hello and welcome back to week four of applied immunology this is lecture 4b where we will be covering t cell development we will talk about the processes involved in the maturation or development of t cells in an organ called the thymus and these include things like beta selection positive selection and negative selection which all work by testing the functionality of tcrs that are generated through vdj recombination mechanisms today's talk will build off of our first lecture from this week on antigen receptor generation and lead nicely into our next lectures centered on learning how mature t cells undergo signaling transduction pathways in order to become activated in response to cognate antigen we've seen this differentiation plot on the left a few times by now which shows that hematopoietic stem cells in the bone marrow give rise to common lymphoid progenitors or clps from which all major lineages of lymphocytes are derived from again t cells express a tcr that can only bind to foreign peptide presented by a self-mhc molecule and upon activation often proliferate and then exert effector functions the first question that we have today is how exactly does a common lymphoid progenitor transition from a multipotent state to differentiate into a functional t cell we're going to answer that question today but it's important to keep in mind that the functionality of a given tcr expressed by a t cell must be determined by two main selection criteria the first is that the tcr must be capable of recognizing foreign antigen presented by peptide mhc as we've established in the previous few lectures in order to do this the tcr must also weakly recognize self-mhc since the tcr binds to both the presented peptide as well as a portion of the mhc molecule itself this is referred to as mhc restriction which we first learned about in our week three lectures covering antigen presentation the second criterion for functional t cells is that they must be non-reactive to self-antigens that are expressed in healthy tissue meaning that they must not be autoreactive so a given tcr must be able to weakly recognize self-mhc but it can't react strongly to self-peptide presented by self-mhc in order to avoid autoimmunity and this avoidance of self-reactivity is the concept of immune tolerance that we've introduced before in this course and is a key aspect of successful adaptive immune development now that we've established these two essential criteria of a functional t cell let's learn more about the specific stages of t cell development from a common lymphoid progenitor stem cell let's first look at a schematic that summarizes what must happen to a multi-potent lymphoid stem cell in order for it to develop into a functional t cell that meets these two requirements this begins in the bone marrow which if you remember back to our week one lectures is categorized as a primary lymphoid organ hematopoietic stem cells in the bone marrow differentiate into common lymphoid progenitors or clps which are also primarily retained within the bone marrow these clps can give rise to any lineage of lymphoid cell which is why we refer to them as multi-potent stem cells a clp undergoes commitment towards a b-cell lineage when it expresses the transcription factors e2a and ebf in combination with the sensing of a cytokine called il-7 which we're going to discuss in more detail next week during our module on b-cells we've already learned that a clp can also undergo commitment towards an innate lymphoid cell lineage upon expression of the transcription factor id2 which suppresses t and b cell development and is required for the generation of ilcs including classical ilcs as well as nk cells lastly a clp can differentiate into a t cell upon egress from the bone marrow into an organ called the thymus if you remember back to our week one introduction to lymphoid organs the thymus is another primary lymphoid organ and is a butterfly-shaped gland that is located above the heart as we will learn today the vast majority of t cell development takes place in the thymus and interestingly when t cells were first discovered they were called thymus-dependent lymphocytes which was abbreviated to t lymphocytes we'll learn more about the subanatomical structures of the thymus and how they can facilitate t cell maturation in a few slides now once a clp travels from the bone marrow to the thymus commitment towards the t cell lineage is made once the clp senses cues in the thymic microenvironment which are capable of activating a receptor called notch 1. so notch signaling induces a gene expression program that differentiates the clp into something called a thymic progenitor cell this is the first step towards development of a t cell there are several developmental stages that must be met in order to take a thymic progenitor cell and successfully turn that into a functional t cell as a quick naming convention immature t cells are called thymocytes during their development and they're only referred to as t cells once they've become fully mature and exit the thymus we will also refer to these thymocytes as clones sometimes and this refers to a population of identical daughter cells that are all derived from the same progenitor cell so the term t cell clones refers to t cells that all express the same t cell receptor or tcr so the first developmental stage of t cells is also called a double negative thymocyte or dn thymocyte we call these double negative thymocytes because they don't express the co-receptors cd4 or cd8 double negative thymocytes can be further subdivided into four different double negative stages which we'll briefly discuss in the upcoming slides but for this first introduction i want you to primarily focus on double negative thymocytes with respect to the fact that these cells are subjected to the first checkpoint of t cell development