Peripheral Blood Mononuclear Cell Subsets Research Articles

IL-11 induces NLRP3 inflammasome activation in monocytes and inflammatory cell migration to the central nervous system

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes, and IL-11R+ neutrophils in comparison to matched healthy controls. IL-11+ and granulocyte-macrophage colony-stimulating factor (GM-CSF)+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65+, NLRP3+, and IL-1β+ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

Regulation of the PD-1/PD-L1 axis in relapsing-remitting Multiple Sclerosis (P2-3.001)

<h3>Objective:</h3> The PD-1/PD-L1 (programmed cell death protein 1/programmed cell death ligand 1) axis plays an important role in the adaptive immune system and has influence on neoplastic and inflammatory diseases. The role of the PD-1/PD-L1 axis in multiple sclerosis (MS) has not yet been investigated in detail. We aimed to delineate distinct expression patterns in peripheral blood mononuclear cell (PBMC) subsets and to evaluate the role of soluble PD-1/PD-L1 in the context of MS. <h3>Background:</h3> PD-L1/PD-1 also exist in soluble forms (sPD-L1/sPD-1) shedded from cellular surfaces. In systemic autoimmune diseases, sPD-1/sPD-L1 serum levels and membrane-bound PD-1/PD-L1 on T and antigen-presenting cells are linked to disease activity and treatment response. In MS, the PD-1/PD-L1 axis and in particular the role of soluble PD-1/PD-L1 is poorly understood but may offer relevance as disease marker and potential target for therapeutic intervention. <h3>Design/Methods:</h3> In depth immunophenotyping by flow cytometry was performed on PBMCs of 49 patients with relapsing-remitting MS (RRMS) and 27 controls. sPD-1/sPD-L1 serum levels were analyzed in a cohort of 84 patients with RRMS and 36 controls. To evaluate the therapeutic potential of soluble PD-L1, 600 μg/kg recombinant protein was systemically administered in Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. <h3>Results:</h3> Patients with RRMS displayed distinct cellular PD-1/PD-L1 expression patterns in PBMCs and an association of sPD-L1 with MS subtype and disease severity. Systemic administration of recombinant PD-L1 in EAE resulted in ameliorated disease severity and reduced infiltration of peripheral immune cells into the CNS. <h3>Conclusions:</h3> The marked expression of PD-1/PD-L1 in immune cell subsets of patients with RRMS, and the correlation of sPD-L1 with disease-severity offers promising potential for the use as a serum marker in the context of MS. Furthermore, enhancement of coregulatory PD-1/PD-L1 signaling by exogenous supplementation of sPD-L1 may serve as a novel therapeutic strategy for severe stages of acute neuroinflammation. <b>Disclosure:</b> Dr. Tsaktanis has nothing to disclose. Mr. Linnerbauer has nothing to disclose. Mrs. Loesslein has nothing to disclose. Prof. Hemmer has received personal compensation in the range of $0-$499 for serving as a Consultant for Vaccitech. Prof. Hemmer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Prof. Hemmer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Polpharma. Prof. Hemmer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for TG Therapeutics. Prof. Hemmer has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Allergy Care. The institution of Prof. Hemmer has received research support from Regeneron. The institution of Prof. Hemmer has received research support from German Research Foundation. The institution of Prof. Hemmer has received research support from German Ministry for Education and Research. The institution of Prof. Hemmer has received research support from European Commission. Prof. Hemmer has received intellectual property interests from a discovery or technology relating to health care. Prof. Hemmer has received intellectual property interests from a discovery or technology relating to health care. Mark Mühlau has nothing to disclose. Dr. Quintana has nothing to disclose. Prof. Rothhammer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Prof. Rothhammer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Merck. The institution of Prof. Rothhammer has received research support from German Research Foundation. The institution of Prof. Rothhammer has received research support from European Research Council.

Abstract 4430: Sacituzumab-Interferon beta mutein fusion protein, ABN202 (anti Trop2-interferon beta mutein), is a potent therapeutics for Trop-2-positive urothelial cancer

