with other viruses/vaccines, it could provide a good tool for identifying MS patients undergoing treatment who will develop strong cellular responses to anti-virus vaccines.

Single-cell RNA sequencing reveals the transcriptomic characteristics of peripheral blood mononuclear cells in hepatitis B vaccine non-responders.

The emergence of a vaccine against hepatitis B has proven to be an important milestone in the prevention of this disease; however, 5%-10% of vaccinated individuals do not generate an immune response to the vaccine, and its molecular mechanism has not been clarified. In this study, single-cell RNA sequencing was performed on peripheral blood mononuclear cells (PBMCs) from three volunteers with a high immune response (HR) and three with no immune response (NR) to the hepatitis B vaccine. We found that the antigen-presenting activity scores of various antigen-presenting cells, the mitogen-activated protein kinase (MAPK) pathway activity scores of naive B cells, and the cell activity scores of three types of effector T cells were significantly decreased, whereas the cytotoxicity scores of CD3highCD16lowKLRG1high natural killer T (NKT) cells were significantly increased in the NR group compared with those in the HR group. Additionally, the expression levels of some classical molecules associated with distinct signaling pathways-including HLA-B, HLA-DRB5, BLNK, BLK, IL4R, SCIMP, JUN, CEBPB, NDFIP1, and TXNIP-were significantly reduced in corresponding subsets of PBMCs from the NR group relative to those of the HR group. Furthermore, the expression of several cytotoxicity-related effector molecules, such as GNLY, NKG7, GZMB, GZMM, KLRC1, KLRD1, PRF1, CST7, and CTSW, was significantly higher in CD3highCD16lowKLRG1high NKT cells derived from non-responders. Our study provides a molecular basis for the lack of response to the hepatitis B vaccine, including defective antigen presentation, decreased T cell activity, and reduced IL-4 secretion, as well as novel insight into the role of NKT cells in the immune response to the hepatitis B vaccine.

Free fatty acids and peripheral blood mononuclear cells (PBMC) are correlated with chronic inflammation in obesity

Obesity-related chronic inflammation is closely related to the ability of immune cells to adapt to the body’s needs, research has shown that excess FAs can further activate pro-inflammatory transcription factors in the nucleus by interacting with various receptors such as CD36 and TLR4, thereby affecting the inflammatory state of cells. However, how the profile of various fatty acids in the blood of obese individuals is associated with chronic inflammation remains unclear.ObjectiveThe biomarkers associated with obesity were identified from 40 fatty acids (FAs) in the blood, and analyze the relationship between the biomarkers and chronic inflammation. Furthermore, by analyzing the difference in the expression of CD36, TLR4 and NF-κB p65 in peripheral blood mononuclear cells (PBMC) between obese and standard weight people, understand that immunophenotype PBMC is associated with chronic inflammation.MethodsThis study is a cross-sectional study. Participants were recruited from the Yangzhou Lipan weight loss training camp from May 2020 to July 2020. The sample size was 52 individuals, including 25 in the normal weight group and 27 in the obesity group. Individuals with obesity and controls of normal weight were recruited to identify biomarkers associated with obesity from 40 fatty acids in the blood; correlation analysis was conducted between the screened potential biomarkers FAs and the chronic inflammation index hs-CRP to identify FA biomarkers associated with chronic inflammation. Changes in the fatty acid receptor CD36, inflammatory receptor TLR4, and inflammatory nuclear transcription factor NF-κB p65 in PBMC subsets were used to further test the relationship between fatty acids and the inflammatory state in individuals with obesity.Results23 potential FA biomarkers for obesity were screened, eleven of the potential obesity biomarkers were also significantly related to hs-CRP. Compared to the control group, in monocytes the obesity group expressed higher TLR4, CD36, and NF-κB p65 in lymphocytes, the obesity group expressed higher TLR4 and CD36; and in granulocytes the obesity group expressed higher CD36.ConclusionBlood FAs are associated with obesity and are associated with chronic inflammation through increased CD36, TLR4, and NF-κB p65 in monocytes.