Signal Transduction & Lymphocyte Activation Leitenberg January 22, 2014 3:00pm-4:00pm NT #6 I apologize for going fast.

It would help to read the outline and watch the videos and if you need to you can read the textbook. I also try and underline and bold the molecules or concepts that I think you should know. Q: Can you summarize the sequence of phosphorylations? 1. Ag binds 2. SRC family tyrosine kinases (which are different for B & T cells) phosphorylate ITAM motif. 3. Other tyrosine kinase enzymes are recruited to this site. SRC family tyrosine kinases phosphorylate and activate these enzymes. 4. These enzymes will go on to phosphorylate other enzymes to produce signaling cascade. For example, they will phosphorylate PLC, which cleaves PIP2 into IP3 and DAG. Q: When do the co-receptors come in? Co-receptors are a way of explaining how the SRC family tyrosine kinases access the Ag receptor when it is being ligated. Cells without these co-receptors do not work as well. There is a defect in how quickly they initiate these different cascades. Its a graded process (its not all or none) but this works A LOT better with co-receptors. Q: What are some examples of co-receptors? For T-cells the co-receptors would be CD4 & CD8. For B-cells the co-receptors are CD21 & CD19. Slide 40: What happens after activation of PLC? Phospholipase C cleaves this lipid (PIP2) into IP3 and DAG. IP3 engages with an IP3 receptor on the ER and on the plasma membrane, opens up Ca2+ channels at those sites and causes an increase of Ca2+ in the cytosol. Slide 41: Cyclosporin A is a drug that inhibits this pathway by interfering specifically with T-cell activation. Another related drug is FK5O6. Here you can see PLC is activated, cleaves PIP2 into IP3 and DAG and then you can see there is an influx of Ca2+. Then we have this molecule called calcineurin, which is a phosphatase that dephosphorylates NFAT, which is usually highly phosphorylated in the cytosol. When it interacts with calcineurin it becomes dephosphorylated and triggers NFAT to move from

the cytosol to the nucleus where it can affect gene transcription. If you give a patient this drug it will inhibit calcineurin activity blocks NFAT from getting to nucleus negative affect on gene transcription and T-cell activation. This drug has revolutionized transplantation. You can do kidney transplants now without being too careful about matching HLA type as long as you treat with cyclosporine, which will decrease T-cell activity. Slide 42: G protein signaling is another signaling pathway. These G proteins are so called because they are active when bound to GTP and inactive when bound to GDP. So this is sort of a molecular switch. There are other factors in the cytosol that determine the amount of GDP/GTP available for G protein. For example guanine nucleotide exchange factor will actually displace GDP from inactive GDP-G-protein complex and replace it with GTP to activate the G-protein. One of these G proteins is called Ras. This happens in lots of cells, but in lymphocytes it happens as a consequence of Ag binding. What I want you to know about this is that small G proteins play a role in signal transduction and that they catalyze the reaction of GTP to GDP. Slide 43: A downstream consequence of activating a G protein, like Ras, is the activation of MAP Kinase cascade. ERK is a result of MAPK enzymatic cascade and it will go on to phosphorylate a transcription factor in the nucleus, which will regulate gene transcription and protein expression. Slide 44: What is the end result of all of these signaling cascades? Through these signal cascades we can activate a variety of transcription factors. When these factors bind on the DNA in the nucleus they direct gene transcription. One important gene that is transcribed as a result of all of this signaling is IL-2 cytokine gene. This is really important for Tcells. When T-cells make IL-2 it can bind to itself or cells in the neighborhood and initiate proliferation. So we think that IL-2 is really important in the initial clonal expansion, survival, and differentiation into effector cells. Slide 45: The T-cell receptor is not shown in this picture but when the ligand binds to the T-cell receptor we will make IL-2 and also make more and better IL-2 receptor. Since IL2 can act in an autocrine (self) manner, the IL-2 will bind on those enhanced receptors and signal them to induce clonal expansion and proliferation. This is the first step in adaptive immunity and these IL-2 are also critical for differentiation of cells with different effector function. Slide 46: Why is A wrong? The question is specific for calcinuerin receptor. B is wrong because NFAT would be localized to the cytosol not the nucleus. D is wrong because this step is upstream from the calcinuerin effect. E is wrong because this is also upstream. We wouldnt expect anything upstream to be affected by what were doing in this question. The answer is C. I just told you this is what calcinuerin does. Slide 47: What weve talked about so far is really based on the Ag point of view. But there are other things that are also necessary for T-cell activation. The most important molecule for nave T-cell stimulation is called CD28. These are on the T-cell and they bind with a ligand called B7 (also called CD80) on the antigen-presenting cell. There is a

