Multiparameter Flow Cytometry Research Articles

High expression of PD-L1 on conventional dendritic cells in tumour-draining lymph nodes is associated with poor prognosis in oral cancer

IntroductionOral squamous cell carcinoma (OSCC), while common and with a favorable prognosis in early stages, presents a marked reduction in survival rate upon metastasis to lymph nodes. Early detection of lymph node metastasis via biomarkers could enhance the therapeutic strategy for OSCC. Here, we explored dendritic cells (DCs) and cytotoxic T-cells in tumour-draining lymph nodes (TDLNs) as potential biomarkers.MethodDendritic cells and cytotoxic T-cells in 33 lymph nodes were analyzed with multi-parameter flow cytometry in TDLNs, regional non-TDLNs surgically excised from 12 OSCC patients, and compared to 9 lymph nodes from patients with benign conditions.ResultsOur results displayed a higher proportion of conventional cDC1s with immunosuppressive features in TDLN. Further, high PD-L1 expression on cDC1 in TDLNs was associated with metastasis and/or recurrent disease risk. Also, elevated levels of memory CD8+ T-cells and terminally exhausted PD-1+TCF-1−CD8+ T-cells were observed in TDLNs and non-TDLNs compared to healthy lymph nodes.ConclusionsWe conclude that TDLNs contain cells that could trigger an anti-tumor adaptive response, as evidenced by activated cDC1s and progenitor-like TCF-1+ T-cells. The detection of high PDL1 expression on cDC1s was indicative of TDLN metastasis and an adverse prognosis, proposing that PD-L1 on dendritic cells in TDLN could serve as a predictive biomarker of OSCC patients with a worse prognosis.

Cytology or Multiparameter Flow Cytometry Positivity in the Cerebrospinal Fluid Before Transplantation is Predictive of Poor Outcomes After Allotransplantation in Acute Myeloid Leukemia Patients.

Central nervous system leukemia (CNSL) remains a serious complication in patients with acute myeloid leukemia (AML) and an ambiguous prognostic factor for those receiving allo-geneic hematopoiesis stem cell transplantation (allo-HSCT). It is unknown whether using more sensitive tools, such as multiparameter flow cytometry (MFC), to detect blasts in the cerebrospinal fluid (CSF) would have an impact on outcome. We retrospectively analyzed the clinical outcomes of 1472 AML patients with or without cytology or MFC positivity in the CSF before transplantation. Abnormal CSF (CSF+) was detected via conventional cytology and MFC in 44 patients at any time after diagnosis. A control group of 175 CSF-normal (CSF-) patients was generated via propensity score matching (PSM) analyses according to sex, age at transplant, and white blood cell count at diagnosis. Compared to those in the CSF-negative group, the conventional cytology positive and MFC+ groups had comparable 8-year nonrelapse mortality (NRM) (4%, 4%, and 6%, p=0.82), higher cumulative incidence of relapse (CIR) (14%, 31%, and 32%, p=0.007), lower leukemia-free survival (LFS) (79%, 63%, and 64%, p=0.024), and overall survival (OS) (83%, 63%, and 68%, p=0.021), with no significant differences between the conventional cytology positive and MFC+ groups. Furthermore, multivariate analysis confirmed that CSF involvement was an independent factor affecting OS and LFS. Our results indicate that pretransplant CSF abnormalities are adverse factors independently affecting OS and LFS after allotransplantation in AML patients.

Changes of circulating tumor cells expressing CD90 and EpCAM in early-phase of atezolizumab and bevacizumab for hepatocellular carcinoma

Circulating tumor cells (CTCs) are noninvasive biomarkers that can indicate the therapeutic response and prognosis. The study aimed to investigate the cellular characteristics of CTCs focusing on monitoring during atezolizumab and bevacizumab (Atezo-Bev) therapy in patients with hepatocellular carcinoma (HCC). Peripheral blood samples were collected from 10 healthy controls and 40 patients with HCC. CTCs enriched using RosetteSep™ Human CD45 depletion cocktail were analyzed by multiparametric flow cytometry. CTC isolation was based on PanCK(+)CD45(−) cells, and CTCs exhibiting markers CD90, CD133, EpCAM, or vimentin. The total number of CTCs and the number of CTCs expressing CD90, CD133, EpCAM, and vimentin were correlated with the BCLC stage of HCC. The change in total CTC count accurately reflected the initial response to Atezo-Bev therapy. The numbers and mean fluorescence intensity of the CTC subsets expressing CD90 and EpCAM molecules decreased in patients with partial response/stable disease, and increased in patients with progressive disease and were markedly correlated with overall survival. CD90(+) and EpCAM(+) CTCs may be candidate biomarkers for the early prediction of the treatment response and the overall survival of patients with HCC receiving Atezo-Bev therapy.

