Immunotherapy Lecture Notes PDF

Lecture 1 – introduction/ Single cell technologies, modalities Immunotherapy = type of treatment in which the immune system is modulated (enhanced or suppressed). There is emphasis on cellular heterogeneity, particularly in solid tumors, where a small fraction of molecularly distinct cells such as...

Lecture 1 – introduction/ Single cell technologies, modalities Immunotherapy = type of treatment in which the immune system is modulated (enhanced or suppressed). There is emphasis on cellular heterogeneity, particularly in solid tumors, where a small fraction of molecularly distinct cells such as cancer stem cells contribute to tumor progression and therapeutic resistance. - Single cell techniques allow you to study rare cells which is important in tumors for example to understand their biology. o Single cell applications ▪ Immune monitoring and immunophenotyping: identification and tracking of the immune cell populations involved in tumor responses ▪ Cell migration and tumor killing: tracking how immune cells move and interact with tumors ▪ Structural and functional characterization: using advanced imaging techniques to understand cellular and subcellular dynamics in real-time - Examples of technologies: o Cytometry and sorting which includes high-dimensional flow cytometry and live-cell sorting o Microscopy: Advanced imaging techniques like confocal microscopy, super-resolution imaging (light- sheet microscopy), and live-cell imaging. o An emerging method is multidimensional data analysis which is key for handling large datasets generated from cytometry and microscopy The life sciences are now moving towards the trans-omics field in which we can integrate all the information of each level Cytometry = a technology that measures properties of single cells. It involves the quantitative analysis of physical and chemical properties of cells or other biological particles, typically at the single-cell level. - Parameters it measures: o The cell surface markers are measured by fluorescence o Light scatter measures the cell size and shape o Using fluorescent dyes and antibodies it can also measure intracellular molecules o And other properties of cell - It provides correlated data that links different population profiles. Flow cytometer = technology used to measure properties of single cells. It is a system that measures and analyzes signals resulting from flowing particles in a liquid stream through a beam of light. It does not always deal with cells but also chromosomes, vesicles, latex beds, or any particles that can be suspended in a fluid. - The work comes from the Greek “cyto” (Cell) and “metry” (measurements). Early cytometers, known as FACS (fluorescence activated cell sorters) were designed to sort cells based on fluorescence. They were the first ever instruments made from the principle of separating cells without measuring what type of cells they really are. - What can we do with a flow cytometer: o Count cells o Distinguish between biological and non-biological o Separates live from dead o Evaluate 105 to 107 particles in less than a minute  o You can measure particle-scatter, autofluorescence, or fluorescence associated to other reagents (antibodies and non-antibodies) o Sort single particles/cells for subsequent analysis o But also, more advanced capabilities: ▪ Obtain images ▪ Measure rare metals ▪ Measure the entire spectrum of light Now, there are more technologies that use the principle to analyze one cell at a time but looking at different particles: - Drop-seq dingle cell analysis This is a platform to profile thousands of cells by encapsulating them into individual droplets. Uniquely barcoded mRNA capture microparticles and cells are co-confined through a microfluidic device within the droplets where they undergo cell lysis and RNA hybridization (https://pubmed.ncbi.nlm.nih.gov/31028633/) - Imaging mass spectrometry (CyTOF) = measure 40 parameters of a single glass slide by using rare metal isotopes instead of fluorochromes for labeling, allowing for highly multiplexed cellular analysis. You measure immunophenotype in tissue to measure special connections In the field of multiplex immunohistochemistry there is a lot of fast improvement of technologies used --> the limits of cytometry are getting blurred! - There are now more advanced techniques o CODEX: imaging technique allowing multiplexed imaging of tissue by cycling through different markers o MIBI (Multiplexed Ion Beam Imaging): Utilizes mass spectrometry to image multiple targets in a sample. o Imaging Mass Cytometry: Combines mass cytometry with spatial resolution to analyze tissue architecture at the single-cell level. o Nanostring Technologies: A method for high-throughput RNA and protein detection at the single-cell level. Benefits of high dimensional microscopy 1. Multiplex tissue images can be mapped using spatial composition analysis and the presence of a certain interested epitope is then checked  2. Single cell segmentation can be done by single cell analysis, where the different cell types are found, and the comprehensive cell states can be reported because we can get information on where certain epitopes lay in the cell of interest. 3. Neighborhood analysis can be done to find neighboring cells of the interested cell: here the neighborhood properties are measured and the higher the relation, the closer they are. What will the future bring? - FLIM Cytometry: integrate imaging with flow cytometry - Single cell sequencing o Expensive technology but if it continues to evolve and transform and becomes affordable, this will be used more and slowly replace flow cytometry - Oil droplet encapsulation o Take advantage of beads that capture mRNA in tiny lipid droplets o Pushing cells at a certain concentration through an oil Single cell sequencing current causes them to be pushed into droplets o It is possible to combine it with DNA binding antibodies ▪ Antibodies are DNA barcoded and label cells -> cells gel partition into droplets -> capturing both the DNA barcode and all RNA molecules present in cell -> added probes specific barcode = each barcode tells you which transcript it was and from which cell it comes from Oil droplet encapsulation Elements of cytometry - steps o Cells in suspensions o Push cells into a stream to get them into a single cell stream o We direct these cells to interrogation point. Here we illuminate them with a specific beam of photons o These excite the fluorochromes on cells o These will change their energy state and when they relax they will emit other photons o These photons are directed through filters into detectors → into electronics → analyzed - Elements needed: o Cells o Fluidics: the system that moves cell through the instrument o Illumination and optic to excite fluorescent markers attached to the cells and to collect o Detector and electronics: sensors (e.g. PMTs, APDs, CCDs) that capture the emitted light and convert into data  o Chemistry: fluorochrome-conjugated antibodies, dyes, and proteins are used to label cells ➔ Each technology uses different elements Conventional flow cytometry = measures physical and chemical properties of cells based on how they scatter light and emit fluorescence. You loose a lot of material so it is not good for rare cell detection because you might lose these cells - Chemistry o Fluorochrome conjugated antibodies, proteins, or dyes - Illumination by lasers - Fluidics: o Hydrodynamic focusing area = push cells into laminar flow, you achieve that the cells are in the middle of the screen o Acoustic = Put a device in middle of the stream and let it vibrate → the acoustic waves in the stream pushes the cells in the center of the screen o Hydrodynamic and acoustic combination is best - Detectors and electronics o Photon counting devices can be PMT, APD, CCD. ▪ PMT allow you to tune the sensitivity of the detectors up or down depending on your needs. They are not very sensitive to red or infra-red, they work very well in violet and blue range. ▪ CCD are a little bit less sensitive, but they allow you to do capturing of images at the same time Mass cytometry (CyTOF) = uses metal isotopes attached to antibodies instead of fluorochromes. These isotopes are detected by a time-of-flight mass spectrometer. o Chemistry ▪ Metal-conjugated antibodies = you cannot measure these with PMT, ADP, or CCD, so now you need mass spectrometry to detect them. o Illumination by ICP: this is an ionization source to excite them and prepare them for flow cytometry o Fluidics ▪ To get a single cell expansion, here we use a nebulizer = gives gas to push liquid into a spray and each droplet withing the spray has a cell o Detector and electronics: ▪ TOF-MS: a detector in mass cytometry that measures the time it takes for ionized metal isotopes (bound to cell makers) to travel to the detector. The distinct masses of these isotopes allow for high-resolution, multiplexed detection of cellular features, making mass cytometry ideal for complex immune profiling and single-cell analysis. Single Cell sequencing = Analyzes the genetic material of individual cells to provide insights into gene expression (RNA sequencing) or DNA variations (whole-genome or exome sequencing). - Chemistry o Uses DNA barcoded antibodies: The chemistry of single-cell sequencing focuses on using DNA barcodes to capture information about both RNA (gene expression) and proteins for a more multidimensional understanding of cellular behavior. - Fluidics o Microfluids: Microfluidic systems in single-cell sequencing facilitate the isolation and individual processing of cells, ensuring that each cell's genetic material can be studied separately. - Detection o Sequencing: Detection in single-cell sequencing is primarily done via RNA or DNA sequencing technologies, providing insights into gene expression (transcriptome) or genetic variations (genome or exome). Instruments differ in terms of spatial resolution, number of markers (multicolor possibilities), multiplexed acquisition, fluorescence, and metal-based