Programme

Abstract: The course will cover the technical aspects of flow cytometer and sample preparation for the detection and analysis of particles below 200nm, emphasizing the use of appropriate controls. Also, we will show the practical issues of Extracellular Vesicles Immunostaining. The course will have a short practical demo.

Abstract: The aim of this course is to present examples of B and T CLPD that illustrate the identification of pathological lymphocytes in different types of samples. It is recommended to both beginners in the field and more experienced flow cytometrists.

The cases will present a wide range of complexity, from straight forward peripheral blood identification to small clusters in cerebrospinal fluid.

The contents will cover the recommended pathway, from screening to strategies applied to B and T cells to evaluate clonality, followed by a more comprehensive panel to characterize the abnormal population(s).

Interaction between the faculty and the audience will be encouraged.

Abstract: The analysis of the huge amount of data currently generated by flow cytometry experiments is not trivial, with specialized tools and skills becoming critical to help the interpretation of large batches of information without losing the biologic sense in both clinical and basic research fields. The application of conventional analysis strategies to massive cytometry data (Cytomics) analysis is extremely time-consuming, introduces a big subjective bias and does not allow to explore the full diversity potentially revealed by a multi-color design. In the last years, several strategies have been developed in order to extract a maximum of first-class information from cytomic data.

In this workshop, we will firstly learn about semi-automated pre-processing, quality control and data cleaning to avoid artifacts during staining and acquisition (data cleaning, doublets and dead cell removal) or batch effects (transformation, normalization). Then several non-supervised data analysis strategies (clustering, trajectories, principal component analysis) and representation (dimensional reduction) or interpretation (trajectories, modules). Finally, we will test these approaches on real-life using high-content cytometry data and different user-friendly softwares.

Abstract: In this workshop we will cover the rules of systematically designing a multicolor immunophenotyping panel for polychromatic (conventional) and spectral cytometers. We will focus on both theoretical and practical aspects that are relevant for panel design and highlight the differences between the different technologies. Furthermore, we will provide a step-by-step guidebook, discuss which controls are needed and how to assess the final panel performance. While part of the workshop will be lectures, we will have an interactive session during which we will go through several examples of panels with plenty of discussion.

The overarching goal is to provide all participants with the tools they need to successfully design and troubleshoot their multicolor flow cytometry experiments irrespective of the platform used.

Abstract: Mesurable residual disease (MRD) is a strong prognostic factor in patients with BCP-ALL and is used to guide treatment. MRD detection by multiparametric flow cytometry has evolved in the last decade to improve sensibility and to increase standardization. So, technical modifications of sample processing have allowed reaching higher sensitivity (≥10-5), comparable to molecular biology methods, and the standardization has allowed to the development of automated methods to identify and classify cells improving the speed and quality of the analysis. Antigen combinations for MRD detection in BCP-ALL are based in CD19 antigen with the inclusion of markers aberrantly expressed in blasts cells. Targeted therapies, mainly anti-CD19, had shown high efficacy in the treatment of BCP-ALL patients, but they may impact on the performance of flow cytometry test as CD19-negative relapses are observed in a proportion of patients.

Therefore, the knowledge of normal B-cell maturation, the expression of other B-cell markers in B-cell blasts, the risk factors for CD19-negative relapses, and the use of alternative combinations for MRD detection in patients receiving targeted therapy are open questions.

The course will review the phenotypic profile of immature B-cells, technical aspects of sample processing, antigen combinations, standardization of test, and analysis of samples in patients receiving either standard treatment or targeted.