CD4+ T-cell subsets are associated with chronic stress effects in newly diagnosed anxiety disorders

AimPrior research has indicated a connection between CD4+ T cells and the development of anxiety, but the specific CD4+ T cell subsets linked to anxiety disorders remain uncertain. Our study seeks to investigate the relationship between distinct CD4+ T cell subsets and anxiety, as well as to explore whether CD4+ T cell subsets mediate the effect of chronic psychological stress on anxiety. Methods56 eligible matched participants were recruited in Peking Union Medical College Hospital. The diagnosis was made based on DSM-5 diagnostic criteria. The severity of anxiety and depression symptoms was assessed using the Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale, respectively. The Life Events Scale (LES) evaluated the chronic stress level. CD4+ T cell subsets were characterized using multiparametric flow cytometry. To assess the impact of CD4+ T cells on the effect of chronic psychological stress on anxiety, Partial Least Squares Structural Equation Modeling (PLS-SEM) analysis was employed. ResultsWe discovered fifteen notably distinct CD4+ T-cell subsets in anxiety disorder patients compared to healthy controls. Multiple linear regression analysis unveiled an association between anxiety severity and CD27+CD45RA− Th cells, CD27+CD28+ Tregs, and the total Life Events Scale (LES) score. The PLS-SEM analysis demonstrated that CD4+ T cell subsets and LES could explain 80.2% of the variance in anxiety. Furthermore, it was observed that CD27+CD28+ Th/Treg cells acted as inverse mediators of the effects of LES on anxiety (P = 0.031). ConclusionsDrug naïve anxiety disorder patients exhibited significant alterations in numerous CD4+ T-cell subsets. Specifically, the memory subset of CD27+CD45RA− Th cells and the naïve subset of CD27+CD28+ Treg cells were found to be independent factors associated with the severity of anxiety. Additionally, the CD27+CD28+ Th and Treg cell subsets played a significant mediating role in the influence of long-term psychological stress on anxiety.

Blood immune cell profiling in adults with longstanding type 1 diabetes is associated with macrovascular complications.

There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity. Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects. Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications. Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications.

Comprehensive Flow Cytometric, Immunohistologic, and Molecular Assessment of Thymus Function in Rhesus Macaques.

The critical importance of the thymus for generating new naive T cells that protect against novel infections and are tolerant to self-antigens has led to a recent revival of interest in monitoring thymic function in species other than humans and mice. Nonhuman primates such as rhesus macaques (Macaca mulatta) provide particularly useful animal models for translational research in immunology. In this study, we tested the performance of a 15-marker multicolor Ab panel for flow cytometric phenotyping of lymphocyte subsets directly from rhesus whole blood, with validation by thymectomy and T cell depletion. Immunohistochemical and multiplex RNA expression analysis of thymus tissue biopsies and molecular assays on PBMCs were used to further validate thymus function. Results identify Ab panels that can accurately classify rhesus naive T cells (CD3+CD45RA+CD197+ or CD3+CD28+CD95-) and recent thymic emigrants (CD8+CD28+CD95-CD103+CD197+) using just 100 µl of whole blood and commercially available fluorescent Abs. An immunohistochemical panel reactive with pan-cytokeratin (CK), CK14, CD3, Ki-67, CCL21, and TdT provides histologic evidence of thymopoiesis from formalin-fixed, paraffin-embedded thymus tissues. Identification of mRNAs characteristic of both functioning thymic epithelial cells and developing thymocytes and/or molecular detection of products of TCR gene rearrangement provide additional complementary methods to evaluate thymopoiesis, without requiring specific Abs. Combinations of multiparameter flow cytometry, immunohistochemistry, multiplex gene expression, and TCR excision circle assays can comprehensively evaluate thymus function in rhesus macaques while requiring only minimal amounts of peripheral blood or biopsied thymus tissue.

EBV T-cell immunotherapy generated by peptide selection has enhanced effector functionality compared to LCL stimulation.

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRβ) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches.