acquisition, autofluorescence, and specialized features such as small particle detection and rare event analysis. Popular instruments at MCCF: - Aurora: known for full spectral cytometry - CyTOF: Excels in metal-based acquisition and single-cell sequencing. - Fortessa: Conventional cytometry with high throughput. - Influx/Fusion: High-performance sorting cytometers. - Nadia: A platform for single-cell sequencing, RNAseq, TCR profiling, and DNA barcoding. Lecture 1 - immunotherapy modalities The pillars of cancer care Only since the introduction of immunotherapy, it was seen that it is possible to cure cancer. Questions that remained were how we do not overactivate the immune system using immunotherapy for example. There are two types of immunotherapies based on their ability to (re-)activate the host immune system against malignant cells. - Passive immunotherapy = therapies that have an intrinsic antineoplastic activity. o Examples are Tumor-targeting monoclonal antibodies and adoptively transferred T cells ▪ Tumor-targeting monoclonal antibodies = antibodies, not made by our own body, directly targeting tumors. ▪ Adoptively transferred T cells = T cells are collected from tumor after extraction by surgery. These are expanded and contain expanded antitumor activity. These are given back to patient and because the patient itself did not expand the T cells, this is passive - Active immunotherapy = exert anticancer effects only upon the engagement of the host immune system o Anticancer vaccines o Checkpoint inhibitor = you release the breaks in an immune system that allow you to expand immune response Immunotherapies can also be classified as targeted vs broad - Targeted = antigen specific o Could be active (vaccines) or passive (CAR-T cells) - Broad = non antigen specific o Stimulate entire immune system and you hope to push the immune response towards anti-tumor response instead of an autoimmune response o Could be immunostimulatory cytokines or checkpoint blockers There are many modalities of immunotherapy: Currently there are 10 classifications of current anticancer immunotherapies - Tumor targeting antibodies = they either target signaling functions of receptors on malignant cells, neutralize signals or recognize tumors based on tumor specific antigens -> then can be used to deliver load to target the tumor cells o Types of antibodies (mAbs): ▪ mAbs that specifically alter the signaling functions of receptors expressed on the surface of malignant cells such as Anti-EGFR: EGFR is constitutively activated in cells, and it is a pathway that it is good for the growth of tumor cells There are multiple antibodies that target EGFR and target different pathways. The antibodies all block functioning of EGFR ▪ mAbs that bind to, and hence neutralize, trophic signals produced by malignant cells or by stromal components of neoplastic lesions such as Anti-VEGF:  Anti-VEGF is an antibody that blocks binding sight of VEGF --> blocks angiogenesis --> blocks growth of tumor There are 3 VEGF receptors. They interact with plasma nuclear growth factor. Antibodies neutralize VEGFR --> cannot bind receptor --> no angiogenesis ▪ mAbs that target tumor associated antigen (TAA) such as an antigen specifically expressed by transformed cells but not by their non-malignant counterparts such as Anti-CD20 in CLL o Functional variants of antibodies: you can use them in many ways ▪ Use a naked monoclonal antibody (mAb) that inhibits signaling pathways required for survival. This can be for example cetuximab which is a EGFR-specific mAb. ▪ Use naked mAb to activate potentially lethal receptors expressed on surface of malignant cells. Example: tigatuzumab which is a TRAILR2-specific mAb ▪ Immune conjugates: bring a molecule that the cell internalizes and kills the cell Example: gemtuzumab ozogamicin which is an anti-CD33 calicheamicin conjugate ▪ Antibodies can also be used to help immune system to identify cells and kill them. This can be mAbs that induce cytotoxicity, phagocytosis Example: rituximab which is CD20-specific. It uses cell death and is able to induce phagocytosis, complement activation, and cytotoxicity. Hence, it is a multifunctional antibody. The function of rituximab can be enhanced to make a antibody conjugate by including auristatin E. This way you can lower the dosage of the drug to get the same result ▪ Make antibodies with double specificity = one side binds to tumor and other binds to immune cells and this way it helps immune cell to find tumor cell and kill it, activating immune system. These antibodies are called Bispecific T-cell engagers (BiTEs) Example: chimeric proteins consisting of two single-chain variable fragments from distinct mAbs, one targeting a TAA and one specific for a T-cell surface antigen - Adoptive Cell transfer = passive immunotherapy o It involves collecting cells that already have the property of attacking the tumor. o This variant of cell-based anticancer immunotherapy involves: ▪ Collection of the circulating or tumor-infiltrating lymphocytes ▪ Their selection/modification/ expansion/ activation ex vivo ▪ Their (re-)administering to patients, most often after lymphodepleting pre-conditioning and in combination with immunostimulatory agents o Hematopoietic stem cell transplantation (HSCT) vs Adoptive cell transfer ▪ Adoptive cell transfer = Reintroduce a cell population enriched in potentially tumor reactive immune effectors ▪ HSCT = You are resetting the entire immune system and not to specifically target a tumor. Hence HSCT is not a form of adoptive cell transfer o DC infusion vs Adoptive cell transfer ▪ DC infusion: DCs are harvested from a patient or donor, modified, or “loaded” with tumor antigens, and then reinfused into the patient. this is done with the goal of stimulating the patient’s immune system, particularly T-cells, to recognize and attack cancer cells. Hence, DC infusion is a form of adoptive cell transfer  o CAR-T cells vs adoptive cell transfer ▪ CAR-T cells are a type of T-cell that has been genetically engineered to express a chimeric antigen receptor (CAR) on their surface. This receptor enables T-cells to recognize and bind to specific proteins (antigens) present on the surface of cancer cells. CAR-T cells are a form of adoptive cell therapy because it involves this transfer of genetically modified T cells back into the patient. - Oncolytic viruses = viral strain that are selective and genetically modified to infect cancer cells and trigger cell death o They kill in such a way that it stimulates an anti-tumor immune response o Mechanism ▪ Cytopathic effect: the viral infection causes lethal overload of cellular metabolism ▪ Endogenous or exogenous gene products that are potentially lethal for the host cell irrespective of their capacity to massively replicate and cause a cytopathic effect ▪ Viruses also promote release of TAAs in an immunostimulatory context. - DC based immunotherapies o 2 types of DC based immunotherapies ▪ Ex vivo DC generation: Collect Spinal cord precursors, differentiate them in vitro and load them with tumor antigens --> now you have DC cells that have seen the antigen --> given back to patient The purpose of this strategy is to create targeted immune response by ensuring that the DCs see the tumor antigens and stimulate the cytotoxic T cells to eliminate cancer cells in patients. The advantage is that the DCs focus on the tumor cells expressing those antigens, making it a specific approach. However, the effectiveness can vary depending on the patient’s immune environment and the quality of the DCs generated. ▪ In vivo antigen delivery: Inject the tumor antigen in the form of a vaccine close to the DC cells --> DC cells capture antigen --> activated, differentiated --> migrate to lymph node --> immune response This strategy aims to leverage the body’s natural immune system by guiding dendritic cells to pick up tumor antigens and initiate an immune response, leading to the activation of T-cells that can fight cancer. This approach takes advantage of natural immune processes, but it relies on the endogenous DCs which can be influenced by the patient’s existing immune environment - Peptide and DNA based anticancer vaccines o This therapy aims to teach the immune system to recognize and destroy cancer cells by using the TAA or neoantigens with adjuvants. It helps T cells fight the cancer effectively and safely. The DCs take in the TAAs, break them down into smaller parts called epitopes and show these parts to the T-cells. This tells the T cells to attack cells with those markers (usually cancer cells). ▪ Neoantigens = Neoantigens are new markers created by mutations in cancer cells. They are unique to cancer and not found in normal cells, so they are great for targeting tumors specifically. They can make the immune response stronger because the body sees them as totally foreign, meaning they are only on the cancer cells and not healthy cells. ▪ TAA = special proteins or peptides found more in cancer cells than in normal cells. They help the immune system and recognize and attack cancer. o TAA therapy is given by injecting them into the body through: intramuscular (into muscle), subcutaneous (under skin), or intradermal (into the skin). o Two types of TAAs ▪ Synthetic long peptides: man-made versions of TAA pieces that are easier for DCs to break down  ▪ DNA vaccines: DNA that tells the body to make the TAA itself which helps the immune system - Immunostimulatory cytokines o Cytokines regulate virtually all biological functions. However, the administration of most immunostimulatory cytokines to cancer patients as standalone therapeutic interventions is generally associated with little, if any, clinical activity o Do not always work well because cytokines have many different function ▪ Some exceptions: IL-2 and IFN-a2b that showed success especially in melanoma. Melanoma has high mutational rate so activating immune system will trigger tumor specific responses to multiple antigens, therefore research is done most often in this specific type of cancer - Immunomodulatory