Abstract: Anti-tumor immunotherapies have achieved remarkable successes in the treatment of cancer, but major challenges remain. An inherent weakness of current treatment approaches is that the therapeutically targeted pathways are not restricted to tumours, but are also found in other tissue microenvironments.
Despite great efforts to define inflammatory processes in the tumour microenvironment, our understanding of tumour-unique immune alterations is limited by a knowledge gap regarding the immune cell function in inflamed human tissues. Here, in an effort to identify such tumour-enriched immune alterations, we used complementary single-cell analysis approaches to interrogate the immune infiltrate in human head and neck squamous cell carcinomas and site-matched non-malignant, inflamed tissues.
Our analysis revealed a large overlap in the composition and phenotype of immune cells in tumour and inflamed tissues. Computational analysis identified tumour-enriched immune cell interactions, one of which yields a large population of regulatory T cells (Tregs) that is highly enriched in the tumour and uniquely identified among all haematopoietically-derived cells in blood and tissue by co-expression of ICOS and IL-1 receptor type 1 (IL-1R1). We provide evidence that these intratumoural IL-1R1+ Treg cells had responded to antigen recently and demonstrate that they are clonally expanded with superior suppressive function compared with IL-1R1- Treg cells.
Overall, our work highlights how combining several single-cell techniques with subsequent validation experiments can provide critical new insight into the function of immune cells in human tissues.

Abstract: There are a wide range of diseases that are caused by dysregulation of the immune system. This imbalance can lead to an exacerbated response of certain immune populations and a severe inflammatory process. The existing arsenal of biological therapies that can block or inhibit these immune processes at different levels is extensive, but in most cases, there is no precise diagnosis to identify the appropriate target for each patient. In the case of pediatric patients this limitation is even greater, given the small volume of blood that can be safely obtained from these patients. For this reason, advanced analysis protocols are needed to enable a correct diagnosis and to correctly prescribe these therapies. Advances in immunological knowledge and in the technologies that allow its study, open the possibility of making a more precise diagnosis and developing personalized medicine treatments.
Taking advantage of the multi-parameter flow cytometry technology, our researchers in the Laboratory of Immune-regulation at the IISGM have developed a protocol for exhaustive immune analysis in pediatric samples. This technique allowed us to analyse frequencies and absolute counts of more than 100 subpopulations of lymphocytes, granulocytes, monocytes and NK cells in less than 0.5 ml of blood. This tool has been employed with success for the diagnosis and treatment of an infant in serious danger of death caused by a rare dermatologic disease.
The thorough study of the immune system employing our protocol allowed us to identify in the patient an immune profile characterized by very high levels of both Th17 cells and IL-17A producing CD4+ T cells. In consequence, the patient was treated with a new biological treatment in order to diminish the inflammation. After the treatment, the patient showed remarkable clinical improvements, notably a weight gain, which has rescued her from grave danger of death.
The use of flow cytometry allows the identification of the immune profile of pediatric patients with immune related diseases. This precision diagnosis could provide insight of the severity of the inflammation; the potential existing treatments that could help improve the symptoms and, therefore, the quality of life of these patients.

Abstract: TranspoCART is a project conceived to develop an innovative CAR-T cell therapy for lymphoma patients based on a viral vector-free gene-transfer strategy. A Sleeping Beauty transposon minicircle (SBmC) carrying a CAR consisting of the 4-1BB CAR endodomain, an anti-CD19 ScFv and a truncated EGFR -as a safety switch- was designed for this clinical trial. Our optimized manufacturing protocol showed transposition efficacies ranging between 30 and 60% in both healthy donors and patients’ T cells, with efficient cell expansion rates (between 29.6x and 31.8x), compatible with clinical applications. Extensive in vitro characterization of TranspoCART19 cells showed an enriched stem-cell memory/central memory phenotype, no signs of cell exhaustion and high level of specific cytotoxicity against tumour cell lines and primary tumour cells expressing CD19. No detectable levels of both the SBmC and the transposase protein were found at the end of the expansion period. NALM6 xenograft models using immunodeficient NSG mice were used to evaluate the in vivo antitumoral efficacy of TranspoCART19 cells. Treated animals showed an improved survival compared to untreated mice, similar to the one observed in mice treated with lentivirally-produced CAR-T cells. Biodistribution and toxicity studies revealed that TranspoCART19 cells administration is safe. Additionally, cetuximab administration in CAR-T cell treated NALM6 xenograft mice depleted TranspoCART19 cells, proving its efficacy. Finally, two independent GMP facilities carried out TranspoCART19 cell manufacturing GMP validations, and showed that the manufacturing procedure is highly efficient and reproducible. These encouraging results support the initiation of a clinical trial with the TransproCART19 cell product.