Abstract Background: Trop2, trophoblast cell-surface antigen 2, is a type 1 transmembrane glycoprotein overexpressed in many solid tumors such as breast, lung, pancreatic, gastric, colon, urothelial cancer. Overexpression of Trop2 is associated with aggressive disease progression and poor prognosis. Recently, Trop2 targeting antibody-drug conjugate such as sacituzumab-govitecan (Trodelvy®) was approved for the treatment of triple-negative breast cancer and urothelial cancer (UC) patients. Interferon-beta (IFN-beta), a pleiotropic cytokine, can induce direct/in-direct tumor killing and activate anti-tumor immune response in tumor microenvironments. However, it is challenging to develop IFN-beta as an anti-cancer agent due to its low biophysical properties, short half-life, and the systemic toxicity. Previously, we developed IFN-beta mutein (ABN102), which showed improved stability and biophysical properties compared to wild-type IFN-beta. We hypothesis to reduce any toxicity of ABN102 by fusing with tumor targeting antibody. In this study, we generated antibody cytokine fusion protein (ABN202), anti Trop2-interferon-beta mutein, and evaluated the efficacy of ABN202 against Trop2-positive UC cell line models. Method &amp; Result: We aimed to determine the direct anti-cancer efficacy of ABN202 using Trop2-positive UC cell lines and the indirect immune cell mediated effect of ABN202 using Trop2-positive UC cell lines co-cultured with human peripheral blood mononuclear cell (PBMC). The in vivo efficacy of ABN202 was further confirmed in Trop2 positive tumor mouse model. Our results revealed that ABN202 directly inhibited proliferation and activates the apoptosis-related signaling mechanisms via Type 1 IFN signaling, showing better efficacy than either anti Trop2 antibody (sacituzumab) or ABN102 alone. ABN202 induces the activation of PBMC subsets and enhanced immune cell-mediated cancer cell killing when UC cell lines were co-cultured with PBMC. The anti-cancer efficacy of ABN202 were confirmed in Trop2-positive tumor mouse model. Conclusion: We evaluated the direct and indirect anti-cancer efficacy of ABN202 (anti Trop2-interferon-beta mutein) in Trop2-positive urothelial cancer. Our results support that ABN202 is a potent drug candidate in Trop2-positive urothelial cancer by directly inhibiting tumor growth and activating anti-cancer immune responses through type 1 IFN signaling. Citation Format: Jiyang Lee, Myeung-Ryun Seo, Heegeon Park, YeongMun Kim, Saehyung Lee, Na Young Kim, Chan Gyu Lee, Hae Min Jeong, Jun Young Choi, Young kee Shin. Sacituzumab-Interferon beta mutein fusion protein, ABN202 (anti Trop2-interferon beta mutein), is a potent therapeutics for Trop-2-positive urothelial cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4430.

Regulation of immune responses in primary biliary cholangitis: a transcriptomic analysis of peripheral immune cells.

In patients with primary biliary cholangitis (PBC), the serum liver biochemistry measured during treatment with ursodeoxycholic acid-the UDCA response-accurately predicts long-term outcome. Molecular characterization of patients stratified by UDCA response can improve biological understanding of the high-risk disease, thereby helping to identify alternative approaches to disease-modifying therapy. In this study, we sought to characterize the immunobiology of the UDCA response using transcriptional profiling of peripheral blood mononuclear cell subsets. We performed bulk RNA-sequencing of monocytes and TH1, TH17, TREG, and B cells isolated from the peripheral blood of 15 PBC patients with adequate UDCA response ("responders"), 16 PBC patients with inadequate UDCA response ("nonresponders"), and 15 matched controls. We used the Weighted Gene Co-expression Network Analysis to identify networks of co-expressed genes ("modules") associated with response status and the most highly connected genes ("hub genes") within them. Finally, we performed a Multi-Omics Factor Analysis of the Weighted Gene Co-expression Network Analysis modules to identify the principal axes of biological variation ("latent factors") across all peripheral blood mononuclear cell subsets. Using the Weighted Gene Co-expression Network Analysis, we identified modules associated with response and/or disease status (q<0.05) in each peripheral blood mononuclear cell subset. Hub genes and functional annotations suggested that monocytes are proinflammatory in nonresponders, but antiinflammatory in responders; TH1 and TH17 cells are activated in all PBC cases but better regulated in responders; and TREG cells are activated-but also kept in check-in responders. Using the Multi-Omics Factor Analysis, we found that antiinflammatory activity in monocytes, regulation of TH1 cells, and activation of TREG cells are interrelated and more prominent in responders. We provide evidence that adaptive immune responses are better regulated in patients with PBC with adequate UDCA response.

Photodynamic Effects with 5-Aminolevulinic Acid on Cytokines and Exosomes in Human Peripheral Blood Mononuclear Cells from Patients with Crohn's Disease.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) which is the precursor of the photosensitizer protoporphyrin IX (PpIX) is an available treatment for several diseases. ALA-PDT induces the apoptosis and necrosis of target lesions. We have recently reported the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). This study has investigated the ALA-PDT-mediated effects on PBMC subsets from patients with active Crohn's disease (CD). No effects on lymphocyte survival after ALA-PDT were observed, although the survival of CD3-/CD19+ B-cells seemed slightly reduced in some samples. Interestingly, ALA-PDT clearly killed monocytes. The subcellular levels of cytokines and exosomes associated with inflammation were widely downregulated, which is consistent with our previous findings in PBMCs from healthy human subjects. These results suggest that ALA-PDT may be a potential treatment candidate for CD and other immune-mediated diseases.