Cataloging circulating CD3+CD56+ NKT-like cells through a series of stimulating (NKG2D and DNAM-1) and inhibitory (PD-1, TIGIT, and Tim-3) immune checkpoint receptors in women diagnosed with precancerous cervical lesions or invasive cervical carcinoma

Persistent human papillomavirus infection is associated with the development of premalignant lesions that can eventually lead to cervical cancer. In this study, we evaluated the expression of activating (NKG2D, DNAM-1) and inhibitory immune checkpoints receptors (PD-1, TIGIT, and Tim-3) in peripheral blood NKT-like (CD3+CD56+) lymphocytes from patients with cervical carcinoma (CC, n = 19), high-grade lesions (HG, n = 8), low-grade lesions (LG, n = 19) and healthy donors (HD, n = 17) using multiparametric flow cytometry. Dimensional data analysis showed four clusters within the CD3+CD56+ cells with different patterns of receptor expression. We observed upregulation of CD16 in CC and HG patients in one of the clusters. In another, TIGIT was upregulated, while DNAM-1 was downregulated. Throughout manual gating, we observed that NKT-like cells expressing activating receptors also co-express inhibitory receptors (PD-1 and TIGIT), which can affect the activation of these cells. A deeper characterization of the functional state of the cells may help to clarify their role in cervical cancer, as will the characterization of the NKT-like cells as cytotoxic CD8+ T cells or members of type I or type II NKT cells.

Monitoring measurable residual disease in paediatric acute lymphoblastic leukaemia using immunoglobulin gene clonality based on next-generation sequencing

BackgroundAssessment of measurable residual disease (MRD) is an essential prognostic tool for B-lymphoblastic leukaemia (B-ALL). In this study, we evaluated the utility of next-generation sequencing (NGS)–based MRD assessment in real-world clinical practice.MethodThe study included 93 paediatric patients with B-ALL treated at our institution between January 2017 and June 2022. Clonality for IGH or IGK rearrangements was identified in most bone marrow samples (91/93, 97.8%) obtained at diagnosis.ResultsIn 421 monitoring samples, concordance was 74.8% between NGS and multiparameter flow cytometry and 70.7% between NGS and reverse transcription-PCR. Elevated quantities of clones of IGH alone (P < 0.001; hazard ratio [HR], 22.2; 95% confidence interval [CI], 7.1–69.1), IGK alone (P = 0.011; HR, 5.8; 95% CI, 1.5–22.5), and IGH or IGK (P < 0.001; HR, 7.2; 95% CI, 2.6–20.0) were associated with an increased risk of relapse. Detection of new clone(s) in NGS was also associated with inferior relapse-free survival (P < 0.001; HR, 18.1; 95% CI, 3.0–108.6). Multivariable analysis confirmed age at diagnosis, BCR::ABL1-like mutation, TCF3::PBX1 mutation, and increased quantity of IGH or IGK clones during monitoring as unfavourable factors.ConclusionIn conclusion, this study highlights the usefulness of NGS-based MRD as a routine assessment tool for prognostication of paediatric patients with B-ALL.

MRD in Philadelphia Chromosome-Positive ALL: Methodologies and Clinical Implications.

Measurable residual disease (MRD) is integral in the management of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). This review discusses the current methods used to evaluate MRD as well as the interpretation, significance, and incorporation of MRD in current practice. New molecular technologies have allowed the detection of MRD to levels as low as 10- 6. The most used techniques to evaluate MRD are multiparametric flow cytometry (MFC), quantitative reverse transcription polymerase chain reaction (RT-qPCR), and high-throughput next-generation sequencing (NGS). Each method varies in terms of advantages, disadvantages, and MRD sensitivity. MRD negativity after induction treatment and after allogeneic hematopoietic cell transplantation (HCT) is an important prognostic marker that has consistently been shown to be associated with improved outcomes. Blinatumomab, a new targeted therapy for Ph + ALL, demonstrates high efficacy in eradicating MRD and improving patient outcomes. In the relapsed/refractory setting, the use of inotuzumab ozogamicin and tisagenlecleucel has shown promise in eradicating MRD. The presence of MRD has become an important predictive measure in Ph + ALL. Current studies evaluate the use of MRD in treatment decisions, especially in expanding therapeutic options for Ph + ALL, including tyrosine kinase inhibitors, targeted antibody therapies, chimeric antigen receptor cell therapy, and HCT.