mAbs o Modulate immune response in such a way that it stimulates or stops immune response in tumor. These molecules are in charge of preventing autoimmunity. o When releasing the breaks, 2 things are likely to happen: ▪ If the tumor is using the immune response, then stopping this will cause killing of the tumor ▪ If you have auto reactive T / B cells, these will cause an autoimmune response o Multiple ways immunomodulatory mAbs work to interact with components of the immune system: ▪ Inhibition of immunosuppressive receptors expressed by activated T lymphocytes, such as CTLA4 and PD-1, or NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family. ▪ The inhibition of the principal ligands of these receptors, such as PD-L1; ▪ The activation of co-stimulatory receptors expressed on the surface of immune effector cells such as TNFRSF4 (OX40), TNFRSF9 (4-1BB), and TNFRSF18 (GITR); ▪ The neutralization of immunosuppressive factors released in the tumor microenvironment, such as transforming growth factor β1 (TGFβ1). o There are many checkpoint antibody inhibitors. Immune checkpoints are regulators of the immune system. These pathways are crucial for self-tolerance, which prevents the immune system from attacking cells indiscriminately. However, some cancers can protect themselves from attack by stimulating immune checkpoint targets. - Inhibitors of immunosuppressive metabolism o Tumors use metabolic pathways to their own advantage and one of them is to convert L-tryptophan by IDO1 into L-kynurenine (Kyn) ▪ 2 functions of the conversion of tryptophan: stimulating growth of tumor cells: breaking down of tryptophan causes less available tryptophan for immune cells --> weakens their ability to fight cancer --> tumor cells grow and survive suppressing immune response: the breakdown product kynurenine (kyn) and its derivatives promote development of Tregs --> less immune response --> tumor cells grow and survive o By metabolically inhibiting this pathway, you stop this. This is done by using: small chemical inhibitors for these specific metabolic pathways - PRR agonists o The activation of various PRRs ignites a signal transduction cascade with potent pro-inflammatory outcomes, including the activation of NF-κB, and the secretion of immunostimulatory cytokines, like type I IFNs and TNFα o They activate immune system and often used in combination with vaccines to make sure that the vaccine is seen as a pathogen and start immune system - Immunogenic cell death inducers o They induce cell death in such a way that the immune system recognizes the molecules that come out of the death cells as a vaccine to start an immune response against that cell type. These are called DAMPS: they can bind on the surface of APCs, including TLR4, and therefore boost their ability to take up material and causes maturation/activation o Types: ▪ Some can be used as chemotherapeutic agents such as doxorubicin ▪ Some can be combined photodynamic therapy Lecture cytometry instrumentation Flow cytometry measures the intensity of different fluorescent colors to measure cellular components such as markers, proteins and so on. It couples speed with multiplexed marker single-cell detection at the cost spatial and most morphological information, leading to quantitative data. So the advantages are that flow cytometry can measure a lot of cells with multiple markers/colors, which is more than a fluorescent microscope. - Flow cytometry can gather quantitative data on cells using forward scatter (FSC) and side scatter (SSC) o FSC: measures cell size o SSC: measures cell granularity or internal complexity Cytometry uses these things to indirectly measure: - Antibody against antigen: antibody binds to antigen on cell --> attached fluorescent dye emits light that can be detected by the cytometer - Fluorescent proteins: these glow when exposed to light of a specific wavelength - DNA binding dyes: these are chemicals that binds to DNA and fluoresce when exposed to light, allowing the cytometer to detect them. They help to measure the amount of DNA in a cell. This way you can determine the cell cycle stage or detect dead cells. - Probes for detecting PH, Calcium flux etc.: specialized chemical probes that when there is a change in cellular environment, emit light. It is used to study cellular functions Each fluorescent molecule has a specific excitation spectrum and emission spectrum (the light that it emits when returning to the ground state). By using specific lasers, the flow cytometer can excite fluorophores and detect the light they emit to gather quantitative data. Hence: you excite the electron at one or multiple wavelengths --> looses a little bit of energy because there is always some heat produced --> when it comes back to the ground state, it emits the light we see - Higher wavelengths have lower energy that is why we emit a different color than at which it is excited In cytometry the emission and excitation states are collected: the emission extra are probabilistic. This means that the emission spectra are not fixed. Although a fluorophore has a preferred emission wavelength, it can emit light across a range of wavelengths. This variability allows cytometers to detect multiple fluorophores that emit at slightly overlapping or distinct wavelengths. - Example o DAPI: When we excite with 50 nm we collect 98% of its emission. If we emit with 405 nm, we collect 7.1%. this means that we will generate light, in flow overlap, in other channels in which we do not want the signal to be. Flow cytometer has 45 components - Fluidics: fluidic system forming a single cell suspension. First the sample is introduced by removing the pressurized air. This way the pressure goes up. This sample goes into the flow cell in which hydrodynamic focusing happens: sheath fluid, for example PBS, pushes the samples through the flow cell and forces it into one laser point. o If we cut in the flow cell, we see that the sheath is surrounding the sample with the sample in the middle of the stream o This should be consistent; therefore we make sure that the pressure stays constant o The sheath fluid makes a constant laminar flow o Acoustic wavelengths can also be used to focus the cells into the middle which is more robust and therefore faster to collect the data - Optics: lasers provide energy to excite fluorophores, and emitted fluorescence is detected using highly sensitive detectors. Once the cells have gone to the laser point, they go to waste. o L.A.S.E.Rs: amplification by stimulated emission of radiation  ▪ The emission stays the same in a laser in which they do not cross each other: the lasers are therefore coherent. ▪ Lasers are always monochromatic = always one specific wavelength. This is why we can excite different molecules with different lasers, making flow cytometry multiplex ▪ The cells are also excited at high powers because they are being excited very fast, in a very short time o Some cytometers have co-linear lasers = multiple lasers aligned along the same axis or path within the flow cytometer. Their light beams follow a single straight line to allow multiple wavelengths of light to excite different fluorophores as the sample passes through ▪ This can be a limitation: if we excite with low wavelength at exactly the same point, we cannot distinguish which excitation comes from which source (laser) ▪ Fixing this limitation by putting the lasers in equally separated distances of which each color goes to a different detector, separating both signals This introduces a problem: we need to keep the time difference between the two excitations constant in order to know that the signals come from 1 cell but the difference in time is just that the cell passes the first detector first and then the other detector later. We correct for this time! Multiplexing involves the detection of multiple fluorophores simultaneously by using lasers and detectors in quick succession. Meaning that cytometers can detect multiple signals from different fluorophores attached to different markers in the same sample even though only one cell passes through the system at a time o Filters in the cytometer separate light by wavelength, allowing specific emitted signals to be collected by detectors. ▪ Optical filter types Short pass filter: allows the light with a shorter wavelength will go through Band pass filters: wavelengths between two wavelengths can only pas through Long pass filters: allows only higher wavelengths to go through the filters o Conventional detection: detector array example ▪ We have light coming from 1 cell and first It goes through a long pass filter, the other wavelengths will go to other filters and separate different signals coming from different molecules from one cell: this way we obtain a lot of information from just one laser light o Optical detectors: these converts the photons to photo electrons and amplifies the signal ▪ Amplification system: photon enters the system and dynodes reflects these and amplifies it each time before it reaches the detectors ▪ Type of detectors: PMTs: amplify signals but have limited sensitivity in the infrared range APDs: amplify the photoelectric effect thanks to a fixed potential difference applied to the device. The amount of amplification can be controlled and adjusted using gain scale, which is an advantage in detecting varying signal intensities. This detector is better in the far red emission part of the spectrum than PMTs - Electronics: signal generation o A pool of cells is generated that passes through the detector: each cell passes one by one through the laser beam which excites the fluorophores. These emit light and is collected by the detector. The emitted light is converted into an electrical signal which is proportional to the intensity of the fluorescence, indicating how much fluorophore is on the cell Height (H) = intensity fluorescence Width (W) = duration of time the cells remain in laser beam which says something about the size of the cell  o the information of each pool needs to be converted into something we can