13_14 Biochemistry Dr.

Leitenberg 1/27/14 14:00-15:00 Notetaker 20 Lecture 7 - Cell Immunity

Slide 1 The immunology exam is coming, so to orient everywhere for time and space, today is T cell day and Friday is B cell day, then we have a review session next week and the exam is the Friday after the review session. You should start to go through Immuno after biochem to ID things you are really not understanding and ask me questions - you can use the blackboard forum section and you can post them anonymously. This way I dont need to answer the same question over and over.

NOTETAKER NOTE: For the acronyms I will use the full term the first time with the acronym in parentheses, then from there on out I will use the acronym. Ex: I ate a Banana Sandwich (BS). The BS tasted great.

Slide 2 The other part of today is historically different than the T cell lectures that I usually give because it includes some content that I didnt have a chance to give during the first session [that we missed]. So I added content, and to really piss you off, the powerpoint handout that you got has maybe three slides that are different (dont worry, I included them into this noteset). Its new information that is in the newer edition of textbooks, so I decided to include it into this lecture. That being said, this is a fundamental lecture on how the adaptive immune system works, so even if you are not here in person (thats you, lovely reader) I hope you enjoy the video and focus on the steps involved in T cell regulation, and the molecular basis by which T cells develop different cell fates (how they know what effector responses to mediate). So this is a big picture slide just to make the point that it all comes down to initially the recognition of T cell Antigen (Ag) receptors and co-stimulatory signals, and how those signals are stimulated by the T Cell Receptor (TCR) engaging with Major Histocompatability Complex (MHC) peptide complexes on Ag Presenting Cells (APC). This stimulates a bunch of stuff. One of those things is to clonally expand and proliferate. We talked about how a big part of that is to make Interleukine 2 (IL2). Now today we are going to talk about how these cells proliferate and differentiate into different T cell subsets - so how a naive T cell becomes a cytotoxic T cell - and how this process is regulated.

One of the things I am NOT gonna talk about today that we will get to after the midterm is negative regulatory cells that kind of tune this process down to inhibit it so we all become one giant lymph node (LN). The big picture is think about the naive being stimulated that may differentiate into an effector cell phenotype, but the major part is remembering how many of these cells will survive a long time and participate in a secondary response. This is what we refer to as immunological memory (and these are memory cells). Slide 3 T cell mediated immunity This slide is the session objectives. One additional objective that I think was not on here was to review a bit about T cell homing to LNs and a little bit about how T cells find Ag and engage in APCs. Slide 4 How do T cells find antigen? The first question is how do T cells find Ags in the first place? And remember homing and adhesive interactions are really important to mediate recirculation of lymphocytes into secondary LN organs, and they come from the blood and when they enter these secondary lymph organs, they may change their pattern of adhesive molecules and migrate to sites of peripheral interactions. These adhesive interaction mediate cell-cell contact on top of migration to different sites. Remember chemokines are important and Ag are brough to Lymphoid organs via lymphatic drainage. Slide 5

This slide is your secondary Lymph organs like LNs, which are all over your body. And if a cell doesnt see an Ag, it will leave, and it will continue to circulate looking for Ag that will engage the Ag receptor. If it does, then it will drain through the afferent lymphatics into the LN where it will engage with a T cell (in this case), and if it stimulates it, it will leave the LN and make its way back to the site of inflammation on the peripheral tissue.

Slide 6 The Lymph Node The LN has different regions that have follicles (B cells live here) and paracortex (T cells live here). These cells have to interact with each other in ways that we will learn about today and friday, but the bottom line is that the LN is the meeting site for naive cells and Ag.

Slide 7

The spleen is a good example of secondary lymph organ - it filters the blood stream and some Ag find their way into the white pulp areas (lymphocyte areas), which act as ming lymph nodes, and it all works the same way, except there is no lymphatic drainage. Slide 8

This is a cross-section of the white pulp - you have the B cell follicle area, the T cell area around the central arteriole, but otherwise its similar to a LN. Slide 9

This is what a real and cartoon LN look like. There are B cell areas and T cell areas again and a cortex. We will talk about how lymphocytes get into the LN and how Ag gets into the LN.