Abstract: There are increasing and solid records supporting the clinical use of Hematopoietic Stem and Progenitor Cells (HSPC) ex vivo gene therapy to treat several, otherwise uncurable, monogenic diseases. Pioneer trials relied on tamed viral components acting as vectors which provided robust and proficient gene transference at the expense of genotoxicity. Recent strategies have upgraded these tools to safer recombinant vectors or shifted towards implementation of gene editing. The later provides accurate control on the chromosome and locusthat is going to be altered, and the number of therapeutic gene copies that can be inserted. However, it’s novelty requires a thorough characterization of every new tool and clinical application in terms of safety and efficiency.
We aim to use tissue resident macrophages and circulating myeloid cells originated from gene-edited HSPCs for paracrine cross-correction of monogenic diseases, such as Pompe Disease. Therefore, we focused in finding a CRISPR/Cas9 guide that efficiently targeted an intronic region from a gene expressed upon myeloid progenitor commitment: CX3CR1. We demonstrated that our guide efficiently targets the 4th intron while it does not disturb physiological expression of the host gene. Furthermore, we tested this locus as a safe harbor by introducing reporter cassettes within. Using a promoter-less or a strong viral promoter configuration, we documented efficient integration of our cassette and unaltered CX3CR1expression in edited lymphoid and myeloid cell lines. Interestingly, we found increased levels of CX3CR1 after editing cord blood CD34 cells in vitro, along with a skewed reporter expression inclined towards differentiated myeloid lineages.
Preliminary evaluation of gene-edited-HSPC transplantation into a NSG mice suggests that their safety and biodistribution justify further exploratory and translational evaluation of this approach that could provide a clinical platform to treat Pompe Disease.

Background: Inflammatory bowel diseases (IBD) consist of chronic inflammatory disorders that mainly involved the intestinal mucosa. Current treatments for IBD have significant side effects as well as a lack of response in a significant number of patients. Mesenchymal stem/stromal cells (MSCs) may be a therapeutic alternative due to their immunomodulatory and tissue regeneration properties. Among the mechanisms participating in the inhibition of intestinal inflammation, the induction of regulatory immune responses has been involved in the beneficial effects of MSC-based therapy.

Aims: Characterization of myeloid populations associated with the beneficial long-term effects of MSC therapy in a clinically relevant model of colitis.

Methods: MSCs were infused into dextran sulphate sodium (DSS)-induced colitic mice during the induction phase of the disease. Following a latency period, mice were re-challenged with DSS. Myeloid populations were analysed by flow cytometry at different time points in different organs and tissues.

Results & Conclusion: After the latency period, increased levels of Ly6G+CD11b+ myeloid cells were noticed in the peritoneal cavity (PC), spleen (SP), and bone marrow (BM) in MSC-treated compared to untreated colitic mice. Ly6G+CD11b+ cells from PC co-expressed F4/80, CD206, and CX3CR1, markers of alternative-activated macrophages from BM origin. Based on the expression of Ly6C and Ly6G markers, the Ly6G+CD11b+ cells from SP were defined as myeloid-derived suppressor cells and, from BM, immature myeloid cells. These phenotypes were also confirmed by the intracellular expression of iNOS, Arg-1, IL-10, and by inhibition of T-cell proliferation. During the 2nd challenge and in parallel to a reduction in the disease activity index, an increase of IL-10+Arg1+F4/80+Ly6G+CD11bdim regulatory cells and a decrease of GM-CSF+TNFα+IL-6+iNOS+IFN+Ly6G+/dimCD11b+ inflammatory cells were observed in the colon in MSC-treated compared to untreated colitic mice. These findings obtained by flow cytometry technology suggest that MSC-based therapy can imprint innate immune memory-like responses that confer sustained protection against intestinal inflammation in the long term.