which is called beta selection at the double negative stage these thymocytes express a tcr beta chain that has undergone vdj recombination and beta selection is a process that tests the functionality of that beta chain which we will learn more about shortly next thymocytes that successfully pass beta selection upregulate expression of both cd4 and cd8 co-receptors and become something referred to as a double positive thymocyte or dp thymocyte importantly along with cd4 and cd8 expression these thymocytes also recombine the v and j regions of their tcr alpha chain this means that they can pair their new tcr alpha chain with their existing tcr beta chain that already passed the beta selection checkpoint so double positive sinocytes express a candidate t cell receptor for the first time however this tcr isn't automatically allowed to be carried through as a fully mature t-cell clone it must first pass a rigorous set of two checkpoints that correspond with the two criteria of functional t cells that we outlined in the first slide of this lecture the first of these checkpoints is positive selection which tests whether the tcr can recognize self-mhc to ensure the mhc restriction which we discussed during week three's lectures on anagen presentation again mhc restriction is the fact that tcr's from one individual can only recognize presented antigen on mhc molecules from that same individual or another individual that has the same exact nhc haplotype and the reason for this is because it is a requirement for thymocyte's survival through the positive selection checkpoint once mhc restriction is confirmed through positive selection the tcr is then subjected to the second checkpoint which is negative selection this process removes tcr clones which reacts strongly to self-peptide presented on mhc so this checkpoint gets rid of any potentially autoreactive t-cell clones that are present in the tcr repertoire this prevents autoimmunity and is a key mechanism in promoting something called central tolerance which is the immunological tolerance towards self-antigen that is established in primary lymphoid organs such as the thymus once the thymocyte survives these three checkpoints of t cell development it can proceed to the final step where it becomes a single positive thymocyte or sp thynocyte at this stage these cells are considered mature naive t cells once they leave the thymus which means that they express a functional non-self-reactive tcr but they haven't encountered cognate antigen yet these cells are referred to as single positive because they express either the cd4 co-receptor or the cd8 co receptor and this step is called lineage commitment since the t cells are fully committed towards either helper cd4 positive or cytotoxic cd8 positive subsets of t cells the selection of one core receptor over the other has to do with whether the t cell passed positive selection by binding to mhc class 1 this is restriction for cd8 t cells or mhc class 2 for cd4 t cells at this stage mature naive t cells have been generated and upon completion of their development they exit the thymus and are exported to secondary lymphoid organs secondary lymphoid organs consist of structures such as the lymph nodes and spleen if you remember back to our week one lectures secondary lymphoid organs are sites of high lymphocyte density where t and b cells are appropriately positioned to undergo activation for t cells this means that they are optimally positioned to meet up with antigen presenting cells that could potentially present cognate antigen on mhc in this case the t cell would become activated in the secondary lymphoid organ and undergo proliferation into identical daughter cells or clones some of which could then migrate to the site of infection and non-lymphoid tissues by following cues such as chemokine gradients as a guide please note that the schematic refers to the developmental processes of t cells that express alpha and beta chains to form their tcr there is another specialized set of t cells that form a tcr using gamma and delta chains and we call these gamma delta t cells but for the purposes of this introductory course we won't be discussing gamma delta t cell development or function now with this slide we've established some basic properties of canonical t cell development and how this process takes clps from the bone marrow and eventually makes t cells in secondary lymphoid organs that are capable of fighting infections next let's learn a little bit more about how the developmental stages of thymocytes correspond to sub-anatomical locations within the thymus this slide depicts the anatomical organization of the thymus in two ways the one on the left labels sub-anatomical structures in the thymus while the one on the right shows where the different stages of t cell progenitors are localized within the thymus first on the left schematic you can see that the human thymus lies above the heart and is comprised of several lobes or lobules each of which can be separated into cortical regions at the surface of the organ and the cortex covers medullary regions that are located deeper towards the center of the organ so when referring to different locations in the thymus we often refer to cells being in either the thymic cortex or the thymic medulla importantly each of these regions contains different cell types and the spatial organization of cell types within the thymus corresponds with what needs to be accomplished during the different stages of thymocyte development from the diagram on the right you can see that the cortex is filled primarily with immature thymocytes denoted by the blue cells clps enter the thymus by venous circulation at the junction between the medulla and the cortex and upon entering the thymus they first interact with thymic epithelial cells to trigger notch one signaling and