measure: ▪ we will represent each pool of cells in the certain channel with a specific intensity: we can create as many channels as we want. So if one intensity (the channels) reaches pool 5, we put it in pool 5 of the data graph ▪ now each channel has its own intensity: represented by different colors in the graph o it might be that we have two cells with the same intensity in the same channel: now we add In this cell on top of the other in the graph. This way we know how many cells we have for each intensity ▪ Density of the clouds is how many cells have same intensity o Now we have also flow cytometers that collect every spectrums coming from any laser lights ▪ This way we can have the whole spectrum of just one fluorophore ▪ Now we can separate the markers better → more information - Data o We can collect in which step of the cell cycle the DNA is and we can collect information from every single cell which gives a lot of data. This way we can analyze high dimensional data o Data conversion ▪ Bit resolution: Higher bit resolution (e.g., 10-bit or 1024 channels) allows for finer distinctions in fluorescence intensity, improving the accuracy of the data. ▪ Voltage: The strength of the fluorescence signal is converted into a digital value based on voltage, which corresponds to the intensity of the signal. New developments in flow cytometry: - Imaging coupled with flow cytometry: instruments like Attune CytPix and FACSDiscover combine brightfield or fluorescence imaging with standard flow cytometry data. This way we get parameters of the cells and morphological information o Advantage of Imaging flow cytometry (IFC) ▪ Interesting phagocytosis information ▪ Useful to see the quality of the sample: sometimes you have cells that explodes ▪ Some microscopes can also get the fluorescent information of each cell on top of the pictures Depending on the distribution of the fluorophores within the cell, you can know the hematopoietic stage of the cells - Modern flow cytometers can sort cells into different populations based on their fluorescence or scatter properties o Cell sorters allow us to separate up to 6 different populations at ultra high speed  530/30 means that it is excited at 530 and 30 around Lecture Spectral compensation in flow cytometry this wavelength Compensation: in flow cytometry, compensation corrects for fluorescence spillover, which is when a fluorochrome emits light that is detected in more than one channel (detector). This must be corrected to ensure that each channel detects only the fluorescence intended for that channel. - Example: FITC is excited at blue and emits at green but there are also other photons emitted. The problem with this is that some of the photons that are emitted also go into the detector. This causes the wrong information to be obtained. Therefore, we need compensation There are three main causes of spillover which can happen during panel design and optimization 1. Spectral overlap (Adjacent overlap): dyes excited with the same laser and they are so close to each other in the spectrum, that the tail of one goes into the other 2. Tandem dyes: fluorochromes resulting from generation of FRED within one molecule o You can couple two dyes but if this is done wrongly, it can become uncoupled due to temperature, causing more direct emission from the donor fluorochrome and its normal emission wavelength o In other words: If the coupling goes wrong, the peak of the other dye can also emit, which is not what you want 3. Cross laser excitation: Two fluorochromes with similar emission spectra but excited with different lasers, will show spillover when one of the fluorochromes Is also excited by the other laser. The excitation of a fluorochrome by the wrong laser can happen on accident. Compensation for the spillovers: - Single staining controls for every color with cells/bead stained with that one color. When the amount of FITC increases, the signal of PE spreads. This is called spread and can be a problem. Because if we are looking for cells positive for PE and for FITC, that would be around the big cloud on the right and separating them would be difficult. This happens with orange and red emitting dyes and it is intrinsic of the dyes: we cannot change this. You need to avoid combination of markers that generate this effect in your panel design: use CD8 with FITC for example First, single-color controls are measured for each fluorochrome to determine the amount of spillover. A compensation matrix is then created using this information. This matrix mathematically adjusts for spillover and ensures the proper separation of signals in multicolor experiments. These figures show that small changes in compensation factor can have very large effects in the data Earlier manual compensations were used that involved sequential subtraction of signals which is prone to error. Automated compensation is the standard in modern cytometers and is more accurate than manual compensation, which was done on older machines like the FACSCalibur. 3 rules of compensation: EXAM QUESTION 1. The single-color control needs to have the exact same spectral characteristics as the one you mix in your experiment o If you do not do this, compensation would be impossible o This is particularly important for tandem dyes, where lot-to-lot variability can affect compensation accuracy. Polymer dyes are sometimes build on top of antibody and do not have the information: there can be a lot of differences between the two same dyes (slightly different spectra), so even when using the same dye, you need to check the characteristics 2. The brightness of the single-color control (positive population) must be as bright or brighter than the positive cells in the experimental sample: if you go beyond the range of your determined values, the accuracy will drop and you will risk under- or overcompensation o With the single-color control you are determining the maximum of your positive cells and minimum fluorescent that you expect for your negative cells o You can cause over or under compensation if you use less brightness in the single-color controls. You need the same brightness in your control as your sample 3. The negative population in the single-color control must have the same autofluorescence as the positive population. o Make sure to place the negative control on the same cell type as the one you are interested in Further considerations - Single-color controls should be treated the same way as experimental whether they are beads or cells because treatments can alter the spectral characteristics of fluorochromes and affect spillover. o Fixation is the procedure in which you give cross-linkers to keep molecules rigid so you do not lose them. This cross-linking will also limit the vibration of the fluorochromes: the fluorochromes will have slightly different property, given an impact on the spectrum. Keep this in mind! - Beads are often used In compensation because in some cases some markers are rare. Then you need to run samples for a long time for good compensation. Hence, one approach that has been used is to use polystyrene beads coated with antibodies the FC part o 50% are coated and 50% not: so In the same tube you always have a positive and negative pick o But sometimes the fluorochromes change properties on these beads: giving slightly different spectra, then you cannot use this o Do single staining control with beads and with cells and see how many antibodies work well with beads and how many with cells: then choose which one you use for your other experiments. Troubleshooting: N X N plot to check data quality - NXN plot is a tool for troubleshooting the compensation matrix. It allows you to see potential issues like over- compensation or under-compensation as well as transformation errors. o You need to remove them and reapply compensation Over-compensation occurs when too much signal is subtracted, leaving less Left top = under compensation signal than actually exists. Left bottom = Overcompensation Under-compensation occurs when not Right top = Under compensation enough signal is subtracted, leaving Right bottom = transformation/Compensation more signal than actually exists. - The right bottom transformed graph shows uniformly distributed data. This should happen in the negative population after compensation and bi-exponential transformation o Super-negative effect (red arrow) = effect of data transformation where the data is pushed to negative. Typically happening when here is not good compensation in another dimension However, in the graph on the right we still see some super-negatives after compensation. This is possibly due to other errors such as fluorochrome aggregates  o Fluorochrome aggregates: high-order complexes of fluorochromes that non-specifically bind to cells, causing random, punctate staining. They can lead to inaccurate data and affect the identification of cell sub-populations. To remove these from your data analysis you can use gates or spin down the staining cocktail. - Flag the suspicious staining patterns: look in the literature what the expected pattern is of your panel. If you do this well then you know how to identify pattern that are not correct and how to re-adjust the data o Sometimes this is caused by biology: Ki67 also flags for HIV+ donors, having influence on your data - If everything is good but you still see problems in your panels, then you need to check for whether the specificity of the antibody is correct: check this by using a clone ➔ You need to do this before you experiment in order to optimize the panel design! Spectral cytometry - Spectral Flow Cytometry: Unlike conventional flow cytometry, where each fluorochrome is assigned to a specific detector, spectral flow cytometry uses full-spectrum detection. It collects all emitted light and generates a unique spectral fingerprint for each fluorochrome. Moreover, spectral allows for autofluorescence extraction. o More measurements also means better resolution - Spectral unmixing = This process involves separating the signals from overlapping