Background: Anti-PD-1/PD-L1 immunotherapy (IT) is indicated in non-small cell lung cancer (NSCLC) stages III-IV without driver gene mutations, and has improved the survival of patients. However, the 5-year survival rate is still only 26%. Myeloid-derived suppressor cells (MDSCs) are circulating cells that express PD-L1 and can infiltrate and proliferate in the tumor environment, inducing immunosuppression.

Aims: In this work, we have studied the conformational changes of PD-L1 in MDSCs and their potential value in assessing prediction of escape from IT in NSCLC patients receiving checkpoint inhibitors.

Methods: Peripheral blood from stage III-IV NSCLC patients (n=37) was processed immediately prior to IT, using minimal sample perturbation protocols. Briefly, blood was stimulated with phorbol esters (PMA), which resulted in conformational changes of PD-L1 and increased epitopic target accessibility. MDSCs were identified as HLA-DRlo/-CD33+CD11b+ and analyzed on the Attune™ NxT flow cytometer (Thermo Fisher). Comparative analysis of the conformational dynamics was assessed using the PD-L1 index (PD-L1i), obtained by calculating the difference of PD-L1 mean fluorescence intensity between stimulated PD-L1+MDSC and non-stimulated MDSCs, divided by two times the standard deviation of the non-stimulated populations.

Results & Conclusion: Responders and non-responders were classified according to PD-L1i and progression-free survival (PFS). With PD-L1i >5.88, disease progression occurred in 58.33% patients, showing significant differences when comparing with a PD-L1i of ≤5.88, where only 7.69% underwent disease progression (p-value = 0.0042 and 95% CI = 3.17-NA). Overall survival (OS) was significantly worse in the group with higher PD-L1i (58.33% vs 23.07% deaths, respectively; p-value = 0.035; 95% CI = 7.53-NA). The conformational dynamics of PD-L1 in MDSCs has been shown to be a good biomarker for predicting NSCLC progression in patients receiving anti-PD-1/PD-L1 IT. Importantly, this approach uses a non-invasive and rapid flow cytometry assay with a promising biomarker for the evaluation of immunotherapy escape.

Background: Malignant melanomas are characterized by a high potential to metastasize and evade immune surveillance. Immunotherapy has revolutionized treatment in melanoma, still, a significant fraction of melanoma patients displays intrinsic or acquired immune resistance. Thus, a pending question in the field is to understand how immune resistant environments are generated. We have recently identified MIDKINE (MDK) as promoter of melanoma metastasis, and a driver of resistance to immunotherapy thought the rewiring of macrophages.

Aims: We aimed to further characterize the roles of MDK in the regulation of the immune system, specifically dendritic cells (DCs). DCs are professional antigen-presenting cells whose main function is to regulate the establishment of efficient anticancer responses. However, aggressive tumors may impair DC function. The precise aims of this project were: i) assess whether MDK interferes with DC differentiation and ii) function. iii) Further study the effects of MDK on antigen presentation and in consequence, its impact on the immune response against melanoma. iv) And finally, to validate our results in melanoma patients.

Methods: In this project, we combined transcriptomic and computational analyses, flow cytometry immunophenotyping and in vivo and in vitro functional analyses to comprehensive profile dendritic cell populations rewired by MDK.

Results & Conclusion: Here, we have unveiled the impact of MDK on DCs at four levels: MDK reduces DC infiltration in melanoma and lymphoid organs by blocking their differentiation at the bone marrow; MDK inhibits DC-mediated antigen presentation by blocking DC key functions. Consequently, MDK-educated DCs trigger a dysfunctional T response that results in an increased resistance to ICB. Finally, we have identified an MDK-educated DC gene signature that stratifies melanoma patients with differing overall survival and deficient response to ICBs. In conclusion, MDK hinders anticancer immune responses by interfering with DC biology facilitating resistance to immunotherapy. Together our results may hold translational relevance for therapeutic intervention in melanoma.