P048 Deep phenotyping of the peripheral immune cell compartment in Crohn‘s Disease

Abstract Background Crohn’s disease (CD) is characterized by chronic inflammation that can discontinuously affect any segment of the gastrointestinal tract. New findings concerning clinical behaviour, epidemiology, genetics, and gut microbiota suggest that CD in the ileum (iCD) and the colon (cCD) should be considered as two distinct subtypes of IBD. As systemic immune cell signatures differ between CD and UC, we propose a similar strategy for the differentiation between iCD and cCD. The aim of this study is to analyse and compare peripheral blood mononuclear cell (PBMC) subsets of iCD and cCD patients. For a robust analysis of the peripheral immune compartment in inflammatory bowel diseases (IBD), PBMC subsets of ileocolonic CD (icCD), ulcerative colitis (UC) and healthy controls (HC) were included as well. Methods In this study a total of 50 adults, 10 subjects per subgroup – iCD, cCD, icCD, UC and HC – were included. From each individual, both naïve and PMA/Ionomycin stimulated samples were stained for phenotypic and functional characterization using mass cytometry. For data analysis pre-gating was performed in Cytobank followed by debarcoding, compensation, normalization, clustering, and analysis on differential abundances edge R (DA_edgeR) and differential states limma (DS_limma) with CATALYST in R. Results FlowSOM allowed us to cluster CD45+ PBMCs of all samples into 16 major cell subtypes as visualized via UMAP (Fig. 1). DA testing showed significant differences between iCD and cCD, iCD and icCD but not cCD and icCD (Fig 2). Significantly different abundances among CD subgroups and HC included cluster 1 CD33+ myeloid cells, 7 CD45RO+ CD16+ cells, 8 CD11b+ cells, 9 CD4+ IL7R+ T cells and 12 CD27+ CD38+ CD45RA+ B cells (Fig. 3). When focussing on differences between iCD, cCD and icCD, DS testing revealed significantly higher IFNgamma and TNFalpha production in cluster 9 (CD4+ IL7R+ T cells), 15 (CD8+ T cells) and 16 (CD8+ CD45RO+ T cells) in iCD compared to icCD. Moreover, cluster 9 showed significantly higher levels of IFNgamma in iCD compared to cCD, and for TNFalpha cluster 16 revealed significantly higher levels in cCD compared to icCD (Fig. 4). Fig. 1 UMAP clustering of stimulated cells. Fig. 2 Differential analysis of stimulated samples with DA_edgeR and DS_limma. Fig. 3 Relative population abundances with DA_edge R testing. Fig. 4 Relative state marker abundance within clusters with DS_limma testing. Conclusion Deep phenotyping of the peripheral immune cell compartment in CD might serve as a confirmatory marker for disease location and therefore as predictor of clinical behaviour.

Association between increased peripheral blood CD86-positive plasmacytoid dendritic cells and immune-related adverse events in patients with non-small cell lung cancer.

The occurrence of immune-related adverse events (irAEs) after immune checkpoint inhibitors (ICIs) is unpredictable. Profiles of peripheral blood mononuclear cells (PBMCs) represent the host immune system and have the potential to predict irAEs. We analyzed PBMC subsets using multicolor flow cytometry before and at weeks 2 and 8 after the start of ICIs in patients with non-small cell lung cancer. Sixteen eligible patients were evaluated. The irAEs occurred in 6 patients (37.5%): diarrhea in 2, diarrhea and a rash in 1, pituitary dysfunction in 1, cholangitis in 1, and pneumonitis in 1. Patients experiencing irAEs had higher levels of CD86+plasmacytoid dendritic cells (pDCs) at the baseline and weeks 2 and 8 after the ICIs than those not experiencing irAEs (p = 0.005, 0.038, and 0.050, respectively). In patients experiencing irAEs, the levels of CD86+pDCs significantly decreased at weeks 2 and 8 compared to the baseline (p = 0 .034 and 0.025, respectively) but did not change in those not experiencing irAEs. The levels of other PBMC subsets were not significantly associated with irAEs. Higher levels of natural killer (NK) cells were significantly associated with an overall objective response (p = 0.024). In conclusion, higher levels of CD86+pDCs at the baseline and a reduction in those levels 2 and 8 weeks after ICIs were associated with the occurrence of irAEs. Higher levels of NK cells were associated with an objective response to ICIs. Evaluation of PBMCs may help to predict the efficacy and safety of ICIs.

Dynamic analysis of tuberculosis specific mononuclear cells in peripheral blood of HIV patients with tuberculosis

