Mimm 465 Lecture 3 Krawcyk

Front 1

Listeriosis

Back 1

1. rare but high fatality rate(20-25%)

2. high risk grp:

-pregnant women

-immunocompromised people

-elderly

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Listeria monocytogenes

Back 2

1. gram positive

2. non-spore forming

3. intracellular

4. found in meat, dairy, fruits, vegetables(un cooked, unpasteurized), sewage, water, soil

5. opportunistic pathogen

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Listeria infection of cells

Back 3

1. enters cell, internalized in vacuole or phagosome (phagocytes)

2. Escapes vacuole/phagosome

3. hijacks cellular actin polymerization machinery to move within the cell

Front 4

Escape of phagosome/vacuole

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1. Lowering of pH results in Listeriolysin ) oligomerization (D1, D2, D3)

2. formation of pre initiation beta sheet

3. pore oligomer

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ActA

Back 5

1. ActA is responsible for the hijacking of actin polymerization machinery

Front 6

Innate immune response to listeria

Back 6

1. internalization of bacteria --> LLO dependent destruction of vacular membrane --> MyD88 dependent innate immune response

2. MyD88-independent induction of CCL2 and CCR2 mediated recruitment of monocytes.

3. MyD88-dependent, TLR-mediated activation of monocytes

4. Differentiation of monocytes into TipDCs and iNOS induction and bacterial killing

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TLR NLR signaling pathways Listeria

Back 7

Listeria that cannot escape vacuole do not induce protective immunity

1. Peptidoglycan --> MDP(muramyl dipeptide --> NOD2 --> AP1

2. Peptidoglycan--> iE-DAP--> NOD1 --> NFkB

3. MDP/(L. monocytogenes) --> NLRP1/(3) --> caspase 1(converts pro IL-1beta to IL-1beta

4. PAMPs --> NF-kB --> IL-1beta, IL-18

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CLR, RLR and CDS signaling pathways of Listeria

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1. dsRNA--> AIM2--> caspase 1

2. dsRNA--> c-di-GMP/c-di-AMP + DDX41--> STING--> ISRE3 and NFkB

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Adaptive immune response to listeria

Back 9

1. Humoral immunity ineffective

2. CD8+ more protective immunity than CD4.

3. CD4+ Th1 cells contribute to protective immunity

4. Killed or mutant listeria that can not escape vacuole do not provide protective immunity

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Antigen presentation

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1. CD8+ T cell prefered (for intracellular pathogen)

2. CD4+ T cell for extracellular

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Antigen presentation to CD8+ T cells of L. monocytogene

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1. classical pathway(MHC-class-Ia-restricted presentation)

-LLO,p60 and Mpl peptides fragmented by proteasome

-MHC-class-Ia-restricted presentation T cells more effective for secondary infection)

2. Non-classical pathway(H2-M3-restricted presentation)

-N-formylated peptides

-More effective for primary infection

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Characteristics of model organism listeria

Back 12

1. pratical, grows easily, easy to infect, recombinant strain easy, safe.

2. requires innate immune for clearance, adaptive for protection against lethal challenge

3. good for inducing adaptive immunity/memory T cells

4. NOT GOOD FOR B CELL BIOLOGIST

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Reasons to genetically manipulate an organism

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1. Make recombinant strains to express model antigens to examine Ag-specific response

2. Recombinant strains to modify virulence factors to examine their effect on bacterial growth, pathogenesis, immuntiy

3. Can make untargeted mutations to identify novel pathogenesis factor, host response

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Model antigens

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1. They have well-defined epitopes

2. T cell antigens have defined cognate TCR and transgenic TCR

3. B cells have well-characterized Ab response

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Lymphocytic choriomeningitis

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model organism used by immunologist/microbiologists

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3 examples of model antigens

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1. Ovalbumin

2. LCMV gp

3. LLO190

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Reasons why model antigens important

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1. allow to study Ag-specific responses through various means

-microbes expressing model Ag

-Immunization w/ proteins containing Ag/adjuvants

-Protein-pulsed/peptide-coated dendritic cells

2. Tetramers used to identify Ag specific T cells

3. Peptides corresponding to Ag can be used to stimulate Ag-specific T cells

4. TCR transgenic T cells express TCRs with known specificity can be used to measure Ag-specific responses

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Detect Ag-specific T cells

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Those in upper right cadran are the Ag specific T cells we looking for in flow cytometry.

1. rlMova: recombinant strain of Listeria expressing ova. (ActA and WT most commonly used strain)

2. Kb/Ova257-264(Y axis) tetramer that detects class I restricted CD8+ T cells(recognizing ova 257-264 in the context of Kb MHC molecule.

3. B6 is strain of mice, express Kb

4. CD44(X axis) is activation marker of CD8 T cell

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Phenotyping Ag-specific T cell

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1. Abundance/presence

2. Expression of cell surface molecules

3. Proliferation/survival

4. Sort using multiple assays

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Analyze cytokine production by Ag-specific T cells

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1. Splenocytes from infected mice incubated with peptide

-LLO190-201(MHC II) stimulates CD4

-Ova257-264(MHC I) stimulates CD8

2. Brefeldin A added to prevent cytokine secretion. allow intracellular staining

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Analyzing phenotype of Ag-specific T cells

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1. Tetramer stain or peptide restimulation can be used to analyze.

-not both because tetramer binds to MHC receptor too.

2. Cytokine production can measure stimultaneously cell surface receptors for phenotypic and functional analysis

3. Polyclonal responses can be measured as well as transgenic TCR responses

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Th1 or Th2 memory cells provide help to CD8 T cells

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Th1 cells

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How to elicit specific T cells in mice

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Immunization with

1. Control DC

-no LLO-190 specific memory T cells

2. DC/P.acnes LLO

-no Th1 memory

3. DC/SEA LLO

-no Th2 memory

Then infect mic with Listeria-ova