Immunology Exam 4 Module 10.3/4

Flow Cytometry & Vaccines

10.3

Flow Cytometry

Leukocyte subsets and lineages can be distinguished by expression of distinct surface markers

All Leukocytes Express CD45

All myeloid cells

express CD11b

• Monocyte

• Macrophage

• DC

• Neutrophil

Most subsets of leukocyte express unique markers

look

CD14

Monocyte

F4/80

Macrophage

tCD11c

DC

Ly6G

Neutrophil

Fluorescently conjugated antibodies can be used to label and identify unique leukocyte populations

1. Harvest Tissues of Interest

2. Digest and Process into a single cell suspension

3. Stain with cocktail of antibodies with distinct specificities and fluorophore conjugates

Flow cytometry uses

direct immunofluorescence to label leukocytes

• Fluorophore conjugated to FC portion of antibody

• Each antibody will ONLY bind to its specific antigen

Leukocytes express surface markers associated with their function

go on what they have - both positive for CD3+

CD8+ = cytotoxic

CD4+ = helper T cells

Leukocyte populations in a heterogenous sample can be resolved using flow cytometry

Stained cell suspension loaded into flow cytometer

• laser —> emission —> detection

  • size of cells

  • granularity (represents the internal complexity, density, or cytoplasmic organelle content of a cell, measured by Side Scatter (SSC).)

  • fluorescence

Flow cytometry allows visualization and quantification of surface marker expression for individual cells

• In a dot plot visualization, every dot represents a cell

• Expression of two distinct markers can be visualized along the X and Y axis

• Cells expressing more of a given marker will fall further along the axis representing its associated color

Example flow cytometry analysis of T cells in distinct lymphoid organs

look at SLOs understand

Flow cytometry can be used to measure changes in leukocyte abundance and phenotype in research and clinical samples

Mice infected with Zika and leukocytes seen after infection - clear increase in CD4 and CD8 cells

Flow cytometric labelling of intracellular targets requires cellular permeabilization

Antibodies cannot cross the plasma membrane in intact cells

• permeabilization allows labeling of intracellular targets

• intracellular labeling can also be performed for cytokines (effector function)

  • use detergent or organic solvent

Fluorescence activated cell sorting (FACS) uses principles of flow cytometry to physically sort cell types of interest for downstream uses

Sorted cell populations can be used for specific cell biology studies (effector function, gene expression, etc.)

• load heterogenous sample containing cell type to sorted

  • positive charged plate - tube containing nontarget cell types

  • negatie charged plate - tube containing pure population of target cell type

Summary 10.3

Flow cytometry uses leukocyte antigens to label cells

• Samples must be digested before being labeled

• Leukocytes are then labeled using direct immunofluorescence

A flow cytometer resolves cells based on their size, granularity, and fluorescence

• Labeled antigens can be used to identify and count subsets of leukocytes

• Intracellular staining can be achieved following permeabilization with detergents

Summary 10.3 pt 2

FACS can be used in conjunction with flow cytometry to sort cells

- Target population is sorted using fluorescence and electrodes

- Sorted cell populations can be used for further studies

10.4

Vaccines

Childhood immunizations represent one of the most powerful tools in modern public health

Several pathogens have been eradicated or eliminated in the United States in part due to vaccination campaigns

• Polio eliminated in 1979, measles in 2000*

Concern for the safety and efficacy of vaccines is observed through modern history

Original findings associated with negative consequences of vaccination are now retracted

• Several studies have failed to replicate findings associated with Wakefield’s paper

Vaccines must meet several criteria to be considered successful

• Safe

• Protective

• Gives sustained protection

• induced neutralizing antibody

• induces protective T cells

• Practical considerations

MEMORIZE THIS

Formation of immune memory is a primary goal in vaccine development

1. Induces neutralizing antibodies

2. Induces protective T cells

Vaccines function by stimulating a comprehensive immune response without risks associated

with natural infection

Effective vaccine responses stimulate all parts of an immune response, including immunological memory

• Some vaccines use adjuvants, which typically strengthen immune responses by activating innate immune sensors

Live attenuated vaccines typically induce the most robust immune responses

Live attenuated vaccines use replication competent pathogens that have been attenuated (weakened)

• Live attenuated vaccines strongly activate both CD8 and CD4 responses

• Attenuation is typically achieved by growing the pathogen in non-human cells or genomic editing to reduce infectivity

Inactivated vaccines use pathogens that are unable to replicate or cause disease

Inactivated vaccines use “killed” pathogens that must be taken up by APCs

• Inactivated vaccines rely on cross presentation and more strongly stimulate humoral immune responses

• Inactivation can be achieved by UV treatment, heat, or chemical means

Subunit vaccines contain proteins associated with the pathogen

Subunit vaccines use pieces of the pathogen to elicit an immune response

• Subunits from different strains can be used together in one vaccine (Gardasil-9)

• Subunit vaccines typically require knowledge of immunodominant antigens

Nucleic acid (mRNA) vaccines encode for target antigen to stimulate immune responses

mRNA vaccines do not use any native part of the pathogen

• mRNA in lipid carrier enters cells locally and uses cellular machinery to make antigen

1. mRNA injected intramuscularly (from synthesized encoding SARS-COV2 viral spike protein)

2. Viral spike protein synthesized within transfected muscle cells

3. Immune response to viral spike protein produces neutralizing antibodies

Vaccines contain other components in addition to immunostimulatory agent

Vaccines may also contain trace components from manufacturing such as detoxifying agents and egg protein

Component: Adjuvant

Purpose: Stimulate innate immune responses

• ex. aluminum salts, oil in water emulsions

Component: Antibiotics, Perservatives

Purpose: ensure vaccine does not become contaminated

• ex. gentamycin, neomycin, phenoxyethanol

Component: Stabilizers

Purpose: improves shelf life in varying conditions

• ex. gelatin, sugars, magnesium salts

Beyond vaccine design, other factors influence vaccine efficacy

Host factors include age at vaccination and how well we understand host immune

responses

Pathogen factors include variability of pathogens (seasonally, strains, etc.), unknown immunodominant antigen (malaria, gonnorhea, syphilis), lack of rigorous testing models (HIV)

Non-biological barriers include infrastructure/access, prioritization, vaccine hesitancy

Vaccination efforts provide herd immunity to protect those that can’t be immunized

Certain individuals such as the very young, immunocompromised, and cancer patients cannot receive vaccines

Vaccination may reduce risk of non-infectious conditions such as cancer, dementia

HPV vaccination decreases risk of cervical cancer: 

Herpes zoster vaccination may lower risk for dementia:

Summary 10.4

Vaccines are powerful tools to limit the spread of pathogens

• Vaccination campaigns have successfully prevented millions of cases of disease

• Vaccines must generate cellular and humoral immune responses, memory

Vaccines take a wide array of approaches to generating immune responses

• Live and inactivated vaccines use whole pathogens

• Subunit and mRNA vaccines focus on specific pieces of pathogen