Slide 10 The first part is lymphocytes enter secondary lymphoid via the bloodstream. One way they know theyre going through the LN is that there are specialized epithelial cells called High Endothelial Venule (HEV). This is a scanning electron micrograph of a HEV with lymphocytes stuck on the surface. Slide 11

This process works similarly to how neutrophils leave the blood stream and enter the peripheral sites. It has similar molecules like selectin, with integrins interacting with members of the Immunoglobulin Supergene Family. Its the same kind of three step process you have rolling, stopping mediated by chemokine-chemokine receptor interaction making the molecule bind with higher affinity, and then in response to chemokine gradient, the cell gets into the parenchyma of the liver.

Slide 12 This is a summary of the three major classes of molecules. Selectin - the ones that are important for mediated lymphocyte homing to secondary lymphoid organs are L-selectin (L for Lymphocyte). And what we will learn in a second is that whether or not cells express this will regular whether or not they will go into the LN. If they dont express it, they are less likely to go into LNs and more likely to go to other places. One way the selections interact with carbohydrate ligands are through addressins. Integrin - one type is the 2 integrins (ex: LFA-1, which binds to ICAM). Another example not a member of the 2 family is the VLA-4, which are expressed on lymphocytes only activated cells are activated and will mediate homing to sites of peripheral inflammation.

A lot of times these molecules interact with ICAM - its a member of the Immunoglobulin Supergene family (it has a conserved immunoglobulin loop structures formed by intrachain disulfide binds).

Slide 14

Here is a LN, and there is a whole network of lymphatic vessels that drain the space under the barrier surface into the LN. These vessels going into the LN are Afferent lymphatic vessels. It brings not just Ag, but also cells, and these cells often internalize the Ag and bring it into the lymphatic tissue.

The other lymphatic vessel leading out is the Efferent lymphatic, and thats how cells get out. Lymphocytes will drain out here if they dont react, which will get back into the blood.

Slide 15 When you get a splinter, you induce inflammatory responses and you activate inflammatory cells, and a consequence is you activate APC. And one of these cells thats particular good at this is Dendritic Cells (DCs), and in response to pattern recognition signals they get activated and will phagocytose Ag that it sees here and bring them through the afferent lymphatics into the LN.

Slide 16

This shows some of the changes that DCs undergo once they get activated by pattern recognition receptors. One thing that is characteristic is they are really good phagocytic cells, but they are really crappy at presenting Ag. Part of the reason they are so crappy is because they dont express high levels of MHC molecules (I or II), and in green we see a micrograph of MHC expression on DCs, and one thing we see is that when they are sitting in the tissue and they are not activated, a lot of the MHC II is intracellular and not expressed. But when stimulated, the cell makes more MHC II and presents more than it has on the surface when it migrates from the peripheral to the LN. It expresses lots of MHCII which makes it better to stimulate Naive T cells. These cells express both MHCI and MHCII. Remember that all nucleated cells express MHC I.

Slide 17 This is a cartoon of the molecules involved in regulating this process. A critical one is Pattern Recognition like Toll-Like Receptor (TLR) or Lectin-like molecules that engage with pathogen associated molecular patterns which allows it to get activated. One consequence of this is the production of chemokine expression that allows it to move from the peripheral sites to the LN in response to the chemokine gradient. A critical chemokine receptor to know is CCR7 and when that engages with its ligand it allows cells to move to the LN from the peripheral. Slide 18

This is a cartoon of the DC once its in the LN. Now it has all it needs to stimulate the T cell. It needs: High levels of MHC and co-stimulatory molecule (B7-1 (also known as CD80), B7-2 (also known as CD86)), which engages with CD28 on the surface of the T cell. The combination of these signals plus cytokines, results in the activation of this T cell. Slide 19

Here it is all together. T cell gets into LN and samples the environment in the paracortex trying to find a Ag for its Ag receptor. The DC are expressing an peptide MHC complex on its surface, and if the T cell expresses a receptor that recognizes that complex in a sufficiently strong way, it will stimulate the cells and it will proliferate. However, the T cells will leave the LN through the efferent vessels if it does not find a receptor and go back into circulation.