Abstract: Non-human primates (NHP) are essential models for the development of therapies and vaccines. They also represent the only animal model for studying the physiopathology of HIV infection. One major anatomical reservoir of HIV in humans and of SIV in NHP are lymph node (LN) B cell follicles (BCF) where HIV/SIV persists through replication in follicular helper T cells. Natural hosts of SIV, such as African green monkeys (AGM) do not progress to disease. We demonstrated that natural killer (NK) cells mediate a strong control of SIVagm infection in secondary lymphoid organs. In order to study the underlying mechanisms of this potent NK-cell mediated control in SIVagm-infected AGMs, we developed a broad range of tools to analyze NK cells in distinct NHP species, and in particular in AGMs and cynomolgus macaques. We determined combinations of cell-surface markers that allow to identify NK cells in distinct NHP tissues, as well as protocols for sorting NK cells from blood and tissues of distinct NHP species and for NK cell in situ imaging analyses. This allowed us to identify a unique CXCR5 and IL-15 dependent NK cell localization within BCF and the expansion of terminally differentiated NK cells in secondary lymphoid organs of SIVagm-infected AGMs. These terminally differentiated NK cells displayed an adaptive transcriptional profile with increased MHC-E restricted cytotoxicity in response to nonamer peptides derived from the Env leader sequence of SIV. In contrast, such NK cell differentiation was found to be impaired in SIVmac infection of macaques (MAC). Given the potential importance of this HLA-E/peptide/NK axis in the control of SIVagm infection, we currently study the role of the SIVagm nonamer Env leader sequence peptide in vivo. Furthermore, we compare the NK cell profiles identified in the NHP to that in a human cohort of early anti-retroviral treated people living with HIV.  

Abstract: T cells are chief white blood cells that protect our organism from infectious diseases and cancer. As we age, T-cells become less effective in fighting infection and they respond less well to vaccines. T cells are generated in the thymus, which makes this organ an essential element in establishing T cell immunity. Given the present interest in T cell-mediated immunotherapy, there is a growing need to understand how T-cell development is controlled in the thymus. The development of immunologically competent T cells is promoted by inductive microenvironments formed by thymic epithelial cells (TECs). Still, how TECs select functionally diverse T cells capable of responding to foreign threats is not fully comprehended. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 is a well-recognized mediator of autolysosome maturation. I will discuss experimental evidence that points to a new role for LAMP-2 in T cell differentiation. Genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing. These alterations in TECs led to a profound reduction in CD4 T cells and a restriction in the diversity of their TCR repertoire, limiting the immune response of the host to pathogenic Listeria. This study reveals a new molecular axis that regulates the production of T cells and that, as such, is deterministic of protective anti-microbial T cell responses.

Background: Flow cytometry is a widely used technique to diagnose several immune-related diseases in humans in peripheral blood (PB). However, in murine models, it presents several limitations, such as the meager amount of PB available , narrow variety of commercial antibodies and fluorochromes and high inter-subject variability in immune populations

Aims: Define novel cytometry panels to analyze extensively immune cell populations in mice from 100uL of PB and test the panels in lipopolysaccharide (LPS) lung inflammation model.

Methods: Twelve C57BL/6J mice were randomized into two groups: LPS, (n=6) undergoing intratraqueal inoculation of LPS (5mg/kg) in 100 µL of saline; and control (n=6). Fresh blood was collected from the maxillary venous sinus on day 1 pre-inoculation, and days 1 and 3 post-inoculation (p.i). Whole blood was labeled with 27 antibodies distributed in two panels, and acquired in a 16 channels Flow Cytometer. Peripheral populations were assessed overtime, overlapping innate and adaptive immune subsets.