fluorochromes by mathematically "unmixing" the spectral data using reference spectra. The algorithm distinguishes each fluorochrome based on its unique spectral signature. o Example: An assay using two fluorochromes (e.g., FITC and PE) generates mixed signals. Spectral unmixing uses reference spectra for each fluorochrome to calculate the contribution of each to the total signal (= vector for each color). This process allows you to isolate the true signal for each fluorochrome o This unmixing is usually done with computers and not by hand because flow cytometer uses a lot of colors at the same time - Unmixing VS compensation o Compensation: Corrects fluorescence spillover by subtracting signals from neighboring detectors. o Spectral Unmixing: Uses full-spectrum data to distinguish between fluorochromes, even if their spectra overlap significantly. ➔ When the panel is designed properly, compensation or spectral unmixing is easy. If you are struggling with the data, it may indicate that the panel design needs revision. Lecture the Cancer Immunity Cycle The cells in the immune system can recognize non-self and danger and thus identify infection and cellular stress/death. The cells are educated to ignore “self”. - 2 immune systems: innate vs adaptive o Innate = first responders ▪ Present at birth ▪ Inherited ▪ Rapid ▪ Limited potency and lower than adaptive ▪ Non-specific and there is no memory o Adaptive = B lymphocytes become plasma cells and secrete antibodies ▪ Develops during life ▪ Slow response (1-2 weeks) ▪ Higher potency than innate ▪ More specific ▪ There is memory B cells can help tumor cell killing but T cells are more important in the tumor cell cycle NK cells: innate effectors - They can recognize “missing self” which are cells that lack MHC class I molecules o When a cell has MHC class I the NK cell does not kill these because they are healthy cells o Tumor down regulates MHC class I to avoide T cell activation, however the NK cells can still recognize these due to removal of MHC Class I and kills the tumor cells - NK cells have a cytotoxicity called: ADCC o This can be used in therapy but also in the body when NK cells generate cytotoxicity against the tumor cell o Steps: antibodies bind antigens on the surface of target cells → trigger cross linking of FC receptors in the NK cells → degranulation into lytic synapse → tumor cells die by apoptosis ▪ Tumor antigens can be produces in therapy and given to patient to kill Dendritic cells and T cell priming - Uptake of antigen by DC o DCs can present endogenous antigens to CD8+ T cells on MHC-I o They can also present exogenous antigens to CD4+ T cells on MCH-II or cross present to the CD8+ T cells on MHC I - Depending on the signal the DC receives when taking up antigen, they will give signal to the Naïve T cells  o The different types of pathogen-associated molecular patterns (PAMPs) or pattern-recognition receptors (PRRs) from tumor cells can trigger different signaling in the DC and based on that they can secrete for example IL-12p70, which will produce the T helper 1 cells Regulatory T cells - When T cells get activated we always need a balance of stopping this as well. The regulatory T cells can inhibit effector T cell receptors, which is good in the case of autoimmunity. The regulatory T cells can also frustrate the activation of DCs which causes not good activation of DC’s. If DCs aren’t properly activated they cannot cause immune response anymore - In case of tumor-immunity, this is a bad thing. The regulatory T cells can kill mature DCs via granzymes and thereby prevent the priming of tumor-specific cytotoxic CD8+ T cell, inhibiting a good immune response against tumor o So in tumor immunity you want low regulator T cell activity - CTL4 is expressed highly on regulatory T cells. The T regulatory cells can disrupt CTL metabolism, inhibiting T cell priming. Types of T cells - In the lymph nodes, migratory DCs, that come from the peripheral tissues, will carry antigens and present them to CD4 or CD8 T cells, dependent on the type of DC: CDC1 present mostly to CD8+ T cells and the CDC2 present antigens mostly to CD4+ T cells through MHC-I. However, this is not enough to get a full blown T cell response. - To activate T cell response more than antigen presentation is needed: the migratory cDC1 needs to transfer its antigen content to a lymph node resident cDC1, which is recruited by the activated CD8+ T cell --> lymph node resident cDC1 interacts both with CD4+ and CD8+ T cells. --> Activated CD4 Th cell secretes IL-2/IL-12 to support CD8 proliferation These interations are need for a full blown T cell response. Missing one will cause problems ▪ CD4: TCR binding to MCH class II with CD40/ CD40L binding --> release of IL-12 and IL-2 which bind to receptors on CD8 T cells. ▪ CD8: TCR binding to MHC class I with CD80/CD86 to CD28 binding. Cancer immunity cycle - The cancer immunity cycle shows the steps required to reach T cell mediated anti-tumor immunity. There are multiple good and bad players that can influence each step. These specific players per cancer type need to be identified to personalize the treatment for each patient. - Defects in the cancer immunity cycle can create different types of Tumor microenvironment (TMEs). These can affect how T cells interact with tumors and they ability to kill cancer cells. The goal of immunotherapy is to restart or enhance this cycle to promote effective anti-tumor immunity. Different types of TMEs when there are defects in immune cycle can be: o If there is not good release of antigen or no DC cells, we end up with a tumor without T cells = called immune desert. Hence, something went wrong early on. The problems in the TME which cause the infiltration problem for T cells could be: ▪ Problem in the starting of migration through vessels ▪ The presence of nutreints/chemokine gradients in TME is in bad shape so the T cells migrate back  ▪ There might be cells that ae suppressive to the T cells, preventing them to move forward. They can form a ring and block T cells ▪ The ECM is structured in such a away that the cells are removed away from the tumor o Immune excluded = there are T cells but they are stuck in the outside environment of the tumor, they cannot infiltrate the T cells o Inflamed Tumors: A lot of T cells in the tumor but the Tumor cells are still alive. This means that there is something wrong later in the cycle (the checkpoints). There might be tertiary lymphoid structures in the tumor environment might cause the T cells to increase its number! This is an updated version of the cancer immunity cycle The role of the tumor microenvironment is very important There are also DC cells that sustain the antitumor response and not only DCs in the lymph nodes! Step 5 shows the entering of T cells into the tumor and stroma. When entering, the T cells can be redirected to create desert/excluded tumors. Difference between immune-excluded and stroma-restricted immune infiltrated tumors - Immune excluded tumors: T cells remain at the border - Stroma-restricted = T cells are in the tumor area in the stromal field but not able to infiltrate within the tumor field ➔ This distinction is impossible to make when only having the tumor biopsies and not the tumor resection How can we make the cancer immunity cycle work properly? - Central tolerance o There must be an antigen for the T cell to recognize as non-self in order to be activated o A suitable antigen for T cells in cancer could be: ▪ Viral proteins ▪ Mutated proteins (neoantigens) = changed proteins that are different in tumors than healthy cells ▪ Cancer-testis antigens = antigens expressed in spermatocytes (or other tumors) and not expressed din other tissues of the bodies. ▪ Post-translational modification proteins that are specific for tumors ▪ Overexpression of an antigen that is present on our normal cells but they are overexpressed in tumors - Peripheral tolerance o In order to kickstark cancer immunity cycle and keep T cells response going. The tumors need to activate the DCs. o Tumors can use this: they can activate pathway to prevent recruitment of right DCs. Tumor cells stop production of a specific cytokine, which causes the needed DC to not migrate to the tumor → causing immune dessert o The metastatic site might be different in microenvironment from the primary tumor area: one treatment might only work in the primary tumor area but not in the metastatic site. This can be explained by: Lack of systems for T cell trafficking to the metastatic site because they lack homing receptors for that tissue.  - Tumor-associated immunosuppression: CTLRs are suppressed in the TME o TME has many infiltrating immune cells which causes different types of TME for each cancer cells ▪ The type of TME can therefore determine prognosis and response to therapy → different immune cells have been linked to poor or good prognosis o Within the TME, APCS and tumor cells can express inhibitory ligands that suppress T cell functioning. We can therapeutically target these with agonistic or antagonistic antibodies. Promoting tumor specific T cell responses 1. Use drug to stimulate DC T cell response 2. Vaccination to get more or better antigens 3. Destruct the tumor with chemotherapy, radiotherapy or oncolytic viruses 4. We could expand and reinfuse T cells to the patients to get better T cell responses Here the cancer immunity cycle is used and shows the treatment in these steps: In step 1 we can reduce local immunosuppression in the tumor In step 2 we can activate dendritic cells, to in vivo generate a t cell response In step 6 we an try to (re)activate the existing anti-tumor T cell response or provide tumor- specific T cells if the activation of the cancer immunity cycle does not work. Lecture flow cytometry Flow cytometry has high speed, statistical power, but lacks imaging. Microscopy lacks speed and statistical power. However, when combining those (ImageStream) offers high speed, statistical power, and the ability to image cells, leading to objective statistical discrimination of cells based on appearance Imaging flow cytometry optical layout: - Camera 1 and 2: capture images from different fluorescence channels - Brightfield and Lasers: Light sources that allow for both brightfield and fluorescence imaging. - Flow Cell: The channel through which cells pass for analysis. - Spectral Decomposition: Breaks down the emitted fluorescence into various channels, allowing multiple colors (fluorophores) to be detected. - Magnification Options: 20x, 40x, 60x magnification, used to capture detailed images of cells at different scales. - Lasers: each laser has a specific wavelength which is used to excite different fluorophores. o Examples: The 488 nm laser excites FITC (a common green fluorophore), and the 642 nm laser excites APC (a red fluorophore). o These lasers allow for the simultaneous detection of multiple fluorophores, creating multi-dimensional data. There are different channels available in ImageStream for fluorescence and brightfield detection - There are up to 12 channels: Two brightfield channels (for cell morphology) and up to 9 fluorescence channels (for detecting different fluorophores). Each channel corresponds to a specific filter that selects light of a certain wavelength, ensuring accurate detection of different fluorophores One of the key features of imaging flow cytometry is the Time Delay Integration (TDI) technology used in the CCD cameras. As cells move through the instrument, the TDI system ensures that the speed of the camera’s detection is perfectly synchronized with the flow of the cells. This allows for clear, high-resolution images without motion blur, even as cells pass rapidly through the flow cell. The TDI system integrates light across the entire height of the detector, ensuring that even weak fluorescent signals are captured accurately. - This feature is essential for capturing sharp images of cells in motion, making it possible to analyze both morphological features (such as cell size and shape) and molecular characteristics (like the intensity and location of fluorescence). The experimental workflow in imaging flow cytometry follows a systematic process that ensures accurate data collection and analysis. First, data is collected from the cells flowing through the instrument, with images and fluorescence intensity measurements being captured simultaneously. Next, a compensation matrix is created to correct for any overlap between fluorophore signals, ensuring that each channel only detects the fluorescence intended for it. Researchers then analyze control samples to calibrate the instrument, and these settings are applied to batch process multiple samples efficiently. Once the data is processed, researchers can generate reports and validate the regions of interest, ensuring that the analysis is both accurate and reproducible. The data is saved in a variety of file formats, including the widely used Flow Cytometry Standard (.fcs), which allows for easy comparison and sharing of results. 1. Collect Data: Gather image and flow data. 2. Create Matrix: Develop a matrix for organizing data. 3. Analyze Control Sample: Use controls to calibrate the instrument. To do this SpeedBeads can be used. They test various subsystems (e.g., lasers, cameras) to ensure consistent, reliable data acquisition 4. Batch Process: Apply settings across multiple samples. 5. Generate Reports: Summarize and review the data. o Raw image file (.rif): contains unprocessed data from the instrument o Corrected image file (.cif): after compensating for any errors or aberrations o Flow cytometry standard (.fcs): standard format for flow cytometry data The images captured by imaging flow cytometry are not just for visual inspection; they are also converted into numerical features that can be analyzed statistically. The system applies masks to the images to define regions of interest (e.g., the cell or nucleus), and these masks are used to extract features such as area, aspect ratio, and intensity. For example, the area feature quantifies the size of the cell in square microns, while the aspect ratio provides information about the shape of the cell by comparing its length and width. This ability to quantify features from images is what makes imaging flow cytometry so powerful. It allows researchers to objectively compare cells based on their physical and molecular characteristics, rather than relying solely on subjective visual assessments. The extracted features can then be plotted in histograms or scatter plots to reveal patterns or differences between cell populations. Shortly said the workflow for feature generation includes the following steps 1. Researchers define the morphological characteristics they want to study (e.g., bacterial uptake by immune cells). 2. They create a mask to isolate the area of interest (e.g., intracellular space). 3. Features are then calculated based on the image data, and the results are plotted in histograms or scatter plots for analysis. Case study – Human monocyte cells treated with LPS, leading to the translocation of NFkB from the cytoplasm to the nucleus. - Imaging flow cytometry is particularly valuable in studying processes like cell signaling, internalization, and cell-cell interactions. For example, in the case of NFkB translocation, researchers can track the movement of the NFkB protein from the cytoplasm to the nucleus in response to cellular stimuli, such as treatment with LPS (a bacterial component). By staining the cells with fluorescent markers that label NFkB and the nucleus, imaging flow cytometry can quantify the degree of translocation in hundreds or thousands of cells, providing a statistical representation of the entire cell population’s response. - Another important application is studying phagocytosis—the process by which cells engulf particles or other cells. Imaging flow cytometry can track the internalization of particles by labeling both the particle and the cell, allowing researchers to see where and when phagocytosis occurs. This provides insights into immune cell function, pathogen clearance, and cell-to-cell communication. To assess the differences between treated and untreated cells, imaging flow cytometry uses Fisher’s Discriminant Ratio (Rd), a statistical method that quantifies the separation between two groups based on their mean values and standard deviations. This method is particularly useful for optimizing experimental protocols because it allows researchers to determine how well the treatment differentiates the cell populations. For example, if treated cells show significantly higher nuclear translocation of NFkB than untreated cells, the Rd value will reflect this difference, helping researchers confirm the effect of the treatment. - nuclear translocation of NFkB in THP-1 cells: When these cells are treated with LPS, NFkB moves from the cytoplasm into the nucleus, activating gene transcription in response to inflammatory stimuli. Imaging flow cytometry allows researchers to measure this translocation across thousands of cells, providing both qualitative images and quantitative data on the extent of NFkB movement. - By calculating similarity scores between the NFkB and nuclear markers (7-AAD), the system can statistically assess the degree of translocation. High similarity scores indicate that NFkB is predominantly located in the nucleus, while low scores suggest it remains in the cytoplasm. Important consideration: how many cells need to be collected to ensure reliable results? - Using Rd analysis, researchers can determine that collecting around 500 cells per sample provides consistent results with minimal variability. Collecting fewer cells (e.g., fewer than 100) leads to highly variable Rd values, which can make it difficult to draw meaningful conclusions from the data. Imaging flow cytometry has broad applications across many fields, including hematology, microbiology, immunology, and cancer biology. It is used to study everything from immune synapse formation to intracellular signaling and apoptosis. By combining high-throughput imaging with detailed analysis, this technology provides insights into complex biological processes that would be difficult to study with conventional flow cytometry or microscopy alone. - Cell Signaling: Tracking molecules like NFkB during cell signaling. - Internalization: Studying phagocytosis or how cells take in particles. - Cell-Cell Interactions: Investigating immune synapse formation between T cells and antigen-presenting cells. o Example Tregs: In imaging flow cytometry, the role of Tregs can be studied by observing their interactions with other immune cells, particularly through immune synapses, and by assessing the localization of specific markers (like FoxP3, a key Treg transcription factor) within these cells. The ability  to capture these interactions visually and quantify molecular markers helps researchers better understand how Tregs function in both health and disease. For instance, FoxP3 localization in the nucleus is indicative of Treg activation and their immunosuppressive role. By staining for FoxP3 and other molecules, researchers can use imaging flow cytometry to examine how Tregs influence immune responses within a tumor microenvironment or during immune synapse formation. - Shape changes o Example: Imaging flow cytometry is useful for studying immune synapse formation as it visualizes and measures the spatial organization of proteins like LFA-1 and ICAM-1, which stabilize the synapse, and MHC molecules, which present antigens to T cell receptors (TCR). Fluorescently labeled antibodies allow researchers to observe synapse formation and study its modulation by factors such as Tregs or changes in the tumor microenvironment. In cancer, this tool helps quantify disruptions in immune synapse formation, providing insights into how tumors evade immune detection. DC- based vaccines Dendritic cells (DCs) are a key component of the immune system, particularly in their role of linking the innate and adaptive immune responses. They act as professional antigen-presenting cells (APCs) that capture, process, and present antigens to T cells, triggering specific immune responses against pathogens or cancer cells. - DCs help the body to recognize pathogens such as bacteria, viruses, fungi, and parasites. Specialized immune cells are recruited to respond to these invaders such as macrophages, neutrophils, eosinophils, and natural killer (NK) cells. The immune system tailors its response depending on the pathogen type: - For bacteria and fungi, it recruits neutrophils and macrophages. - For viruses, NK cells and cytotoxic T cells (Tcyt) are critical. - For multicellular parasites, eosinophils and Th2 cells dominate. ➔ This differentiation is guided by the interaction of DCs with pathogen recognition receptors (PRRs), which trigger specific signaling pathways, leading to the appropriate T cell response (e.g., Th1, Th2, Th17). Specialized T cells, such as Th1, Th2, Th17, and regulatory T cells (Tregs), further amplify innate immune responses. Th1 cells are particularly important in combating intracellular pathogens and cancer by secreting interferon-gamma (IFN-γ), which enhances the cytotoxic function of T cells and NK cells. - DCs are crucial in initiating T-cell mediated immunity DC therapies and cancer One crucial consequence of dysfunctional immune system is cancer. Cancer cells can evade the immune system through various mechanisms, such as inducing an immunosuppressive microenvironment that favors immune tolerance rather than destruction. DCs play a role in reversing this immunosuppressive environment by presenting tumor antigens to T cells, enabling an effective immune attack against cancer. - DC-based vaccines aim to harness the power of dendritic cells to stimulate tumor-specific immune responses. Historically, these vaccines have evolved as a treatment for cancers like melanoma, prostate cancer, and renal cell carcinoma. - Antigens for DC based cellular vaccines can be: o mRNA o DNA o Tumor lysate o Viruses o Antigen peptides o Proteins DC vaccine development (Anguille et al 2015). - DC vaccine preparation o DCs are derived from monocytes or stem cells in the lab. o These cells are then loaded with tumor antigens, typically peptides or proteins derived from the patient’s tumor. o The modified DCs are matured and activated using adjuvants like GM-CSF, TNF, or TLR ligands to enhance their immunogenicity. o Finally, the DCs are injected back into the patient to induce a robust anti-tumor immune response. - Example: sipuleucel-T o One of the most successful examples of DC-based immunotherapy is Sipuleucel-T, a treatment for metastatic prostate cancer. Sipuleucel-T uses DCs loaded with a fusion protein of GM-CSF and prostatic acid phosphatase (PAP). Clinical trials have demonstrated a survival benefit, though the therapy has limitations in terms of cost and logistics. - Challenges of DC-based immunotherapy o Cost and complexity: Generating DC vaccines in clean rooms under Good Manufacturing Practice (GMP) conditions is expensive and labor-intensive.  o Variable effectiveness: The response to DC vaccines can vary between patients, and it is often difficult to achieve long-lasting immune memory. o Side effects: While generally well-tolerated, some patients may experience mild to moderate immune- related side effects, such as fever or inflammation at the injection site. Adjuvants in DC vaccines - Adjuvants are substances that enhance the immune response to an antigen. For DC-based immunotherapy, adjuvants are crucial in improving the efficacy of vaccines by: o Increasing the strength and duration of tumor antigen-specific responses. o Enhancing the activation of T cells and reversing immune tolerance within the tumor microenvironment. o Reducing the required dose of tumor antigens. - Examples adjuvants o GM-CSF o TNF o PgE2 o TLR-L The optimal phenotype of dendritic cells for cancer immunotherapy includes high expression of: - CCR7: Important for trafficking to lymph nodes. - CD80/CD86: Co-stimulatory molecules needed for T cell activation. - IL-12/IL-27: Key cytokines that drive Th1 differentiation and cytotoxic T cell activation. The location of administering the DC vaccine is important in interaction with tumor - Injection site of DC vaccine o DC vaccines are typically injected intradermally (into the skin), subcutaneously (under the skin), or sometimes directly into the tumor (intratumorally) or lymph nodes (intranodal injection). o The skin and lymph nodes are primary sites because they are rich in immune cells and have a network of lymphatic vessels that facilitate the migration of DCs to the tumor-draining lymph nodes (TDLNs). o These injection sites are chosen because they mimic the natural pathways of antigen-presenting DCs, which capture antigens and travel to lymph nodes to activate T cells. - DC migration to TDLNs o After being injected into the body, mature DCs migrate to the TDLNs, where they present tumor antigens to T cells. This interaction is crucial for initiating a systemic immune response that can target and kill tumor cells. o DCs express CCR7, a receptor that helps them migrate towards CCL19/CCL21 chemokines produced in lymph nodes, guiding the DCs to the appropriate location for T cell activation. - Intratumorally DC vaccines o Intratumoral injection directly places the DCs into the tumor microenvironment, which can be beneficial for stimulating a localized immune response and overcoming the immunosuppressive factors within the tumor. o This method of injection aims to promote direct interaction between the DCs and tumor cells, allowing the DCs to capture tumor antigens efficiently and then migrate to lymph nodes to activate T cells. T cell responses can be enhanced by combination of DC-based immunotherapy with immune checkpoint inhibitors like anti-PD-1 and anti-CTLA-4 antibodies by: - Boosting the activation of tumor-specific T cells via DC vaccination. - Preventing the tumor from suppressing these T cells by blocking inhibitory signals with checkpoint inhibitors. Combination therapy leads to increased T cell infiltration into tumor DC therapies and Tolerance Tolerance refers to the immune system’s ability to recognize and ignore self-antigens (i.e., the body’s own cells) to prevent autoimmune reactions. This mechanism is vital for maintaining immune homeostasis and preventing the immune system from attacking healthy tissues. - Immature dendritic cells (iDCs) are involved in maintaining immune tolerance. These DCs are found in peripheral tissues where they capture antigens but do not stimulate a full immune response. - In the context of tumors, immature DCs often promote tolerance instead of an immune attack, which allows tumors to evade the immune system. There is a shift from ex vivo manipulation of DCs to in vivo targeting which is more practical and scalable: Nanoparticles and liposomes are used to deliver antigens and adjuvants directly to DCs in the body. - These particles are: o Biodegradable and biocompatible. o Customizable in size and composition to enhance DC uptake and activation. o Capable of carrying both hydrophilic and hydrophobic components, making them versatile tools for delivering vaccines. - VD3/RA-loaded liposomes can modulate DCs to promote the development of regulatory T cells (Tregs) rather than inflammatory Th17 cells. This strategy is particularly useful in diseases where immune tolerance needs to be restored, such as in autoimmune diseases. Lecture Cytometry applications, sample prep, protocols Cytometry - The two golden rules in cytometry are o WYSIWYG (what you see is what you get): The quality of the data you analyze will only be as good as the quality of your experimental setup. o GI GO (Garbage In, Garbage Out): If you fail to handle your samples properly or set up your experiment poorly, the data will be inaccurate and misleading. - It is important to think about how samples are collected and processed. These can have huge impacts: impact viability, loose functionality of cells, and eventually you may loose protein integrity. o Techniques such as using specialized storage solutions for tissues (e.g., MACS Tissue Storage Solution) or flow sorting and cell isolation reagents ensure that cell viability, protein integrity, and other critical characteristics are maintained during experiments. - You need to consider o Sampling methods: for example cell culture harvest o Sample logistics: for example storage solution o Preparation protocol: for example mechanical disruption ➔ These decisions are tissue dependent and there are guidelines for the type of tissue. Blood preparation - Ficoll density gradient separation and hypotonic lysis are methods used to isolate mononuclear cells (MNCs) and granulocytes. - Considerations for blood o Storage of blood: when having hole blood it is not good to put it in room temperature o Avoid mechanical stress: typical mistake is that when blood is collected and brought to the place, the blood has been shaking and you get activated monocytes o Storage of prepared cells need to be at 4 degrees - Preparation of blood o Density gradient: centrifuging blood, the cells go to the buffer with the same density → separating the nucleated cells ▪ Not a good method to collect granulocytes because it is too aggressive and they might get activated o Hypotonic lysis: better for collection of granulocytes o Analysis on whole blood without lysing the red blood cells. This becomes a problem: red blood cells are most abundant but with this experiment, they become rare to collect. - Types of cytometry graphs o Good lysis shows different populations in the graphs o If the blood is older, bad lysis or no lysis at all gets rid of these populations and analysis is difficult Solid tissue preparation - When preparing solid tissues, factors like transport media, temperature, and time before dissociation are crucial. Improper preparation can lead to incomplete digestion, dead cells, and epitope degradation, leading to false positives or non-specific labeling in flow cytometry analysis. Problem with enzymes: depending on quality of the