Skin Deep: Illuminating our body s immune defenses. Vinidhra Mani PhD Candidate in Immunology Mempel Laboratory, HMS Wednesday March 23, 2016

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1 Skin Deep: Illuminating our body s immune defenses Vinidhra Mani PhD Candidate in Immunology Mempel Laboratory, HMS Wednesday March 23, 2016

2 Skin: Our first line of defense

3 Why is this important to study? Fight disease Boost our barrier defenses through vaccination

4 Roadmap for the Evening 1. A Resilient Barrier Defense The immune system in the skin 2. The Immunologist s Toolbox How we study skin immunity 3. Generating Memory What do scientists need to know to make better vaccines? (my project!)

5 Roadmap for the Evening 1. A Resilient Barrier Defense The immune system in the skin 2. The Immunologist s Toolbox How we study skin immunity 3. Generating Memory What do scientists need to know to make better vaccines? (my project!)

6 What would you want your skin immune system to do?

7 The skin immune system Keeps pathogens from invading our bodies Kills infected or abnormal cells Senses potential invaders and alerts the rest of the immune system Cleans debris from war zones or normal cell death Heals wounds Prevents the body from attacking itself and more!

8 Players in skin immunity Epidermis Microbes Dendritic Cell Survey for bugs Educate other cells Memory T Cell Remember bugs Recruit help Dermis Killer T Cell Kill infected cells Bacteria: Pixabay B Cell Make antibodies Helper T Cell Recruit help Instruct other cells Macrophage Clear debris Heal wounds Recruit help

9 Skin memory T cells Important for immune response to pesky pathogens (e.g. Herpes Simplex Virus) Specifically recognize and remember certain pathogens Quickly alert surrounding cells to recruit more help Constantly survey the skin Skin Memory T cell Dendritic cell Long-lived (lifetime?)

10 How do immune cells know what to do and when to do it?

11 Cell structure and function Proteins Carry out functions inside and outside of the cell Cytokine Second Messenger Nucleus Control center Contains genetic material Receptor

12 Immune cells sense local cues through receptors Cytokine Ligand Cell-cell interaction Receptor Each cell has over hundreds of types of receptors!

13 Signals can be relayed by a second messenger Second Messenger Nucleus

14 Skin memory T cell responds to infection Survival signal Infected cell

15 Immune cells sense and respond to local cues Make more/different proteins Cytokines Migrate Kill infected cells Heal wound Become a memory cell???? Cell-cell interactions

16 If a cell gets many different signals through its hundreds of receptors how does it decide what to do?

17 Study of immune cell decision making Wikimedia Commons: Pete Souza, White House

18 How do killer T cells become memory cells?

19 Part I Summary The skin is composed of diverse immune cells which keep us disease-free Memory T cells in the skin are important for quickly responding to and preventing the spread of pathogens All immune cells integrate many signals and respond accordingly- we are studying how this happens!

20 Questions?

21 Roadmap for the Evening 1. A Resilient Barrier Defense The immune system in the skin 2. The Immunologist s Toolbox How we study skin immunity 3. Generating Memory What do scientists need to know to make better vaccines? (my project!)

22 Models to study the immune system Pros in vitro Use of human cells No use of animals Cons Does not capture complexity of signals Pros in vivo Allows us to understand complex dynamics/signals Cons Imperfect correlation with human immunity Wikimedia Commons: Unknown, Kaibara87

23 Studying signals in the immune system 1. Change/modify signals Modify production or receipt of signals or responses by immune cells 2. What happens when you alter signals? Monitor functional changes in immune cells as a result of modulating signals

24 How are we able to alter and study how signals are integrated by immune cells?

25 Recall: Proteins Allow cells to receive signals and carry out functions Can come in the form of second messengers, receptors, ligands, cytokines and more We can alter proteins or their function and study the effects on immune cell function.

26 Genetic manipulation allows us to alter proteins Central Dogma DNA RNA Protein (e.g. second messenger) Wikimedia Commons (scissors)

27 Block protein function by drug or antibody Antibody: protein that binds another protein, alters its function Drug: small chemical that binds a protein to alter its function

28 Studying signals in the immune system 1. Change/modify signals Modify production or receipt of signals or responses by immune cells 2. What happens when you alter signals? Monitor functional changes in immune cells as a result of modulating signals

29 How do we analyze changes in function upon altering signaling?

30 Recall: Immune cell responses to signals Make more/different proteins Cytokines Migrate Kill infected cells Heal wound Become a memory cell???? Cell-cell interactions

31 Flow cytometry lets us analyze cell proteins Fluorochrome 1. Isolate cells 2. Label cells with antibodies in different colors 3. Measure amount of each color

32 Energy Level Fluorescence Phycoerythrin (PE): fluorochrome from red algae Absorption Emission (e.g. Fluorescence) Ground State Green Fluorescent Protein (GFP) from jellyfish Wikimedia Commons: Darekk2/PDB, Zephyris

33 Flow cytometry lets us analyze cell proteins Fluorochrome 1. Isolate cells 2. Label cells with antibodies with different fluorochromes 3. Measure amount of fluorescence

34 Flow cytometry helps us characterize cells based on fluorescence Wikimedia Commons: Kierano, Whimchuck

35 Protein A Fluorescence Immune cells can be identified by specific combinations of proteins they make Memory Cells A B Protein B Fluorescence

36 Proteins can tell us about cell functions Granzyme and Perforin: ability to kill an infected cell Integrins: ability to interact with another cell Cytokine receptors: ability to respond to specific signals and much more!