Objective: To evaluate the response of peripheral blood mononuclear cells (PBMCs) in patients with human immunodeficiency virus (HIV) combined with active tuberculosis (TB) to TB-specific antigen stimulation. Methods: From January to December, 2018, individuals infected with both HIV and TB (HIV/TB group) were taken as the study subjects. Individuals infected with HIV alone (HIV group), individuals infected with TB alone (TB group) and healthy people (Health control group, HC group) were taken as the control groups. PBMCs were isolated and stimulated with purified protein derivative of bacillus calmette-guerin (BCG-PPD). The expression of surface molecules in T cells (CD3+, CD4+, CD8+) and monocytes (CD14+) and the percentages of Interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected by cell surface molecular staining, direct intracellular cytokine staining and flow cytometry (CD3- lymphocytes were mainly B lymphocytes and NK cells). Analysis of non-parametric data was used to compare the data between the two groups, and paired t-test was used to compare the data before and after PPD stimulation in each group. Results: Before PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the peripheral blood of HIV/TB group(mean 0.52%) was significantly lower than that in TB group(mean 0.94%, P=0.010). The TNF-α+cell percentages in CD3+, CD4+, CD8+, or CD14+ cells in the HIV/TB group(mean 19.2%) were significantly lower than those in the HIV group(mean 31.9%, P=0.002). The percentage of TNF-α secreted by monocytes in the HIV group was significantly lower than that in the HC group. The percentages of IFN-γ+ CD8+ and IFN-γ+ CD3- cells in the peripheral blood of the TB group (mean 0.94%) were significantly higher than thoset in the HC group(mean 0.51%, P=0.020), while the percentages of TNF-α+ cells in each subsets of PBMCs were significantly lower than those in the HC group. After PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the HIV/TB group was significantly lower than that in the TB group(P=0.008), and the change was more marked than that before stimulation. The percentage of IFN-γ+ CD8+ cells in the HIV group(mean 0.20%) was lower than that in the HC group (mean 0.52%, P=0.044). The percentage of IFN-γ+ CD3- in the TB group was significantly higher than in the HC group. There were no significant differences in TNF-α+ cell percentages in the 3 groups compared with the control group after PPD stimulation. The percentages of IFN-γ+ CD4+ cells in the HC and the TB groups were significantly increased after PPD stimulation in each group (P=0.002, P=0.001, respectively). However, there were no significant differences of IFN-γ+ CD4+ cell percentages in the HIV/TB group and the HIV group. The percentages of TNF-α production by monocytes were significantly increased after PPD stimulation in all groups. Conclusions: Chronic Mycobacterium tuberculosis (MTB) infection reduced the ability of PBMCs to produce TNF-α. For patients with TB infection, the production of TNF-α was reduced when combined with HIV infection. The capacity of CD8+ and CD3- lymphocytes to produce IFN-γ was increased in TB patients, while the capacity of CD8+ T cells to produce IFN-γ was decreased with co-infection of HIV. Infection of HIV weakened the immune response to MTB infection, which made the clinical diagnosis and treatment of TB more difficult.

Radiation therapy induces immunosenescence mediated by p90RSK.

Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated β-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.

Methylprednisolone pulse therapy promotes the differentiation of regulatory T cells by inducing the apoptosis of CD4+ T cells in patients with systemic lupus erythematosus

ObjectivesTo investigate the differentiation of regulatory T cells (Tregs) induced by methylprednisolone (MP) pulse therapy in patients with Systemic Lupus Erythematosus (SLE). MethodsWe enrolled 30 patients with SLE and analyzed peripheral blood mononuclear cells (PBMCs) before and after MP pulse therapy. Peripheral Tregs, apoptosis of PBMCs subsets, and TGFβ production by monocytes was quantified by flow cytometry. Proliferation and IFN-γ production of CD4+ T cells were measured. Furthermore, TGFβ1 production by human monocyte-derived macrophages (HMDM) stimulated with MP-treated CD4+ T cells were quantified by ELISA. ResultsPeripheral Tregs was significantly increased after MP pulse therapy (6.76 ± 1.46% vs. 3.82 ± 1.02%, p < 0.01), with an expansion of Nrp1− induced Tregs (4.54 ± 0.46% vs. 1.75 ± 0.38%, p < 0.01). Proliferation and IFN-γ production of CD4+ T cells were significantly decreased after MP pulse therapy. MP pulse therapy induced CD4+ T cell apoptosis (early apoptosis, 26.34 ± 3.54% vs. 14.81 ± 2.89%, p < 0.01) and TGFβ expression on monocytes (6.02% vs. 2.45%, p < 0.01). Furthermore, MP induced CD4+ T cell apoptosis in vitro, which stimulated HMDM to produce TGFβ. Moreover, elevated TGFβ level in supernatant from HMDM stimulated with MP-treated CD4+ T cells promoted Tregs differentiation. ConclusionsMP pulse therapy induces CD4+ T cell apoptosis, which promotes monocytes to produce TGFβ and further facilitates Tregs differentiation. Newly-differentiated Tregs suppress proliferation and IFN-γ production of CD4+ T cells and contribute to immunoregulatory milieu after MP pulse therapy.

Abstract 1650: Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers

Abstract Background The preclinical evaluation of novel immune therapies demands 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 transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor 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 NOG mice. 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 (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted on those humanized mice. These models were used to evaluate novel immune therapies. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were functional T-cells, characterized by a high PD-1 expression. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of tumors revealed an increased percentage of tumor infiltrating T-cells. 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 immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors, cell therapies and immune cell engagers. Our 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, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1650.