differentiation into thymic progenitor cells these then form the first stage of double negative thymocytes again the double negative stage can be broken up into four subcategories of double negative cells but the take-home point here is that progenitor cells undergoing the immature stages of thymocyte development are really concentrated within the thymic cortex these immature thymocytes interact with various antigen presenting cell populations expressing mhc class 1 or class 2 which help determine selection checkpoints of tcrs so keep in mind that the cortex is also enriched with various cells that can present antigen to developing thymocytes these include professional antigen presenting cells such as macrophages and dendritic cells but notably there's also a sub type of cells called cortical thymic epithelial cells or tecs and these can also present antigen on mhc class 1 or 2. only once progenitor thymocytes progress through the double negative to double positive stage can they undergo positive and negative selection which yields mature single positive thymocytes which can then migrate to the medulla and eventually leave the thymus by egressing into secondary lymphoid organs another important way of tracking the maturation of thymocytes through these different developmental stages is to consider where these cells are at in the process of tcr formation this figure helps us map these phases of development according to when vj or vdj recombination events occurred in the alpha and beta chains of the tcr not surprisingly the timing of these recombination events on either chain of the tcr corresponds with different checkpoints of t cell selection now i personally think that this is the most helpful way to approach t-cell development since the thymocyte development stages and checkpoints are all based on which components of the tcr have been rearranged and need to be tested for functionality we've established that the first stage of t cell development is the formation of double negative thymocytes which can be further subdivided into four types based on their expression of a combination of cell surface markers one thing worth noting at the double negative stages is that this marker cd25 starts being expressed from the dn2 stage and cd25 is important because it is a subunit of the receptor for a cytokine called il-2 which is an essential cytokine for t cell development survival so at this dn2 stage cd25 expression represents an irreversible commitment step towards the t cell lineage however one of the main things that i want you to remember about double negative thymocytes is that at the dn3 stage they turn on expression of rag 1 and 2 which lets them recombine v d and j segments on the tcr beta chain so at the double negative stage thymocytes only express a candidate tcr beta chain and they haven't rearranged their own alpha chain yet at this point cells are subjected to the first checkpoint that we talked about briefly beforehand which is beta selection the primary goal of beta selection is to test whether a thymocyte expresses a functional tcr beta candidate chain and it does this by pairing the tcr beta chain with something called the alpha surrogate chain abbreviated as pt alpha this protein heterodimer of pt alpha and tcr beta is called the pre-tcr which tests the functionality of the candidate tcr beta chain by undergoing antigen independent activation and signal transduction so the expression of pt alpha allows the cell to be interrogated as to whether vdj recombination successfully generated a functional tcr beta chain if signaling is successfully engaged the double negative thymocyte proliferates and turns on expression of both cd4 and cd8 to progress to the double positive stage of thymocyte development there are a few important things to note about beta selection one is that it requires that only one beta chain be expressed per t cell which means that only one allele of the vdj segments in the tcr beta locus is expressed at a time this is something that we call allelic exclusion which only happens for the beta chain of the tcr also the signaling initiated through the pre-tcr only requires this co-receptor ce3 and is therefore antigen independent so it doesn't require recognition of mhc class 1 or 2. if a thymocyte successfully induces signaling through its pre-tcr it upregulates transcriptional survival programs that allow it to progress to the double positive stage of thymocyte development the double positive stage is when the tcr-alpha chain is rearranged using vj recombination forming the t cell's full candidate tcr consisting of its own rearranged alpha and beta chains at this point the candidate tcr must be subjected to rigorous testing before being allowed to move to the final single positive stage of thymocyte development this testing includes checkpoint number two which is positive selection positive selection tests the functionality of the candidate tcr and it does this by determining whether the vj recombination at the alpha chain created a protein that when complexed with the beta chain can recognize self-nhc again positive selection ensures mhc restriction and because it requires binding of the candidate tcr to self-peptide presented by mhc this is an antigen dependent process most cells will fail positive selection but the ones that pass through this checkpoint will then be subjected to the final checkpoint of t cell development which is negative selection the purpose of negative selection is to remove tcr clones that recognize self-antigens which again is important for the maintenance of central tolerance in order to prevent autoimmunity autoreactive thymocytes are eliminated by apoptosis during negative selection which is we something that we also refer to as clonal deletion since the individual t cell expressing that tcr aka that tcr clone is being deleted from a pool