lot of cell-cell mediated interaction going on here. You have interactions outside of MHC:peptide and T-cell interaction. Slide 48: Now well talk about signaling through co-stimulatory molecules like CD28. We talked about pathogen recognition receptors when we talked about innate immunity. And well talk about signaling through cytokine receptors. Slide 49: This is a cartoon of the immunological synapse. We have talked a lot about MHC interaction with T-cells but we havent talked about this other stuff yet. There are a variety of other molecules that get engaged at the immunological synapse that positively and negatively regulate activation of T-cells. CD28 is one of these and it is constitutively expressed by nave CD8 T-cells. When CD28 recognizes MHC presented by a professional APC (with co stimulatory molecules) it will trigger the cell to express additional molecules on the cell surface that can further engage in interaction with MHC on the APC. CD40 ligand on the T-cell is another one of these molecules at the immunological synapse that interacts with CD40 molecule on the APC. There is bidirectional signaling going on here. Slide 50: Well talk about CD28 first. This is the two-signal model of T-cell activation. Signal 1 is a signal derived from the Ag receptor interacting with MHC-peptide complex. Another signal is required for nave T-cell stimulation and this is done via the CD28 molecule. This is essential to activate virgin T-cells in secondary lymphoid organs. Only professional APCs can do this, like we talked about before, because they express costimulatory molecules like the B7 molecule, which are required for interaction with CD28. This is less important for previously activated effector cells. Slide 51: What does CD28 do? Binding of B7 with CD28 initiates a variety of important molecular events. Binding promotes cell survival, protects the cell from programmed cell death, promotes transcription of IL-2 so the cell can make more and better IL-2 and there is also rearrangement of the cytoskeletal framework to optimize the immunological synapse. Dont worry about the specifics here. CD28 is important for naive T-cell activation. Slide 52: Adhesion molecules also play a key role. LFA-1, presented on the surface of Tcells, interacts with ICAM-1 on APC molecules and maintains a long-lived interaction between these two cells. We talked about these interactions in another context earlier where they were mediating leukocyte migration. Slide 53: When the T-cell is in a lymphoid organ, scanning the microenvironment, the Tcell can get activated by adhesion molecules. Adhesion molecules, like LFA-1, can loosely interact with the ICAM on APC, and if, at the same time the T-cell recognizes an MHC-peptide complex you will get a strengthening of this reaction. This is called inside-out signaling. There is something going on within the cell that changes the interaction of MHC-peptide and T-cell on the outside of the cell. The change in affinity of LFA-1 for ICAM-1 is changed by something intrinsic to the LFA-1 molecule. Slide 54: The 2 signal model also works for B cell activation. The first signal would be the B-cell receptor interacting with Ag and the second signal would be CD40 molecule