Abstract 6179: Increased circulating PD-L1 expressing CD14 high CD16 negative classical monocytes correlate with poor prognosis in cancer patients treated with PD-1 blockade therapies

Abstract Background: Immune checkpoint inhibitors (ICIs), like PD-1 and PD-L1 blockade therapies, are currently considered as one of the most significant breakthroughs in potent cancer immunotherapy. However, many patients are non-responders or develop resistance following an initial response to ICIs. ICIs have no accurate predictive biomarkers. Previously, we showed that increased expression of PD-L1 molecules on CD14+ monocytes was significantly correlated with shorter overall survival (OS) for patients with several cancer types on PD-1 blockade therapies. As monocytes can be further classified into subsets (classical, intermediate, non-classical), we herein investigated the prognostic factors in peripheral blood mononuclear cell (PBMC) subsets and assessed the clinical significance for the PD-L1-expressing subsets of each of these cells including classical, intermediate and non-classical monocyte subsets in patients with various cancer types, along with their clinical implications in PD-1 blockade therapies. Material and Methods: We evaluated 44 patients (20 with gastric cancer, 17 with non-small cell lung carcinoma, and 7 with esophageal cancer) undergoing PD-L1 blockade therapy (pembrolizumab or nivolumab) for PD-L1 expression levels in classical (CD14high, CD16-), intermediate (CD14high, CD16+), and non-classical (CD14low, CD16+) monocytes measured via flow cytometry before ICI treatment. The percentages of PD-L1+ cells in respective monocyte subsets were compared with respect to different clinicopathological conditions and the association with survival time was assessed. Results: The number of PD-1 positive cells in CD14+ monocytes was very low (almost &amp;lt;1%). Also, the monocyte subset expressing PD-L1 had a high percentage of classical monocytes, whereas non-classical monocytes expressed less PD-L1. Higher levels of classical monocytes were correlated with shorter OS (P = 0.044), whereas higher levels of non-classical monocytes were correlated with longer OS (P = 0.027). Focusing on non-small cell lung cancer population (N = 17), higher levels of PD-L1-expressing CD14+ monocytes were statistically correlated with shorter OS (P = 0.025). Furthermore, analysis of a subset of monocytes showed that the higher PD-L1-expressing classical monocytes correlated with the shorter OS, suggesting that they predict poor prognosis (P = 0.0095). Conclusion: Circulating PD-L1-expressing classical monocytes in the peripheral bloods were associated with poor prognosis in patients treated with PD-1 blockade therapies. Among the monocyte subsets, classical monocyte, especially PD-L1-expressing classical monocytes can be a potential biomarker for prognosis in these patients. Citation Format: Ryotaro Ohkuma, Katsuaki Ieguchi, Makoto Watanabe, Tsubasa Goshima, Rie Onoue, Kazuyuki Hamada, Yutaro Kubota, Atsushi Horiike, Toshiaki Tsurui, Risako Suzuki, Nana Iriguchi, Tomoyuki Ishiguro, Yuya Hirasawa, Hirotsugu Ariizumi, Kiyoshi Yoshimura, Mayumi Tsuji, Yuji Kiuchi, Shinichi Kobayashi, Takuya Tsunoda, Satoshi Wada. Increased circulating PD-L1 expressing CD14 high CD16 negative classical monocytes correlate with poor prognosis in cancer patients treated with PD-1 blockade therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6179.

Modeling Inflammatory Immune Cell Recruitment and Response on Human Colon Intestine-Chip

Abstract Objective Immune cell recruitment into tissues is an essential step in inflammatory responses. This occurs in a highly tissue- and stimulus-specific manner, which presents a significant challenge to modeling disease and testing therapeutics ex vivo. We previously developed an advanced primary human vascularized Colon Intestine-Chip model and showed that it recapitulates physiologic cell composition, morphology and barrier function. The goal of this study was to test the ability of this system to model inflammatory bowel disease (IBD)-like immune cell reactions ex vivo. Methods We perfused primary human peripheral blood mononuclear cells (PBMC) across the vascular channel in untreated ‘resting’ or TNFα/chemokine-treated ‘inflamed’ Colon Intestine-Chips. We analyzed total cell recruitment, inflammatory cytokine secretion and barrier function in the following 24–72 hours. Results We show that the perfused PBMC efficiently adhered and transmigrated to the epithelial channel in an inflammation-specific manner. This was followed by an accumulation of proinflammatory cytokines characteristic of IBD (e.g., INFγ, IL1β, IL18), as well as loss of barrier function, the hallmark feature of IBD. We further showed that 1) the recruited cells were strongly enriched in the ‘gut trophic’ α4β7+/CCR9+ PBMC subsets and 2) this recruitment could be blocked with the IBD therapeutic Entyvio, which targets the α4β7-MAdCAM-1 interaction. Conclusion Our findings indicate that our Colon Intestine-Chip can model inflammatory immune cell recruitment and in situ immune reactions that reflect key clinical correlates of IBD. This model may prove effective for development of new anti-inflammatory therapeutics for human intestinal diseases. Supported by Emulate Bio

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients.

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients.