of t cells like with positive selection negative selection requires candidate tcr binding to self-peptide mhc so this is also an antigen-dependent selection process now if you're paying attention you'll have noticed that both positive and negative selection require binding between a tcr and self-peptide mhc but these lead to very different outcomes so this leads to survival and positive selection but clonal deletion and negative selection so how can these two outcomes be so different if they're both based on a tcr interacting with self-peptide mhc and the answer to this problem relates to the strength of the binding interaction between tcr and peptide mhc this is also a property that we refer to as the affinity of an antigen receptor now the affinity of a given tcr is what ultimately determines whether that tcr survives the processes of positive and negative selection this diagram here represents what we call the affinity model of positive and negative selection for t cells and shows a red bell curve that plots the number of thymocytes or tcrs against the affinity or binding strength of that tcr2 self-peptide plus self-image see the majority of candidate tcrs generated in the thymus fall in the left portion of this curve which indicates that they are not capable of binding to self-peptide nhc this essentially means that they are useless tcrs since they are not mhc restricted these cells are eliminated at the positive selection checkpoint through a process called death by neglect death by neglect refers to the process in which thymocytes with a non-functional tcr that can't recognize self-mhc do not receive survival signals in the thymus and this leads to their non-inflammatory death by apoptosis again this is a failure of the tcr to meet the first criterion of mhc restriction the majority of thymocytes do not pass positive selection and so they are eliminated through death by neglect on the left we have a tissue section of the thymic cortex of a mouse which again is the site of immature thymocyte selection this tissue section was stained with a dye that detects apoptotic cells which are dead cells and these show up in red both the lower magnification image on the left and the higher magnification image on the right show that there are large clusters of apototic thymocytes in the cortex and the majority of these are cells that did not pass positive selection and underwent death by neglect notably to handle these large quantities of dying cells there are populations of macrophages in the thymic cortex which phagocytose and break down the apototic thymocytes this is shown in the higher magnification image on the right hand panel of these microscopy photos where you can see that the dead thymocytes stained in reddish brown are surrounded by macrophages which are stained using a marker in dark blue only thymocytes expressing a tcr that binds to self-peptide self-mhc with sufficient strength to induce pro-survival signaling are capable of surviving positive selection these clones bind to self-peptide mhc with moderate to high affinity which reflects their ability to bind to self-mhc although this checkpoint uses self-peptide mhc to test mhc restriction the group of tcrs that passes the selection checkpoint will include non-autoreactive tcrs that will function optimally when presenting foreign peptide on self-mhc so we still have one last selection checkpoint that must be used to isolate only this group of optimal tcrs shown in the blue bar and weed out potentially autoreactive tcrs this is accomplished through negative selection which works to remove autoreactive thymocytes from the tcr repertoire these are defined as tcr's which pass positive selection because they recognize self-peptide mhc but which also have a very strong binding affinity which reflects that the tcr recognizes self-peptide and is therefore autoreactive on this diagram these tcr's would be the ones that fall under the high affinity tail of the tcr bell curve on the right side of the plot these tcrs violate the second criterion of functional t cells that we had established at the beginning of this lecture which is that they must not react to self antigen again removal of these autoreactive tcr clones from the thymocyte pool by clonal deletion is a critical step in the establishment of central tolerance within primary lymphoid organs note that this diagram also has a green bar on it that represents tcr's with stronger binding affinity to self antigen than positively selected clones but lower binding affinity than negatively selected clones these tcr clones will become a type of immunosuppressive t cell called a t regulatory cell or t reg which are cd4 positive and we'll discuss these more in our upcoming lecture about effector functions of t cells i hope that going over this model has helped to demonstrate how a candidate tcr's affinity for self-peptide presented on self-mhc determines the developmental fate of that t-cell clone the affinity of successful tcr candidates really has to fall in this happy middle ground that's shown here in the blue bar where the tcr successfully obeys mhc restriction by recognizing self-mhc but doesn't spill over into this dangerous area on the right side of the affinity bell curve which represents tcr's that are strongly autoreactive to self-antigen now we've seen all this information a few times by now but i still want to end today with this summary schematic which outlines the main questions and outcomes of the developmental checkpoints of developing t cells that we've discussed so far the first checkpoint is beta selection which thymocytes must pass in order to transition from double negative thymocytes that have rearranged their tcr beta chain but don't express cd4 or cd8 towards double positive thymocytes that express both cd4 and cd8 beta selection asks whether the genetic rearrangement