Transient Differentiation-State Plasticity Occurs during Acute Lymphoblastic Leukemia Initiation.

Leukemia is characterized by oncogenic lesions that result in a block of differentiation, whereas phenotypic plasticity is retained. A better understanding of how these two phenomena arise during leukemogenesis in humans could help inform diagnosis and treatment strategies. Here, we leveraged the well-defined differentiation states during T-cell development to pinpoint the initiation of T-cell acute lymphoblastic leukemia (T-ALL), an aggressive form of childhood leukemia, and study the emergence of phenotypic plasticity. Single-cell whole genome sequencing of leukemic blasts was combined with multiparameter flow cytometry to couple cell identity and clonal lineages. Irrespective of genetic events, leukemia-initiating cells altered their phenotypes by differentiation and dedifferentiation. The construction of the phylogenies of individual leukemias using somatic mutations revealed that phenotypic diversity is reflected by the clonal structure of cancer. The analysis also indicated that the acquired phenotypes are heritable and stable. Together, these results demonstrate a transient period of plasticity during leukemia initiation, where phenotypic switches seem unidirectional. Significance: A method merging multicolor flow cytometry with single-cell whole genome sequencing to couple cell identity with clonal lineages uncovers differentiation-state plasticity in leukemia, reconciling blocked differentiation with phenotypic plasticity in cancer.

Analysis of T follicular and T peripheral helper lymphocytes in autoimmune thyroid disease

PurposePeripheral helper T (Tph) cells have an important role in the induction of humoral immune responses and autoantibody production. Accordingly, it is feasible that this lymphocyte subset has a relevant role in the pathogenesis of autoimmune thyroid diseases (AITD). In this study we aim to analyze the levels and function of Tph cells in blood samples from patients with AITD.MethodsWe performed an observational study with cases and controls. Blood samples were obtained from nineteen patients with Hashimoto’s thyroiditis (HT), twenty-four with Graves’ disease (GD), and fifteen healthy controls. In addition, the levels of follicular T helper (Tfh) cells and Tph cells, the release of interleukin-21 (IL-21) by these lymphocytes and the number of plasmablasts were analyzed by multi-parametric flow cytometry analyses.ResultsIncreased percentages of Tfh and Tph lymphocytes were detected in patients with HT and GD. Furthermore, an enhanced synthesis of the cytokine IL-21 by these cells was observed. Accordingly, we detected significant higher percentages of plasmablasts in patients with GD, and these values tended to be also higher in HT patients. Moreover, significant positive associations were observed between the levels of Tfh or Tph and the number of plasmablast or anti-TSHR Ab titers in patients with AITD.ConclusionOur data suggest that Tph lymphocytes may have a relevant role in the pathogenesis of AITD.

Monitoring immunE DysregulAtion foLLowing Immune checkpOint-inhibitioN (MEDALLION): protocol for an observational cancer immunotherapy cohort study

BackgroundCheckpoint inhibitors (CPIs) are widely used in cancer treatment, with transformative impacts on survival. They nonetheless carry a significant risk of toxicity in the form of immune-related adverse events (IrAEs), which may be sustained and life-altering. IrAEs may require high-dose and/or prolonged steroid use and represent a significant healthcare burden. They mimic immune-mediated inflammatory diseases (IMIDs) but understanding of their pathogenesis is limited. The MEDALLION project aims to determine targetable mechanisms of immune dysregulation in IrAE development, employing an immune monitoring approach to determine changes in circulating and tissue resident cells of CPI recipients who do/do not develop them and assessing the contribution of the microbiome in parallel.MethodsMEDALLION is a non-randomised longitudinal cohort study aiming to recruit 66 cancer patient recipients of anti-PD1/PD-L1, anti-CTLA-4 or combination therapy. Eligible participants include those with malignant melanoma in the adjuvant or metastatic setting, mesothelioma and non-small cell lung carcinoma (NSCLC) treated in the metastatic setting. Comprehensive clinical evaluation is carried out alongside blood, skin swab and stool sampling at the time of CPI initiation (baseline) and during subsequent routine hospital visits on 6 occasions over a 10-month follow-up period. It is conservatively anticipated that one third of enrolled patients will experience a “significant IrAE” (SirAE), defined according to pre-determined criteria specific to the affected tissue/organ system. Those developing such toxicity may optionally undergo a biopsy of affected tissue where appropriate, otherwise being managed according to standard of care. Peripheral blood mononuclear cells will be analysed using multi-parameter flow cytometry to investigate immune subsets, their activation status and cytokine profiles. Stool samples and skin swabs will undergo DNA extraction for 16 S ribosomal RNA (rRNA) sequencing and internal transcribed spacer (ITS) gene sequencing to determine bacterial and fungal microbiome diversity, respectively, including species associated with toxicity. Stored tissue biopsies will be available for in situ and single-cell transcriptomic evaluation. Analysis will focus on the identification of biological predictors and precursors of SirAEs.DiscussionThe pathogenesis of IrAEs will be assessed through the MEDALLION cohort, with the potential to develop tools for their prediction and/or strategies for targeted prevention or treatment.Trial RegistrationThe study was registered on 18/09/2023 in the ISRCTN registry (43,419,676).

