Caput Immunoengineering
16 important questions on Caput Immunoengineering
Explain what is ment with "artificial immunotherapy". What is the difference between systemic (in/ex vivo) and local (in/ex vivo) delivery?
Intravenous delivery: (delivery of ex vivo expanded immune cells eg DCs, TILs, CARs ór in vivo acting nanovaccins or checkpoint inhibitors): systemic exposure to the immunostimulatory agent and treatment-associated toxicity.
Local delivery: more effective treatment at lower dosis, preventing systemic toxicity. Scaffolds: near tumor cells to attack and retrain immunsupressive cells.
enables local immunomodulation but also may overcome other limitations of current immunotherapeutic interventions that are related to cell delivery and sustained availability of immunostimulatory agents.
Why is understanding heterogeneity so important in immunoengineering?
Single sair pair interaction studies are therefore needed.
What are the cons of both conventional methods?
- Difficult to isolate cells for further molecular or gene expression analyses
- Require pre-existing knowledge of immune cell responses, in order to know what cytokines to detect
- Only allow cytokine measurements means that they fail to assay other aspects of activation.
- Poor spatial (within-cell) and temporal resolution.
- Higher grades + faster learning
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What is essential for better single cell analysis (extra)?
Microfluidic technologies are microtools to investigate single cells.
What questions can be answered with this technique?
Which aspects of immunology can be measured with microfluid based nanowell technology?
Signalling (cytokine detection)
TCR/BCR (by cell loading on wells, lysis and mRNA capture)
What are the con's of microfluid based nanowell technology?
Pipetteren is lastig
Bij was en incubatie stappen moet statistisch worden berekend hoeveel wellen er zijn gevuld enz.
How does droplet-based microfluidic technology works?
- Cell isolation and modification: Cells of interest are isolated and form droplets together with cytokine capture beats/cytokine catch reagens. Stimulated with CpG (DNA) to stimulate cytokine production.
- Encapsulation of single cells in droplets.
- Incubation in droplets
Cytokines are captured inside the droplets with cells and can after this be measured with fluerescent antibodies....?
Which aspects of immunology can be measured with droplet based microfluidics?
TCR/BCR: co-encapsulate single cells and poly(dT) magnetic beads together with lysis buffer. In individual droplets, cells are lyzed and mRNAs are annealed to poly(dT) beads which are subsequently recovered and emulsified for cDNA synthesis. PCR, sequencing.
Antibody screening: cells are co-encapsulated together with fluorescent ab capture.
(Same as nanowells)
PDC IFN I production can be measured with droplet based microfluidics. What do you already know about pDCs?
They link innate and adaptive immunity.
pDCs are a heterogeneous family (some have innate functions and secrete IFN I, some adaptive immune functions etc).
When disregulated; hyperactivated; auto immune disease.
How can be investigated how the type I IFN response is regulated on the single cell event?
Droplet formation with IFN I capture beads.
Incubation (?) and cooling afterwards to stabilize droplets.
Secreted cytokines bind capture beads confined within the same droplet.
Labeling with antibodies against IL-2, TNF-α, and IFN-gamma
Staining of the beads was measured by flow-cytometry.
What can be concluded about (the heterogeneity in) the pDC derived type I IFN response?
The higher the amount of CpG, the more cytokine production. But not in all cells!
Primed pDCs (with CpG) show a increase in IFNalfa secretion.
Why is pDC heterogenticity important in pDC vaccination
How can the best pDCs for pDC vaccination be selected? Mention microfluid droplets, microscopy and flow cytometry.
Input of CpG --> output of pDC (on CTL) --> output of CTL (tumor).
De cellular interactions between pairs of single cells can be also measured in droplets (tip loading; controlled pairing of CTL + pDC).
Microscopy for longitudinal effect; killing and cytokine secretion.
Flow cytometry for measuring the end-point; high throughput and multicoloar analysis.
Explain what a beadline capture antibodies is and how this contributes to multiplexed and longitudinal cytokine detection.
Positive for cyt1: beadline shown. etc.
Explain how valve based microfluid controls articficial micro-environments.
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