Chicken Peripheral Blood Mononuclear Cells Response to Avian Leukosis Virus Subgroup J Infection Assessed by Single-Cell RNA Sequencing.

Chicken peripheral blood mononuclear cells (PBMCs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Here we performed single-cell RNA sequencing (scRNA-seq) of PBMCs in Avian leukosis virus subgroup J (ALV-J) infected and control chickens at 21 days post infection (DPI) to determine chicken PBMCs subsets and their specific molecular and cellular characteristics. Eight cell populations and their potential marker genes were identified in PBMCs. T cell populations had the strongest response to (ALV-J) infection, based on the detection of the largest number of differentially expressed genes (DEGs), and could be further grouped into four subsets: activated CD4+ T cells, Th1-like cells, Th2-like cells, and cytotoxic CD8+ T cells. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection activated CD4+ T cell was probably inclined to differentiate into Th1-like cells. Compared to the control PBMCs, ALV-J infection also had an obvious impact on PBMCs composition. B cells showed inconspicuous response and their numbers decreased in PBMCs from ALV-J infected chicken. Proportions of cytotoxic Th1-like cells and CD8+ T cells increased in the T cell population of PBMCs from ALV-J infected chicken, which were potentially key mitigating effectors against ALV-J infection. More importantly, our results provide a rich resource of gene expression profiles of chicken PBMCs subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

Abstract PD9-02: Peripheral immune subsets and circulating tumor DNA (ctDNA) in patients (pts) with residual triple negative breast cancer (TNBC) treated with adjuvant immunotherapy and/or chemotherapy (chemo): The OXEL study

Abstract Background: Poor clinical outcomes are noted in pts with TNBC who do not achieve a pathologic complete response (pCR). We characterized peripheral immune subsets and the role of minimal residual disease (MRD) detection via ctDNA in pts who participated in the OXEL study. Methods: OXEL (Opdivo® -XELoda ®) is a recently completed phase II open-label 3-arm randomized study of nivolumab (nivo), capecitabine (cape) or the combination as adjuvant therapy (tx) for pts with residual TNBC after appropriate neoadjuvant chemo. Residual disease was defined as ≥ 1.0 cm of primary tumor and/or nodal involvement. Eligible pts had completed definitive local tx. Pts were randomly assigned to nivo 360 mg iv q3wks x 6 (arm A); cape 1250mg/m2 po bid D1-D14 q3 wks x 6 (arm B); nivo 360mg iv q3wks + cape 1250mg/m2 po bid D1-D14 q3 wks x 6 (arm C). Peripheral blood mononuclear cells (PBMCs) and ctDNA were assessed at baseline (D1 of cycle 1), 6, and 12 wks and at time of recurrence, if applicable. PBMCs were stained with 30 markers and analyzed by flow cytometry to identify changes in 158 immune cell subsets at 6 wks, as a percent of total PBMCs. RaDaRTM, a deep sequencing based, tumor-informed personalized assay was utilized to detect the presence of ctDNA in plasma. Distant disease-free survival (DDFS) and overall survival (OS) were analyzed by the Kaplan-Meier method and Log-Rank test was used to compare DDFS and OS according to baseline MRD results. All pts will be followed for distant recurrence and survival for 3 yrs. Here we report the translational endpoints of the OXEL study. Clinical endpoints according to treatment received will be reported in a future analysis. Results: 45 pts were enrolled between 8/2018 and 6/2021. 29 (64%) were Caucasian and 14 (31%) were African American. Mean age at enrollment was 51 [+/- 12]. 93% of pts received a taxane-anthracycline containing neoadjuvant tx. 15 pts were randomized to each arm. DDFS probability at 1-yr and 2-yrs was 0.71 (+/- 0.07) and 0.66 (+/- 0.08) respectively. At 12 mos of median follow up, 13/45 pts (29%) experienced distant recurrence, none had local recurrence. 43 pts were evaluated for PBMC subsets. Changes in PBMC subsets at 6 wks were different amongst the arms; in arm A, reductions in NK subsets, including a 33% reduction in CD56dimCD16- cells, were observed, while in arm B, increases in naïve CD4+ T cells (+45%) and CD73+CD8+ T cells (+12%) and reductions in ki67+CD8+ T cells (-48%) were noted. In arm C, increases were observed in conventional dendritic cells (+36%), effector memory ki67+CD4+ T cells (+46%), and CD56dimCD16- NK cells (+29%). 33 pts underwent successful MRD analysis. 12/33 (36%) pts were MRD+ at baseline. 2/12 pts MRD+ at baseline subsequently cleared MRD, with undetectable ctDNA on future time points; neither patient has had recurrence to date. The remaining 10/12 MRD+ pts (83%) have experienced distance recurrence. 21/33 (64%) pts were ctDNA negative at baseline; 20/33 remained negative for all follow up timepoints. 10/11 pts experiencing distant recurrence were MRD+ at baseline, compared to 1/11 pt who became MRD+ at wk 6 post initiation of tx. At 12 mos of median follow-up, baseline MRD+ testing was significantly associated with an inferior DDFS ( p&amp;lt;0.0001 Log-rank test, median DDFS 4.0 mos vs. not reached) and OS (p=0.02 Log-rank test, median OS not reached for both groups). Results will be updated at the time of abstract presentation. Conclusions: Changes in PBMC subsets were associated with receipt of chemo and/or immunotherapy. Our results suggest that baseline MRD+ in pts without pCR is a poor prognostic factor. Future trials aiming to optimize adjuvant treatment with chemo and/or immunotherapy in residual TNBC should consider incorporating ctDNA as a selection marker of pts at higher risk of recurrence. Citation Format: Filipa Lynce, Candace Mainor, Xue Geng, Greg Jones, Ilana Schlam, Hongkun Wang, Ute Feger, Renee Donahue, Nicole Toney, Caroline Jochems, Jeffrey Schlom, Christopher Gallagher, Rita Nanda, Deena Graham, Erica M Stringer-Reasor, Neelima Denduluri, Julie Collins, Asma A Dilawari, Ami Chitalia, Shruti Tiwari, Raquel Nunes, Rebecca Kaltman, Katia Khoury, Margaret Gatti-Mays, Sandra M Swain, Heather A. Parsons, Paula Pohlmann, Claudine Isaacs. Peripheral immune subsets and circulating tumor DNA (ctDNA) in patients (pts) with residual triple negative breast cancer (TNBC) treated with adjuvant immunotherapy and/or chemotherapy (chemo): The OXEL study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-02.