enzyme and type of enzyme, some epitopes may be clipped. Enzymes are important because they are proteases and depending on the cell type you need a different combination of the enzyme, however they can also get rid of epitopes which is not good - You need to test beforehand whether the enzymes clip off the epitopes: take a tissue In suspension (blood) that expresses interested epitopes. Without treating the tissue you can see epitopes and after giving the enzyme you still need to see the epitopes. - Enzymatic dissociation is required for high cell yield o To improve the high quality dissociated cells this is needed: ▪ Mechanical disruption: cutting tissue with scalpel and then some devices can grind the tissue in a gentle way to dissociate the tissue fragments with the presence of enzymes  To shorten the time exposure of the tissue to the enzymes ▪ Enzyme digestion Tissue has cells, interstitial tissue, connect tissue and some loss due to dead cells: these need to be removed. Some reagents can help you remove these o For example ◼ brain tissue has myelin. Removing this can be done by myelin removal beads ◼ Dead cells can be removed through density gradients ◼ Debris removal by solution ◼ Endotoxin removal beads for tissue that are not in sterile condition When using rough methods in sample collection, and getting loss of viability → the cells that are dead in the assay remain in the mix and will create an artifact in the analysis - Can create false positive because they become sticky and bind antibodies → creating rare phenotypes - False negative: non-specific labeling of the dead cells Immunomagnetic enrichment before flow: rare cells - Original samples contains a lot of negative cells and little target cells: in order to decrease negative cells you can remove these by magnetic beads targeting the positive cells - This will make our analysis a lot faster --> o Example ▪ Bulk tumor and we are interested in tumor infiltrating lymphocytes (CD8 cells). Normally this will take very long to find these cells and sort these cells in the bulk tumor, which will also change the phenotype of the cells ▪ When doing a pre-enrichment by removing the tumor cells: finding the CD8 T cells is faster Controls in cytometry are important: - To ensure that the instrumentation is performing optimally, and the data is accurate; o Machine characterization/optimization controls: Beads are used to find optimal settings for the instrument, ensuring high sensitivity and accuracy. - Biological controls: Unstained cells help determine autofluorescence levels, and compensation controls (single- color stained cells or beads) are used to correct for spectral overlap between fluorophores o Important because it allows us to see the level of autofluorescence o Do not use these for setting voltages: use positive controls for this - Fluorescence Minus One (FMO) control: help determine the gating strategy by showing the contribution of all reagents except the one of interest. This helps identify whether a particular cell population is truly positive for the marker or just a result of spectral spillover or background fluorescence. o Ethanol = fluorescence – 1 o FMO are used to calculate the difference between stained and unstained - Experimental controls: o Negative control o Positive control: then you can draw a range of values across assay that determines where the experimental sample is o Test sample - Isotype controls: antibody with the same Fc as your interested antibody but its antibody does not bind to your cells o Isotype controls are not good ▪ Our interested antibody binds with high affinity constant to our target with both parts (FaB and FC): this prefers to bind to the target than the isotype control ▪ Isotype control binds wit its FC tail to the FC receptor but this is less strong than our interested antibody You will always overestimated the contribution of the FC to the staining ▪ Fixing this problem Blocking reagents that will block all the FC receptors are a solution for this  Recombinant antibodies: they have mutations in FC so that the FC does not react to the FC receptor anymore Now we do not need to worry anymore about isotype controls Non antibody-based applications: remember the reagents and considerations Viability assays measure whether cells are alive or dead, which is crucial for accurate analysis. Commonly used dyes like propidium iodide (PI), 7-AAD, and SYTOX™ are impermeant to live cells but stain dead cell - Dead cells release DNA and other debris, which can affect flow cytometry results. Dead cell removal kits, endotoxin removal beads, and myelin removal beads are useful for cleaning up samples before analysis. Immunomagnetic enrichment is another technique used to isolate rare cell populations by selectively removing unwanted cells, which improves the quality of the data collected. - Fixable dead cell stains can be used, such as the LIVE/DEAD™ Fixable Dead Cell Stain Kits for dead cell identification. These dyes bind to amines on the cell surface and remain after fixation, making them ideal for flow cytometry experiments that involve fixation, where traditional viability dyes (like PI) would no longer work. o Cells exit anywhere on a continuum between healthy and dead. If you do not have viability indicator, you will find things that do not make sense in biology which needs to be avoided always, for example dead cells o Dead cells have holes in the membrane and are permeable: when using reagents that can cross the membrane and stain nucleus then we can distinguish between dead and alive cells ▪ These reagents are chemical dyes that bind to DNA and you have two types One that is non fixable: Amine-reactive Dyes o If you fix dyes that are non-fixable, all your cells will be positive o Make sure you do not have proteins in the buffer because otherwise the reagent will bind to the protein and the protein not to the cell Reagents that can be fixed to the cells: bind to DNA or proteins in the cell o Once you do the staining with live/dead cells --> fix cells --> remain properties of these cells and they will always stain the live cells a little bit as well because they bind surface proteins o Practical when you have large panels because you make sure the fix afterwards and the assay will be reliable for a long time - Proliferation assays o Tracking proliferation by dyes such as CellTraceTM. These are incorporated into cells and distributed equally between daughter cells upon division --> allows us to track cell proliferation over time. o Doing this by fluorescent DNA binding dyes: ▪ Using a linear scale for the G0/G1 peak, S phase, and G2m peak: these peaks overlap with each other so identifying the DNA expressed in which phase is difficult To overcome this: introducing a reagent, thymidine analog, that incorporates into the DNA. There are different versions thymidine analog: Quantifying by radioactivity BrdU: antibodies that bind to the epitope so you can quantify BrdU EdU: has chemical handle which you can attach to fluorochrome in order to distinguish the different cells with different number of DNA (the different phases) o Generational analysis ▪ Proliferation analysis by dye dilution: the daughter cells contain half of the parent cell - Apoptosis detections: Relative apoptosis timeframe o Apoptosis has multiple phases and you can measure reagent for each of these phases ▪ Annexin V binds phosphatidyl serine and this can be detected using a fluorochrome. Annexin V is combined with a viability dye ▪ You will end up with double negative cells: no annexin V and no viability dye Cells positive for annexin V = apoptotic cells  Double positive cells are necrotic cells o Many more assays for apoptosis: see slides Lecture Panel Design Starting materials for designing a panel 1. Having a scientific question o What are new trying to accomplish with this new panel? And which population? 2. What markers minimally describe this population 3. What markers would be interesting to have in the panel besides your other markers? Which markers should you include: ask the question how we can classify these markers - There are 3 types of markers o Type 1: on/off markers ▪ These are well characterized and are either expressed or not expressed, therefore easily divide positive and negative population ▪ Examples: CD4, CD4, and CD8 o Intermediate expressin markers ▪ Are well characterizes and nicely expressed but they are expressed in a continuum. This means that they form a smear on the panel because they define a differentiation stage of the cells in the populations. They do not separate from the baseline ▪ Examples: CD45RA, CCR7, and IFNg o Dim/rare antigens ▪ Low frequency antigens and dim expression on the cells ▪ Example: CD25 or tetramers which are highly expressed but low frequency - When identifying the research question and determine the markers, you need to avoid spread on dim co- expressed markers. With this, during the optimization phase you know what to look for! So do this beforehand Procedure to match markers to dyes 1. Take fluorochromes that do not overlap with each other o Limitations: multicolor flow will always use dyes that overlap. This overlap is no big deal! 2. Pair overlapping fluorochromes with markers that aren’t co expressed and chose bright dyes for dim markers o When two markers are expressed in the same cell, those need to have non overlapping fluorochromes: CD4 and CD8 for example o Limitations: cells are so heterogeneous that there is usually some co-expression of markers in any phenotyping experiment 3. Choose bright dyes for dim markers o Reagent brightness does not depend only on the dye used for conjugation. Antibodies clones differ in signal strength, too. So, charts and guides ranking dye brightness may be a limited utility Compensation is worrisome because if your perform compensation wrong, your panel is guaranteed to fail - Make sure you follow the three rules of compensation - Hoe can you mess up compensation: o Comp control not as bright as sample stain. o Negative population has different autofluorescen