binding to CD40 ligand on the B-cell. The B cell is activated by Ag binding which results in signaling events. This facilitates Ag presentation of the Ag molecule by a helper Tcell. The Helper T-cell and the B-cell will physically engage and the T-cell will become activated to express CD40 molecule on its surface. This CD40 molecule will interact with the CD40 ligand and will help the B-cell to become further activated via promotion of Ig switching, development of memory function, and germinal center formation. Slide 55: The same principle applies when T-cells interact with macrophages and dendritic cells. These cells also express CD40 ligand. Consequence of activation of this system for those cells might be production of pro-inflammatory cytokines. This is how the adaptive immune response feeds back on the innate immune response. Slide 56: This is the structure of CD40 engaging with CD40 ligand family members. CD40 ligand belongs to the Tumor necrosis factor receptor superfamily. This family is responsible for activating transcription factor, NFK-B. Depending on the cell that NFK-B is activated in will determine the effect. Activation of NFK-B in T cells and B cells promotes cell survival. In macrophages and dendritic cells, activation of NFK-B promotes cytokine production. This is a common way that cells get signaled. We can develop drugs to interfere with this pathway. Slide 57: CD40 signaling is important for B-cell activation and for T-cell help in B-cell activation but there are some Ags that dont go this route. We refer to Ags that activate the cell via PRR and B-cell receptor interaction alone, as T-independent antigens, which can activate these other guys independent of T-cell help. There is no CD40 signaling here. These interactions dont generally promote isotype switching, germinal center formation, or development of memory function. Slide 58: This is an example of how Toll like receptors signal. They use an intracellular molecule, an adaptor protein, that acts as a bridge between the receptor and additional cascade signaling events that ultimately activates NFK-B transcription factor. MyD88 is an example of one of these adaptor proteins, which will result in NFK-B activation. Just know that PRRs can result in NFK-B activation and MyD88 is commonly involved in this. Slide 59: Class I and II cytokine receptors regulate a lot of important immune responses. One example is the receptor for IL-2. Class I and Class II receptors all use the JAKSTAT signaling pathway. Slide 60: The cytokine engages with cytokine receptor, stabilizes the receptor and activates tyrosine kinases called JAK kinases, Janus associated kinases, so named after the mythological creature with two heads. These two JAK proteins oligomerize together to activate each other and the cytoplasmic tail of the receptor via phosphorylation. This recruits a STAT protein to the site of cytokine receptor signaling. The STAT molecules then get phosphorylated and dimerize. Then they get transported to the nucleus where they can act as transcription factors. This is a simple pathway. There are different STAT proteins that regulate different patterns of transcription. Different cytokines will signal different JAKS and STATS and therefore cause transcription of different genes.

Slide 61: Chemokine receptors are important and regulate where cells go in the body. This is done via mediation by large G proteins, as opposed to the small G proteins that we talked about earlier. The large G proteins are assemblies of different subunits of alpha and beta chains but they do the same thing as the small G proteins. Their activity in initiating signal transduction pathways is dependent on that switching of GDP-GTP. Slide 62: There is also negative signal transduction. Ex: the Fas molecule mediates cell death via down regulation of immune responses. This occurs when the Fas molecule trimerizes and initiates a signaling cascade with the result of apoptosis. You end up killing off activated cells. Slide 63: These signal transduction events determine proliferation, survival, and activation. These events are not necessarily distinct from each other. They may be happening at the same time, or over several days and you can have repetitive and simultaneous engagement of these receptors. Slide 64-66: How does this work? Within the cytoplasmic tails of inhibitory receptors there are ITIM motifs instead of ITAM motifs. When these ITIM motifs are phosphorylated as a result of Ag binding at receptor the signaling cascade is inhibited rather than propagated. When ITIM motifs are phosphorylated they recruit phosphatases, which cleave the phosphate from tyrosine kinases to turn them off. Slide 67: Another way to down regulate signaling is that you can degrade the proteins involved in these pathways. One way to do this is to tag these proteins with a protein called ubiquitin, which marks that cell for denaturation, by a proteosome. Ubiquitin ligases (ex: CBL) add ubiquitin to these components. If you add ubiquitin to ZAP70, a signaling molecule, you are marking it for destruction. If cells have less ZAP70 they wont be signaling as much. Down regulation of signaling can be important in autoimmune disorders. Down regulation of CBL, and therefore lower levels of ZAP70 will cause these cells to be less active, and this is example of anergy. You can do this in mice and potentiate autoimmune disease. Slide 68: The answer is A. Slide 69: This is the outline of everything that we talked about today. And some of this stuff we will revisit as we go along in the course.