Effect of Prior ChAdOx1 COVID-19 Immunisation on T-Cell Responses to ChAdOx1-HBV.

There are varying data concerning the effect of prior anti-vector immunity on the T-cell response induced by immunisation with an identical vectored vaccine containing a heterologous antigen insert. To determine whether prior exposure to ChAdOx1-SARS-CoV2 immunisation (Vaxzevria®) impacts magnitudes of antigen-specific T-cell responses elicited by subsequent administration of the same viral vector (encoding HBV antigens, ChAdOx1-HBV), healthy volunteers that had received Vaxzevria® (n = 15) or the Pfizer or Moderna mRNA COVID-19 vaccine (n = 11) between 10 and 18 weeks prior were recruited to receive a single intramuscular injection of ChAdOx1-HBV. Anti-ChAdOx1-neutralising antibody titers were determined, and vector or insert-specific T-cell responses were measured by a gamma-interferon ELISpot and intracellular cytokine staining (ICS) assay using multiparameter flow cytometry. Participants were followed for three months after the ChAdOx1-HBV injection, which was well-tolerated, and no dropouts occurred. The baseline ChAdOx1 neutralisation titers were higher in the Vaxzevria® cohort (median of 848) than in the mRNA cohort (median of 25). T-cell responses to HBV antigens, measured by ELISpot, were higher on day 28 in the mRNA group (p = 0.013) but were similar between groups on day 84 (p = 0.441). By ICS, these differences persisted at the last time point. There was no clear correlation between the baseline responses to the adenoviral hexon and the subsequent ELISpot responses. As vaccination within 3 months using the same viral vector backbone affected the insert-specific T-cell responses, a greater interval after prior adenoviral immunisation using heterologous antigens may be warranted in settings in which these cells play critical roles.

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Immune responses associated with mpox viral clearance in men with and without HIV in Spain: a multisite, observational, prospective cohort study

Since the emergence of the global mpox outbreak in May, 2022, more than 90 000 cases have been diagnosed across 110 countries, disproportionately affecting people with HIV. The durability of mpox-specific immunity is unclear and reinfections have been reported. We aimed to compare mpox immune responses up to 6months after diagnosis in participants with and without HIV and assess their effect on disease severity and viral clearance dynamics. This study was embedded within a prospective, observational, multicentre cohort study of viral clearance dynamics among people with mpox in Spain (MoViE). We included women and men aged 18 years or older, who had signs of mpox, and reported having symptom onset within the previous 10 days at the moment of mpox diagnosis from three sex clinics of the Barcelona metropolitan area. Samples from skin ulcers were collected weekly to estimate the time to clear monkeypox virus (MPXV) from skin lesions. Blood samples were taken at diagnosis, 29, 91, and 182days later for immune analysis. This included quantifying IgG and IgA against three mpox antigens by ELISA, evaluating in-vitro neutralisation, and characterising mpox-specific T-cell responses using interferon γ detecting enzyme-linked immunospot (ELISpot) assay and multiparametric flow cytometry. Of the 77 originally enrolled participants, we included 33 participants recruited between July 19, and Oct 6, 2022. Participants without HIV (19 [58%] participants) and participants with HIV (14 [42%] participants) had similar clinical severity and time to MPXV clearance in skin lesions. Participants with HIV had a CD4+ T-cell count median of 777 cells per μL (IQR 484-1533), and 11 (78%) of 14 were virally suppressed on antiretroviral therapy. Nine (27%) of 33 participants were age 49 years or older. 15 (45%) of 33 participants were originally from Spain, and all participants were men. Early humoral responses, particularly concentrations and breadth of IgG and IgA, were associated with milder disease and faster viral clearance. Orthopoxvirus-specific T cells count was also positively correlated with MPXV clearance. Antibody titres declined more rapidly in participants with HIV, but T-cell responses against MPXV were sustained up to day 182 after diagnosis, regardless of HIV status. Higher breadth and magnitude of B-cell and T-cell responses are important in facilitating local viral clearance, limiting mpox dissemination, and reducing disease severity in individuals with preserved immune system. Antibodies appear to contribute to early viral control and T-cell responses are sustained over time, which might contribute to milder presentations during reinfection. Fundació Lluita contra les Infeccions, IrsiCaixa, and Consorcio Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación e Universidades.