of the tcr beta locus generated a functional beta chain and this is tested by pairing it with a surrogate pt alpha chain to induce pro-survival signaling again this complex is called the pre-tcr in more simplified terms beta selection simply asks whether the candidate rearranged beta chain could possibly work in a functional t cell receptor if the answer to this is no then the cell can try beta chain rearrangement one more time using the other allele encoded on the second chromosome of the cell again only one tcr beta chain can be expressed per cell which is the rule of allelic exclusion for tzr beta also remember that because the pre-tcr signals by itself it doesn't require peptide mhc stimulation so this checkpoint is antigen independent thymocytes that have successfully rearranged a functional tcr beta chain are capable of passing this checkpoint which is shown by their proliferation and expression of the co-receptors cd4 and cd8 which reflects their advancement to the double positive stage of development the next checkpoint of t cell development is positive selection which is the first step required for a double positive thymocyte to progress towards maturation positive selection asks whether vj recombination that occurred in the tcr alpha chain created a protein that can pair with the cell's already established tcr beta chain and also whether this candidate tcr recognizes a complex of self-peptide on self-mhc in simpler terms positive selection asks if the candidate tcr is useful and useful in this context refers to whether the tcr exhibits mhc restriction which again is the ability of a tcr of an individual to bind to mhc molecules from that same individual at this step tcrs are tested using self-antigen presented on mhc class 1 or class 2 by thymic epithelial cells found in the thymic cortex where thymocyte development occurs again because this requires tcr binding to self antigen positive selection is said to be an antigen-dependent process unlike beta selection also note that the lineage commitment of a t cell towards either cd4 or cd8 subtypes is determined during positive selection and this occurs as a function of whether the t cell gets its survival signal following tcr binding to either mhc class 1 for the cd8 lineage or class 2 for the cd4 lineage another important difference between beta selection and positive selection is the fact that while the tcr beta chain only gets two total chances to recombine a successful protein due to allelic exclusion the tcr alpha chain can undergo repeated rearrangements until either the candidate tcr functions properly or the cell undergoes apoptosis by death by neglect whichever one happens first lastly note that the strength of the binding interaction or affinity of the tcr to self-peptide mhc will only yield a successful t cell if this tcr has moderate affinity as high affinity tcrs will be removed in the next checkpoint of thymocyte development negative selection occurs with cells that successfully pass through the positive selection checkpoint and serves as a final checkpoint which determines the safety of candidate tcrs negative selection asks whether the tcr strongly reacts to self-peptide presented on self-mhc which is indicative of an autoreactive tcr that would jeopardize central tolerance in simpler terms negative selection asks whether this candidate tcr is safe or not here tcr safety is determined through the ability of a tcr to bind with high affinity to self-peptide that's presented by professional apcs present in the thymus and these include dendritic cells and macrophages again because this process requires recognition of peptide mhc negative selection is an antigen dependent mechanism just like positive selection if a candidate tcr does bind with high affinity to self-peptide mhc then this cell will die through clonal deletion however if the candidate tcr only binds to self-peptide mhc with moderate affinity then the thymocyte is allowed to progress to the final stage of development where it forms either cd4 or cd8 single positive t cell this mature cell is then free to exit the thymus and travel to the secondary lymphoid organs i want to emphasize the fact that between gene recombination and all of these checkpoints that test the functionality and safety of tcrs that a very small proportion of only about two percent of initial thymocytes that begin t cell differentiation actually mature into single positive t cells so these checkpoints are essential in removing the vast majority of thymocytes which express either non-functional or potentially dangerous as in autoreactive tcrs from the t cell pool to summarize in this lecture we've learned that progenitor cells undergoing t cell maturation in the thymus must survive through three major developmental checkpoints and that these exist to ensure two main criteria of functional t cells that they can recognize self-mhc in a process we call mhc restriction and that they must not react to self-antigen in order to preserve immunological tolerance only the thymocytes that survive through these three checkpoints can then go on to form mature naive single positive t cells we've learned that beta selection positive selection and negative selection all differ with respect to which thymocyte developmental stage they act on the type of testing that they exert on candidate tcrs and their dependence on antigen you've seen this information a few times now throughout this talk but some key facts are written out as bullet points on this slide that's all for today's lecture but please remember to watch the remaining two lectures for week four in which we'll learn more about what mature t cells do once they leave the thymus which includes the signal transduction events that underlie t cell activation as well as the different effector functions that are exerted by different types of t cells