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Lecture 4

MMI436 Lecture Notes - Lecture 4: Influenza A Virus Subtype H3N2, Neutropenia, Neutrophil

Medical Microbiology and Immunology
Course Code
Paige Lacy

of 3
Monday, April 4, y
Neutrophil trails guide influenza specific CD8+ T cells
in the airways
Lim et al. (2015) Science 349:1071
-HKX31 (H3N2): lab derived influenza strain
-IV TPM- infrared penetrates tissue- two infrared photons excite fluorophore- emission
goes through detector- you get a much thicker section analysis- can be use like
immunofluorescence but you can do it with thicker tissues
-Fig 1. Reduced T cell response in HKx31 infected trachea of neutropenia mice
monoclonal antibody 1A8: to make neutropenic mice- get rids of neutrophils and
their functions
epithelial cells were the ones infected with the influenza virus: these cells then
are the ones that have the most neutrophils/CD8+ cells after Day 2-8 of infection
-Blocked neutrophils with 1A8 antibodies and then compared with IgG—> with flow
cytometry measured amount of CD8+ cells after neutrophil block.. CD8+ cells were
reduced when 1A8 was used
tetramer of the class I molecule + the peptide antigen: NP366* this is how the complex
binds directly to the T cells that see it- this is not an antibody but it is MHC + peptide:
these select specific T cells: recognize antigen specific T cells where you wanted to look
Local and systemic effects of neutrophil depletion
in the monoclonal treated mice:
no significant difference in the number of neutrophils in the spleen
but in the local tissues: lung and trachea: less T cells
Figure 2: Migration/Localization of CD8+ T cells
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Monday, April 4, y
CD8+ cells in the epithelial barrier
1A8: less infiltration into the tissue:
two phase microscopy of mouse trachea illustrates…: confirms the fact that the
localization of the CD8+ cells is compromised with the 1A8 treatment
quantification of velocity and displacement of CD8+ cells in tracheal epithelium
average velocity and displacement figure: epithelium- heterogeneity
some cells in IgG treated and 1A8: different treatments but cells have the same results-
why is this
depending where you are in the airway there is heterogeneity
Figure 3:
what is being released?
microarray- detected all the chemokines released: found 9 released from neutrophils, 6
interacted with T cells
CXL12: manipulated the movement of the T cells in a dose response
cxl12: binds cxcr4 on the surface of cells… signal-cascade that leads to migration
antagonist cxcr4: reduces the migration, reduces the amount of CD8 T cells recruited
control for tetramer: use DofB with another peptide and then don’t get any specificity-
negative control tetramer- what about other T cell reactive epitopes? + control
get 3 epitopes> are they going to add up more than the total T cells
—> total t cells: 6,000 total specific T cells: 2,400: very close to total—> weird!! almost
half of the total T cells are specific*
CXCL12 reporter mice: Dsred: shows the cxcl12 producing cells- epithelial cells and
neutrophils both produce it!
of the total amount of the cxcl12 a portion is because of neutrophils
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ensure that cxcl12 is from neutrophils: conditional ko mice: only knocks out CXCL12 in
the neutrophils: because of the cross with the ELA2 gene: elastase- expressed in
neutrophils specifically with cree: associated with a cell type specific protein—> cell type
that the protein is in gets delated
elastase- critical enzyme in neutrophils: targets ko to the neutrophil
effects of inflammatory stimuli on CXCL12 release in neutrophil
why add Heparin sulfate: interacts with ICAM in extracellular matrix: heparin sulphate—
> proteinglycan molecule in the ECM when cells migrate in the ECM these
proteoglycans are part of this—> with their usage they are trying to mimic the ECM
T cell chase and run model:
T cell migration doesn’t depend on neutrophil migration
but then with the coverslips: wash everything off the coverslip and left “trails” behind and
show T cell migration
neutrophil trail development depends on integrin activity
don’t say how integrin help develop the trails
how are the trails made? unknown
CXCL12 conditional ko: decrease in T cell migration- T cells are dependent on CXL12-
neutrophil derived chemokines*
Fig 6:
Fig 7: trail remains until day 8, even though virus is removed until day 8
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