Slide 20

So imagine if the T cell in the region is sampling peptide MHC complexes trying to engage its T cell Ag receptor. Most of the time you can have a low affinity for the reactant, but its not enough so it will move on. But sometimes if it does find something to stimulate it, signal transaction events (that we learned about in the last lecture) will happen, and one of those events is the inside-out signaling that increase the affinity of LFA-1 to the ICAM-1. So its a stronger interaction between the T cell and the APC. This will allow those cells to have a more long-lived interaction at that site. This subinterface is called immunological synapse. This is maintain in part between the high affinity interaction between the two adhesion molecules.

Slide 21 So you have your immunological synapse and youre getting signaling in both directions and this is a central event in regulating what will happen to both the T cell and sometimes the APC. So you get signals through T cell and signals through the co-stimulatory molecule CD28, and you get signals through cytokines that interact with cytokine receptors. And the type of signal that the T cell will see is based on the type of signals the DC sees because it samples the micro environment and its Pattern Recognition Receptors (PRRs) are being activated. Its allowing that cell to do things that is couldn't before the PRR were activated. Slide 22

This is a slide you've seen before. You have different levels of interaction between the T cells and the APC. These things happen quickly because the T cell receptor and the CD28 and constitutively expressed and they engage with MHC and B7 molecules expressed on this APCs. This results in T cell activation. One of the central things that regulates this interaction though is that once the T cell is activated it can express CD40 ligand that engages with CD40 on the APC surface, signaling the APC to do what these cells do (if its a B cell, it will tell it to do B cell things, if its a T cell it will tell it to do other T cell things). Regardless, CD40 signal can be thought of as a secondary or co-stimulatory for the APC, and its an important way that shows how the T cell helps activate the cells that its interacting with. Slide 23 This is all review. Professional APC are professional because they express high levels of MHC (especially MHC II, but also I). Thats a way of providing signal I to the T Cell via T cells Ag Receptor. They also express co-stimulatory molecules like B7 that interacts with CD28 (signal II). And cytokines can also be present (signal III). Think of these not happening in a linear array, although all required, but they may happen at different time points again after activation. Slide 24

DC are really the most important cells in activating Naive cells, and there are a fair amount of studies, mostly on mice, where you can remove different types of APC and see what is most important. This shows that for Ag, DCs are most important for activation of Naive/virgin cells. Its not to say like other cells cant do it, there are many times when the other cells play an important roll. This is a particular example of when a Macrophage (MO) is stimulated. The MO samples the environment and phagocytosed bacteria, then breaks down proteins that are expressed as peptides on the surface. This is activated because PRR and the cell expresses B7 and engages with CD28 and this is sufficient for the T cells to be activated. Slide 25

You can think of B cells in a similar way - they are also considered professional APC and express MHC II and co-stimulatory molecules. One situation this occurs is when B cell Ag receptor is ligated by antigen, which induces signal transduction events to activate the cell. The other thing that happens is that the B cell receptor Ag complex gets internalized into an endozomal compartment, and the proteins that were bound by that receptor get degraded into peptides, presented by MHC II, and then if you have a T cell with a receptor for that molecules, it is stimulated and in term provides co-stimulatory help for the B cell. We will go over this over and over again so you will see how this works.

Slide 26

One cartoon that people like to show is this, and about the different roles here, does it matter where they are? One of the things you can see is that the DCs are prevalent throughout the paracortex of the LN, and thats partly why they are so good at activating naive T cells in addition to having super high MHC molecules and super high costimulatory molecules. MOs tend to not be as good or prevalent at activating naive T cells, but may play a roll and are present in secondary lymphoid organs. B cells are more present in the follicle and we will talk more about how B cells and T cells meet up in the follicle in later lectures and a little bit today.

Slide 27

The central point of this is that if the APC is capable of (it has high levels of MHC and costimulatory molecules) it can activate a naive T cell, whether it is CD4(helper) or CD8 (killer), and this slide shows that one of the initial things that happens when it is activated it makes IL-2 cytokine and expresses the high affinity form of the IL-2 receptor. The cytokine feeds back in an autocrine fashion and can interact with other T cells around and activate them, which is important for clonal expansion/T cell proliferation.