Results & Conclusion: LPS-induced inflammation was rapidly observed at day 1 p.i, by an increase of granulocytes frequency and numbers, correlated with the mice's weight loss. Interestingly, a decline in the pro-inflammatory Th1 and NK subsets was observed in the same group. Rare cellular populations have shown high variability, likely due to very low cells/µL. For all these reasons, our panels allowed us to analyze most physiological peripheral immune cellular subsets from a minimal amount of whole blood and can permit us to detect immune changes due to rapid inflammation induction. This longitudinal study could help in determining the best timing for future experimental designs, using minimal blood and with a minimal impact on the animal status, avoiding unnecessary animal sacrifices and thus, reducing sample size

Background: Current evidence establishes a causality relation between inflammation, and the development of many pathologies, such as CDI, one the most common and severe nosocomial infections. Its prognosis and recurrence prediction are still an unsatisfied need, just based on clinical criteria which are not sufficiently accurate. Therefore, the determination of immune profile could throw relevant information about the progression and gravity of CDI.

Aims: To evaluate the evolution of the immunological profile in peripheral blood (PB) on a mouse model of CDI over time.

Methods: CDI was induced in 20 female C57BL/6 mice following Cussó et al. (DOI: 10.1007/s11307-019-01408-4) protocol. Mice were divided into mild (ribotype 001, low toxin production) and severe (ribotype 027, high toxin production) groups. Clinical signs score (CSS) was obtained for each animal over time. PB samples were collected at basal, pre-infection (0d), post-infection (+2d) and recovery (+15d); and incubated with 2 multiparametric antibodies panels to analyse lymphoid and myeloid populations. Then erythrocytes were lysate and samples were acquired by flow cytometry using a MACSQuant® Analyzer 16 Flow Cytometer, and the results were analysed using Kaluza Analysis Software.

Results & Conclusion: Both groups showed an increase of activated and effector CD4+ T cells post-infection which recovered after 15 days. Also, B lymphocytes values raised progressively according to CDI progression, in particular plasmablasts percentages were higher at recovery, while monocytes (Mo) and dendritic cells (DC) were decreased post-infection, compared to basal levels. However, severe group at recovery presents increased values of activated CD8+ T cells, Th17 cells and pro-inflammatory Mo and DC compared to mild group, indicative of more intense inflammatory responseThis result was consistent with the CSS since severe group failed to recover. In conclusion, our flow cytometry strategy allows us to detect PB immunological changes on CDI mouse models that can discriminate between CDI severity degrees.

Background: Fasting during chemotherapy treatment enhances anti-tumor efficacy in mice and humans.

Aims: In turn, fasting shows a marked sexual dimorphism in its metabolic effects, but little is known about the influence of sex in the benefits of fasting on chemotherapy.

Methods: B16F10 melanoma or MC38 colon carcinoma cells were engrafted in C57BL/6JOlaHsd mice and subjected to two cycles of chemotherapy (doxorubicin or oxaliplatin) plus 48 hour fasting. This protocol was also performed in castrated males and in females implanted with testosterone. For immunophenotyping, inguinal lymph nodes, peripheral blood and tumors were analysed. For classical cytometry we generated four different panels: lymphoid (10 markers), subtypes of CD4 (8 markers), exhausted CD8 (6 markers) and myeloid (9 markers:). For high-dimensional analysis, two panels were generated: B16F10 panel (17 markers) and MC38 panel (19 markers).

Results & Conclusion: Here, we showed that fasting can enhance the anti-tumor effects of certain chemotherapeutic agents in a sex-specific manner in B16F10 or MC38 allografts. Fasting strongly improved the anti-tumor B16F10 effects of doxorubicin only in males, and testosterone levels were critical for this fasting-mediated benefits. In turn, B16F10 tumors treated with oxaliplatin exhibited a beneficial effect in both sexes, more prominent in males. In contrast, fasting was beneficial in both sexes in the treatment of MC38 allografts plus oxaliplatin. Fasting led to different immune responses depending on the tumor and chemotherapeutic agent used. In B16F10 tumor, fasting increased anti-tumoral NK and NKT infiltration in males and increased pro-tumoral exhaustion of CD8 in females. In MC38, fasting increased anti-tumoral effector CD8 T cells and type I CD4 cells, and reduced tumor-associated macrophages in both sexes. These findings highlight the potential for sex-specific differences in response to fasting and the importance of considering tumor type and chemotherapeutic agent when studying the effects of fasting on the immune system and cancer treatment.