The effects of postoperative targeted immunotherapy on peripheral blood cytokines and immune cell profile in lung cancer patients.

Cytokines and cell subsets are important components of the tumor microenvironment. Previous research has revealed that there are differences in cytokines and cell subsets in the peripheral blood of lung cancer (LCA) patients before and after eradication. The purpose of this study is to explore the monitoring value of cytokines and cellular subpopulations as biomarkers in post-immunotherapy monitoring of patients with LCA after surgery. We conducted a case-control study using double-antibody sandwich magnetic microsphere flow cytometry with immunofluorescence technology and fluorescent monoclonal antibody multiparameter flow cytometry to detect differences in peripheral blood cytokines and cell subsets between LCA patients after immunotherapy and healthy controls. Our research results show that there are differences in the levels of IL-4, IL-6, IL-10, IL-17, IFN-γ, TNF-α in the peripheral blood of LCA patients (n=70) after immunotherapy compared to the healthy controls (n=55) (P<0.05), and there are differences in 10 cell subgroups including DP T Cells, AT cells, and NLR in the peripheral blood compared to the healthy controls (n=35) (P<0.05). Further analysis revealed significant differences in the detection data of IL-6, IL-10, IFN-γ, CD56dim NK cells, Total B cells, Total NE cells, CD15+M cells, and NLR between LCA deceased patients (n=25) and LCA surviving patients (n=27) during the same period (P<0.05). The continuous monitoring of cytokines and cell subsets is far more valuable than a single-time test, as abnormal fluctuations in the data of cytokines and cell subsets are often associated with poor prognosis. In addition, IL-6 and NLR showed the strongest discriminative ability between postoperative immunotherapy-treated LCA patients and healthy controls, with AUC values of 0.840 and 0.822, respectively. There was a significant association between IFN-γ and distant metastasis in LCA (P<0.05), as well as between CD56dim NK cells and lymph node infiltration (P<0.05). This research results support peripheral blood cytokines and cell subsets as biomarkers for monitoring the postoperative immune status and predicting the prognosis of LCA patients after immunotherapy. The continuous monitoring of cytokines and cell subsets is far more valuable than a single-time detection.

Measurable Residual Disease Analysis by Flow Cytometry and Correlation With Molecular Measurable Residual Disease in Acute Promyelocytic Leukemia: A Real-World Prospective Study.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia distinguished by its rapidly progressive and fatal clinical course. Measurable/minimal residual disease (MRD) monitoring is vital for the prognosis and clinical management of acute myeloid leukemia. To examine the immunophenotypes of the residual leukemic cells, evaluate the performance of multiparametric flow cytometry (FCM) measuring MRD, and compare it with molecular monitoring in patients diagnosed with APL. Two hundred seventy-seven patients with APL were enrolled. Immunophenotypes were prospectively analyzed by a 1-tube-10-color antibody panel via FCM. MRD of APL with PML::RARα was detected by real-time quantitative polymerase chain reaction (RQ-PCR). The clinical value of MRD as an indicator of survival was also examined. APL showed 5 distinct patterns of residual leukemic cells, based on CD45 and side-scatter scattergram, all with CD9 positivity and a previously unrealized loss of CD117. FCM-based MRD evaluation showed a concordance rate of 87.7% with PCR. At the end of the consolidation therapy, MRD measured by both PCR and FCM could differentiate patients with longer and shorter overall survival (OS) (P = .04 and P = .03, respectively). Patients with APL variant had a shorter OS than patients with APL who harbored PML::RARα (P < .001). CD9 is a reliable marker to differentiate residual leukemic cells from normally differentiating myeloid cells. FCM demonstrated a high comparability to PCR-MRD and an excellent performance in predicting OS, and thus could potentially be used as a routine indicator in the clinical management of patients with APL.