Side Notetaker Memory Tool (ignore when needed): the rule of 8s. MHC I is related to CD8. 1 x 8 = 8 MHC II is related to CD4. 2 x 4 = 8

Slide 28

Which structure is most important for lymphocyte entry into the lymph node? A Lymphocytes get into the LN via the blood stream by recognizing ligands on HEV. Afferent Lymphatics - this is how Ag get into the LN through defects and such. APCs like DCs will also get in through here. I ask question like this on the test. Slide 29 Lymph Nodes (LN) enlarge during an upper respiratory tract infection because: (more than one answer may be correct): What are they feeling these LNs for/ why do LNs get big? They get big because you have activation of lymphocytes there that clonally expand. B is the best answer. There may be a second best answer in that cells can be migrating through all the time, so if they have receptors they can increased migration, but by far B is the best answer.

Slide 30

These cells are clonal expanding, differentiating, then leaving the lymph node through the efferent lymphatic vessels. When they leave they will change their expression so they wont go to another lymph node, but they will go to another site (i.e. where you got your splinter). Slide 31

Now lets talk a little about what decides whether or not lymphocytes stay or go in the LN. This process is in part regulated by the expression of a particular receptor called the Sphingosine 1 Phosphate receptor (S1P) and its interaction with the sphingosine molecule. What happens is that these naive T cells that just got here express low levels of this receptor, which prevents its egress (into the efferent vessels). Thats why it hangs out for a while looking for Ag, but once its been there for a while, then it will up regulate the S1P receptor, allowing it to engage with sphingosine and allowing it to leave the LN. This is something that also happens once the cell gets activated. So either way, if they dont get activated or if they do, they will have higher levels of SP1 receptor, which will allow it to engage with the molecule and leave through the efferent lymphatics. Why even bother telling you about this, it seems like a trivial detail. There are drugs that take advantage of this interaction that are immunomodulatory that are clinically used. One thing that has been developed clinically is actual molecules that interfere with the SP1SP1 receptor, and you would think that blocks lymphocyte exit from the LN. Slide 32

In addition to the SP1 receptor molecule, there is a change a bunch of other proteins are expressed when comparing a virgin to an active molecule. This is a list of those changes. Let me point out some of the key ones: One thing we talked about already is how naive CD4 T cells express L-selectin, which allows it to get into the LN. Once activated, they down-regulated its expression, so once they leave the LN and enter the circulation, they cant get back in very well and are more likely to go somewhere else. This is because they express different adhesion molecules when activated. One example of that adhesion molecule expressed after activation is VLA-4 (Very Late Antigen-4) molecule that engages with the VCAM-1 (Vascular Cell Adhesion Molecule 1) adhesion molecule expressed on activated endothelial cells. Remember we talked about this way back when in the innate immunity lecture - how TNF (Tumor Necrosis Factor Alpha) at sites of peripheral inflammation and some other innate cytokines act on the endothelial cell to make them sticky - one of the examples of that is the VCAM molecule. That allows cells to get into site of peripheral information. Let me skip the rest and go over to this next one, because you often hear people comparing resting and activated cells. Stimulated cells change their ectodomain of the CD45 molecule, so one way to tell how many activated vs resting cells you have in your body is by labeling these sells with Antibodies (Ab) that recognize either CD45RA or CD45RO, and people use that as a rough measure of how many Ag experienced cells you have. Slide 33

This is an adhesion molecule that I talked about before - again you have a naive T cell with L-selectin expressed (also called CD62L, but he always calls it L-selectin) engaged with addressins, which allows it to get into the parenchyma of the LN. If activated, it down regulates L-selectin and up regulates VLA-4, or it can engage with VCAM expressed on the activated, inflamed endothelial cells. So thats how it can regulate where cells go in the body.

Slide 34 In an effort to develop novel approaches to treating multiple sclerosis (demyelinating disease-associated with CNS lymphocyte infiltration), a pharmaceutical company asks you to identify targets that will affect lymphocyte homing to peripheral sites (e.g. CNS), but have little affect on migration to secondary lymphoid organs. Which of the following molecules would be attractive targets for this approach? More than one may be correct (oopsie daysie), but on test questions it will only be one (hmmmWalker?). Correct: D, C Both interfere with lymphocyte homing that are used clinically like Multiple Sclerosis. L-Selectin is wrong because it mediated homing to LNs. LFA-1 is wrong because it mediates homing to LNs as well as other places. Vascular Addressins would be ligands for a variety of selectness including L-selectin, so it wouldnt be specific for these peripheral enflamed endothelial cells.