37 Flow cytometry uses and limitations Responses to Signals: Make more/different proteins Migrate Communicate with other cells Flow Cytometry Flow cytometry gives us a rather static view of a very dynamic (constantly-changing) system.

38 Two-photon intravital microscopy (IVM) Two-Photon: two packets (photons) of energy (light!) Intravital: inside of a living animal Microscopy: visualizing/magnifying small objects

39 Two-photon IVM to study skin immune cells 1. Label cells with fluorescent proteins or fluorochromes. 2. Excite fluorescent proteins deep in the organ using a laser. 3. Take movies with the microscope!

40 It takes up an entire room!

41 What can two-photon IVM show/tell us? Real-time, dynamic readings of: Cell-cell interactions Migration of cells in an environment and more! (if used creatively)

42 Limitations of two-photon IVM Expensive Time-consuming Limited tools/reagents for use currently

43 Part 2 Summary We can manipulate genes or block protein function to modulate signals taken in by immune cells Flow cytometry can reveal the proteins expressed by single cells in the skin Two-photon intravital microscopy allows us to see real-time changes in immune cell function

44 Questions?

45 Brief Intermission

46 Roadmap for the Evening 1. A Resilient Barrier Defense The immune system in the skin 2. The Immunologist s Toolbox How we study skin immunity 3. Generating Memory What do scientists need to know to make better vaccines? (my project!)

47 Recall: Skin memory T cells Important for immune response to pesky pathogens (e.g. Herpes Simplex Virus) Specifically recognize and remember certain pathogens Quickly alert surrounding cells to recruit more help Constantly survey the skin Long-lived

48 How do killer T cells become memory cells?? Generating memory is the basis of most vaccines We want to know how to make better vaccines

49 Useful information for vaccine development 1. What signals do cells receive to become memory cells? 2. Where do these signals come from?

50 Recall: Studying signals in the immune system 1. Change/modify signals Modify production or receipt of signals or responses by immune cells 2. What happens when you alter signals? Monitor functional changes in immune cells as a result of modulating signals

51 What signals are needed to make a memory cell? Hypothesis: A cytokine called TGF-βis needed for killer cells to become memory cells. TGF-β Approach: Block TGF-βor the ability of T cells to respond to TGF-βand see if we make memory cells. TGF-β Receptor?

52 How do we know if we generate memory? Surface-Level Properties: Look by flow cytometry of skin cells for proteins expressed by memory cells A B Function: Upon challenge with a pathogen, see if the animals are able to fend off the bug Wikimedia Commons: Unknown

53 Protein A Fluorescence Recall: Memory cells make protein A and B Memory Cells A B Protein B Fluorescence

54 Protein B Fluorescence What does blocking TGF-βdo to memory cell generation? No blocking of TGF-β Blocking TGF-β Protein A Fluorescence Protein A Fluorescence

55 Useful information for vaccine development 1. What signals do cells receive to become memory cells? TGF-βis one signal 2. Where do these signals come from? Use two-photon IVM to find out!

56 How do we learn more about TGF-βsignals? Require a fluorescent protein/molecule that can report when a T cell receives TGF-βsignals There is a second messenger called Smad which goes into nucleus when a cell receives TGF-βsignals Smad

57 Smad-GFP reports TGF-β signals in T cells Smad GFP *Nucleus marked by red fluorescent protein fused to a nuclear protein* Fusion Protein Reporter Smad GFP

58 Visualizing TGF-β signals in T cells in vivo No signal Signal

59 Visualizing TGF-β signals in T cells Dendritic Cell Smad-GFP/T cell nucleus Control cell nucleus No signal Signal

60 Quantifying the amount of signal over time

61 What can we learn from IVM of Smad? Where and when T cells take up TGF-βsignals How TGF-βsignals might change in different contexts How TGF-βsignals can alter immune cell function Use this information to identify more signals and sources that contribute to creating a memory T cell.

62 Useful information for vaccine development 1. What signals do cells receive to become memory cells? TGF-βis one signal 2. Where do these signals come from? Work in progress!

63 Part 3 Summary TGF-βis an important signal for making memory T cells I study how T cells take in TGF-βsignals using two-photon IVM using fluorescent reporters Studying the nuances of TGF-βsignaling can aid vaccination efforts and beyond

64 Summary Skin immunity is a complex, robust system that maintains a balance and protects from disease Immunologists use a variety of tools to study signaling in the immune system Modulating and studying signaling in immune cells can inform us of approaches to use for vaccination

65 Thank you! SITN would like to acknowledge the following organizations for their generous support. Harvard Medical School Office of Communications and External Relations Division of Medical Sciences The Harvard Graduate School of Arts and Sciences (GSAS) The Harvard Graduate Student Council (GSC) The Harvard/MIT COOP

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