Indeterminate measurable residual disease by multiparameter flow cytometry is associated with an intermediate risk of clinical relapse in adult patients with acute leukaemia

Measurable residual disease (MRD) is useful for prognostication and for monitoring response to treatment in patients with acute leukaemia. MRD by multiparametric flow cytometry (MFC-MRD) utilises the leukaemia-associated immunophenotype (LAIP) and difference from normal (DfN) strategies to identify the leukaemic clone. Difficulties arise when the LAIP overlaps with normal regeneration, there is clonal evolution, or when the abnormal clone population is exceptionally small e.g., <0.01% of CD45+ cells. Such cases are reported as 'indeterminate'; however, there is little international consensus on this reporting. The relationship between clinical outcomes and indeterminate MFC-MRD is unknown. Here we determine the rate of indeterminate MFC-MRD reporting, its relationship to concurrent molecular MRD results when available, and to clinical outcomes to 12 months. We performed an internal audit of all adult testing for MFC-MRD between January and December 2021. A total of 153 consecutive patients with a diagnosis of acute leukaemia were included. Successive MFC-MRD results and clinical outcomes were recorded over a 12-month period from time of inclusion into the study. In total, 460 MFC-MRD tests from 153 patients were reviewed and 73 (16%) MFC-MRD tests from 54 (35%) patients were reported as indeterminate. The majority (70%) were at low levels between 0.01-0.1% of CD45+ cells. Compared to patients with a negative result, acute myeloid leukaemia (AML) was more frequent in patients who had an indeterminate MFC-MRD (70% vs 36%), and B-cell acute lymphoblastic leukaemia was less common (20% vs 55%). In patients with indeterminate MFC-MRD results, one-third had received either chemotherapy or allogeneic haemopoietic stem cell transplant (aHSCT) within the preceding 3 months. Agreement between MFC and molecular MRD testing was low. Patients with indeterminate MFC-MRD had leukaemia relapse rates below patients with a positive MFC-MRD, but greater than those with negative MFC-MRD (positive 33% vs indeterminate 21% vs negative 8%, p=0.038). Overall, these findings indicate that indeterminate MFC-MRD results are more common in adults with AML and also in those who have received chemotherapy or aHSCT within the previous 3 months. We report for the first time that indeterminate MFC-MRD is a finding of potential clinical significance, which associates with a numerically higher median relapse rate within 12 months when compared to a negative MFC-MRD result.

Immune-phenotyping of peripheral blood mononuclear cells from patients with early triple-negative breast cancer: Identification of a systemic immunosuppressive CD3+ CD4+ CD39+ Treg subset.

3040 Background: Cells of the immune system and malignant cells interact in the tumor microenvironment, influencing response to treatment and survival. The current study was undertaken to assess the prevalence of these immune cells systemically in patients with early triple-negative breast cancer (TNBC). Methods: Multi-parameter flow cytometry was used to examine the percentages and phenotypes of cytotoxic T cells, natural killer (NK) cells, monocyte subsets, and regulatory T cell (Treg) subsets in the peripheral blood of 19 TNBC patients (pts) and 10 controls (cnt). Multicolor flow cytometry was carried out using DURAClone IM phenotyping (basic and Treg tubes) with a CytoFLEX Flow Cytometer (Beckman Coulter, California, USA), and data analyzed with FlowJo v 10.10.0 software (BD Life Sciences, USA). The Mann-Whitney U-test was used to compare non-parametric data where appropriate. Results: Significant increases in the percentages of immunosuppressive CD3+ CD4+ CD39+ Tregs cells in the setting of a significant decrease of CD19+ B cells were observed in pts with TNBC (Table).Tumour size, nodal status, age and ki-67 did not correlate with the percentages of any type of circulating immune cell. Conclusions: TNBC is associated with severe immunosuppression. In this context, percentages of circulating CD19+ B cells were significantly lower, while CD3+ CD4+ CD39+ Tregs were significantly higher in early TNBC pts. The peripheral blood immunome of TNBC pts identified a subset of immunosuppressive Tregs. A study is ongoing to identify the prognostic and predictive values of this systemic subset of Tregs for pCR in early TNBC pts undergoing neoadjuvant chemotherapy. [Table: see text]