Photodynamic Effects with 5-Aminolevulinic Acid on Cytokines and Exosomes in Human Peripheral Blood Mononuclear Cells.

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor to the potent photosensitizer, protoporphyrin IX (PpIX), is an established modality for several malignant and premalignant diseases. This treatment is based on the light-activated PpIX in targeted lesions. Although numerous studies have confirmed the necrosis and apoptosis involved in the mechanism of action of this modality, little information is available for the change of exosome levels after treatment. We report from the first study on the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). The treatment reduced the cytokines and exosomes studied, although there was variation among individual PBMC samples. This reduction is consistent with PDT-mediated survivals of subsets of PBMCs. More specifically, the ALA-PDT treatment apparently decreased all pro-inflammatory cytokines included, suggesting that this treatment may provide a strong anti-inflammatory effect. In addition, the treatment has decreased the levels of different types of exosomes, the HLA-DRDPDQ exosome in particular, which plays an important role in the rejection of organ transplantation as well as autoimmune diseases. These results may suggest future therapeutic strategies of ALA-PDT.

Immunomodulatory Effects Associated with Cladribine Treatment.

Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.

Short- and Long-Term Immunological Responses in Chronic HCV/HIV Co-Infected Compared to HCV Mono-Infected Patients after DAA Therapy.

Background: Direct-acting antivirals (DAAs) treatment, although highly efficacious for the treatment of hepatitis C virus (HCV) infection, may not completely reconstitute the HCV-mediated dysregulated immune system, especially in patients co-infected with human immunodeficiency virus (HIV) and HCV. Objectives: We aimed to evaluate the impact of HCV eradication following DAA therapy on the immune system and liver disease improvement through comparative monitoring of 10 HCV mono-infected and 10 HCV/HIV co-infected patients under combined antiretroviral therapy (cART). Early and late longitudinal phenotypic changes in peripheral blood mononuclear cell (PBMC) subsets, T-cell activation, differentiation and exhaustion, as well as inflammatory biomarkers, indoleamine 2-3 dioxygenase (IDO) activity, and liver stiffness, APRI and FIB-4 scores were assessed. Materials and Methods: Samples were obtained at baseline (T0), week 1 (T1), week 2 (T2), week 12 (T3, end of treatment, EOT), and month 9 (T4, end of follow-up, 36 weeks post EOT). Results: All patients achieved a sustained virological response (SVR 12) after DAA treatment. Overall, changes of the T-cell immune phenotypes were greater in HCV/HIV co-infected than in HCV mono-infected, due to an increase in CD4+ and CD8+ T-cell percentages and of CD8+ T-cell activation and memory markers, in particular at the end of follow-up. On the other end, HCV mono-infected showed changes in the activation profile and in the memory CD4+ T-cell compartment. In HCV/HIV co-infected, a decrease in the IDO activity by DAA treatment was observed; conversely, in HCV mono-infected, it resulted unmodified. Regarding inflammatory mediators, viral suppression was associated with a reduction in IP-10 levels, while interferon regulatory factor (IRF)-7, interferon (IFN)-β, and interferon (IFN)-γ levels were downregulated during therapy and increased post therapy. A decrease in liver stiffness, APRI, and FIB-4 scores was also observed. Conclusions: Our study suggests that, although patients achieved HCV eradication, the immune activation state in both HCV mono-infected and HCV/HIV co-infected patients remains elevated for a long time after the end of DAA therapy, despite an improvement of liver-specific outcomes, meanwhile highlighting the distinct immunophenotypic and inflammatory biomarker profile between the groups of patients.

Characterization of a Novel Missense CXCR4 Mutation in a Patient with WHIM-like Syndrome

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare primary immunodeficiency with a heterogeneous presentation of symptoms defining its acronym as well as panleukopenia. The majority of cases are inherited in an autosomal dominant manner, with gain-of-function mutations in the C-terminus of the C-X-C chemokine receptor 4 (CXCR4) (McDermott D, et al. Immunol Rev. 2019;287:91-102). To our knowledge, there are no literature reports implicating CXCR4WHIM mutations outside the CXCR4 C-terminus. Here we report the clinical presentation of a patient with the novel mutation CXCR4D84H and characterize the functional effects of the mutation on CXCR4 trafficking and chemotaxis in in vitro and ex vivo assays. Further, CXCR4D84H is reported in 3 population genetic databases (average allele frequency 3.8 × 10 -5) and is also found in 5 unrelated patients in another rare disease database (CentoMD).C ase Report: A 40-year-old female presented with a history of recurrent vulvovaginal and anal dysplasia and carcinoma in situ requiring multiple surgeries starting at age 20 years. Cytopenia was first documented at age 15 years in the context of mononucleosis, which resolved, although cytopenias persisted through adulthood. To date, the patient has decreased absolute neutrophil counts (600-1100/mm 3 range), WBC counts ~2000/mm 3, and normal immunoglobulin levels. Bone marrow biopsy revealed bilobed neutrophils and granulocyte precursors. The patient has no history of recurrent infections besides HPV/EBV and no family history of warts, immunodeficiency, or squamous cell carcinoma. Genetic testing revealed a heterozygous mutation in CXCR4 (c.250G>C, D84H) that has not been previously reported. This missense mutation occurs in the transmembrane domain of CXCR4 in proximity of the residues involved in signal initiation, and hence, may alter signaling responses.Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from this patient and 2 healthy controls and analyzed for functionality ex vivo including CXCR4 internalization assays and chemotaxis to exogenously added CXCR4-ligand CXCL12. The CXCR4-negative K562 cell line was transiently transfected to express CXCR4D84H, CXCR4WT, and known CXCR4WHIM mutations for further comparison of their functional responses to CXCL12.Results: Flow cytometry data shows abnormal peripheral leukocyte frequency and count. Lymphocyte subpopulations showed 33%-57% reduced CXCR4 internalization in response to CXCL12 compared to controls. Chemotactic response to CXCL12 was increased in PBMC subsets including natural killer T (NKT) cells compared to healthy donor cells. These observations are comparable to previously published responses to pathogenic CXCR4WHIM variants, but, chemotactic responses of NKT cells is a novel finding. In K562-transfected cells, CXCR4D84H demonstrated impaired CXCL12-induced internalization compared to CXCR4WT at a level comparable to the CXCR4E343K variant, the only CXCR4 missense variant known to date to cause WHIM syndrome. Chemotaxis of cells expressing CXCR4D84H in response to CXCL12 is increased by 1.25- to 2.4-fold compared to CXCR4WTdepending on CXCL12 concentration, in line with the observation of increased chemotaxis of cells expressing known CXCR4WHIM variants. Overall, the CXCR4D84H mutation recapitulates the phenotypes exhibited by known CXCR4WHIM variants in both internalization and chemotaxis assays.Conclusions: We show the functional effects of a novel missense mutation CXCR4D84H found in a patient with clinical WHIM syndrome recapitulates the internalization and chemotaxis findings of other CXCR4WHIM mutations. This is the first report of a missense mutation in CXCR4 outside of the C-terminus causing a WHIM phenotype, and additionally highlights defective chemotaxis in NKT cells, which may be relevant in this patient's HPV-associated carcinoma in situ phenotype. Based on an analysis of population databases, and assuming a conservative 5-10% penetrance, there are potentially ~1250-2500 individuals in the United States with disease due to the p.D84H variant alone, highlighting the under-recognition of WHIM syndrome. Patients who have similar clinical presentation as described in this case study should be evaluated for WHIM syndrome and other primary immunodeficiencies by using genetic screening for CXCR4 and diagnostic assays described. DisclosuresPawar: X4 Pharmaceuticals: Current Employment. Zmajkovicova: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wiest: X4 Pharmaceuticals: Current Employment. Nguyen: X4 Pharmaceuticals: Current Employment. Monticelli: X4 Pharmaceuticals: Current Employment. Maier-Munsa: X4 Pharmaceuticals: Current Employment. Sondheimer: X4 Pharmaceuticals: Consultancy. Walter: Octapharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmig: Consultancy, Membership on an entity's Board of Directors or advisory committees. Taveras: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Badarau: X4 Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Tarrant: ThermoFisher Scientific: Consultancy; X4 Pharmaceuticals: Consultancy, Research Funding; Department of Justice: Consultancy; Abbvie: Research Funding; Viela Bio: Research Funding.