Paper : 16 Module : 19 Principal Investigator Paper Coordinator and Content Writer Dr. Sunil Kumar Khare,Professor Dept. of Chemistry, I.I.T. Delhi Dr. M.N.Gupta, Emeritus Professor Dept. of Biochemical Engg. and Biotechnology, I.I.T. Delhi Content Reviewer: Dr. Prashant Mishra, Professor Dept. of Biochemical Engg. and Biotechnology, I.I.T. Delhi 1
Description of Module Subject Name Paper Name Module Name/Title Biochemstry 16 19 Dr. Vijaya Khader Dr. MC Varadaraj 2
1. Objectives To learn about T h and T c cells. To understand how T h 1 and T h 2 cover various kinds of pathogens To learn about immune response in term of T-cells against superantigens To learn about memory T-cells 2. Concept Map 3
3. Description We have referred to helper T-cells (T h ) and cytotoxic T-cells (T c ) occasionally without actually discussing these classes of T-cells in detail. T-cells are needed for effective B-cell response. Most of the antigens evoke response from both kinds of T-cells but one kind of response may dominate. Immune system is a teamwork with a well thought out plan and clear roles assigned to each member. In this module, we focus on these different kind of T-cells Immune responses have broadly three phases which seamlessly segue into each other. Each phase is succeeded by another one wherein the specific response have a greater role to play. In the late phase > 96 hrs, there is practically little innate immunity component and memory lymphocytes form. The formation of effector cells and memory cells for T-cells is in many ways similar to what we discuss in the context of B-cells. While class switching make B-lymphocytes produces different classes of Ig, in case of naïve T-lymphocytes, the place, type and severity of infection dictates their differentiation into various types of T-cells. Table 1: Effect of neonatal bursectomy and thymectomy on the development of immunological competence in the chicken All X- irradiated after birth Peripheral blood lymphocyte count Ig conc. Antibody Delayed skin reaction to tuberculin Graft rejection Intact 14800 ++ +++ ++ ++ Thymectomized 9000 ++ + _ + Bursectomized 13200 + + The neonatal bursectomy and thymectomy performed separately on the chickens was a very valuable approach which not only confirmed the existence of two kinds of lymphocytes, it 4
also pointed out the respective roles of B and T lymphocyte in the phenomenon like antibody production, hypersensitivity and graft rejection. It also pointed out that some of these phenomena depends upon both B-cells and T-cells. With hindsight, we can also see that no antibodies appear in blood without Bursa, removal of thymus also diminished their concentration. So, T cells are involved in antibody production somehow! The existence of Bursa in chicken thus was very valuable in the design of many similar studies. After almost a decade s search, it was realised that in mammals, the B-cell production was in bone marrow itself. Mammals thus, would not have been a good system as even T-cells are produced in bone marrow before migrating to thymus for undergoing thymic education. In fact, as we know, bone marrow is the source of pluripotent stem cells or haematopoetic stem cells which differentiate into all cells present in the blood. T-cells clones are also selected from a pre-existing population upon exposure to antigens. Thus, most of the features of clonal selection theory which are discussed in the context of B- cells, apply to the T-cells as well. Figure 2: Clonal expansion and development of memory T-cells Selected by the antigens (as presented by an APC). Clonal expansion of T-cells takes place. Two kinds of effector T-cells are produced: T h (helper T-cells) and Tc (cytotoxic T-cells). Just like in case of B-cells, some memory T-cells are also produced. 5
The first version of memory T-cells retain homing and chemokine receptors of naïve cells and recirculate to the lymphoid tissues. This clonal reservoir allows swift T-cell response. While most of the memory cells are rested, a small % keep dividing all the time. Perhaps constitutively produced cytokines may be responsible. IL-15 has been implicated in maintaining CD8 memory T-cells. The extent of cell division is such that the number of any antigen specific T-cells remain more or less constant. In fact memory cells corresponding to T h cells appears faster and precede appearance of memory B-cells. This is necessary as B-cells need T h cells for full activation. Memory T-cells have been more difficult to isolate and characterize. All the assays for effector T-cells take several days which can allow memory T-cells to revert to effector T- cells. We will come back to memory T-cells after discussion on effector T-cells. Figure 3: There are two major intracellular compartments, separated by membranes 6
Figure 4: Pathogens and their products can be found in either the cytosolic or the vesicular compartment of cells Cell mediated immunity primarily may have evolved to mount effective response against pathogens which are intracellular. In fact, within cells pathogens can replicate into two different compartments. Viruses and some bacteria proliferate in the cytoplasm or nucleus. On the other hand some other pathogenic bacteria and parasites proliferate in endosomes or lysosomes. Pathogens replicating in cytosol are destroyed by Tc cells. As these have CD8 as coreceptor, these are called CD8 T-cells. These are Tc cells The other class of pathogens require response by another class of T-cells. Presence of CD4 coreceptor molecule results in their being called CD4 T cells. These are T h cells. As the name suggests, T h cells play a helping role. These activate other cells and/or cooperate with other cells of the immune system. On functional basis, T h cells are further divided into T h 1 cells and T h 2 cells. T h 1 cells are also known as inflammatory T-cells. These activate macrophages which in turn kill intravesicular pathogens. T h 2 cells, on the other hand cooperate with B-cells to result in humoral response. 7
It may be useful to look at the respective role of cell mediated immunity and humoral immunity in combating typical pathogens. Important point to note is that both T h 2 or T h 1 cells are required for humoral immunity to fully function against pathogens which invade extracellular fluid in tissues. T h 2 cells in fact are required to transform naïve B-cells to IgM producing B-cells. These cells are also involved in class switching which includes B-cell production of neutralizing and opsonising subtypes of IgG. Cytosolic location results in the peptide antigens (of pathogens) being presented to CD8T cells along with MHC class I molecules. This presentation converts naïve CD8 T-cells into Tc effector cells. Peptides from pathogens located in macrophages and dendritic vesicles provoke differentiation of T-cells into T h 1 cells. In turn T h 1 cells activate macrophages and also provoke B-cells to produce opsonising ab so that opsonised extracellular pathogens undergo phagocytosis. Thus T-cells participate in primary response itself and also generate memory T-cells as a result of encounter with the antigen. Memory cells differ from naïve cells but require exposure to quickly become effector T-cells. Superantigens and T-cell response 8
Figure 7: Superantigens bind directly to T-cell receptors and to MHC molecules. Superantigens are produced by many bacteria, mycoplasms and viruses. These manage to provoke immune response which are helpful to themselves rather than host animal. T-cells respond to these without MHC antigens involvement. These are not processed to produce peptides for T-cell response. These as such bind to outside of a MHC class II molecule with some other antigenic peptide bound to it. These superantigens are also able to bind to Uβ region of many TcR. Each superantigen is specified for one more of Tβ gene segments. A superantigen can bind to about 2-20% of T-cell population. The immune response, however is nonspecific, releasing cytokines from responding CD4 T cells. The result of a very high cytokine concentration results in systemic toxicity and immunosuppression. Bacterial superantigens include staphylococcal endotoxins and toxic shock syndrome toxin- 1. The best characterized viral superantigens are mouse mammary tumor virus superantigens. Superantigens, however, have proved useful in design of experiments with animals which have given insight into deletion of T-cells against self antigens in thymus and for learning how viruses use these to facilitate their transmission. The T-cell development 9
Figure 8: The development of T cells. Most of the T-cell development occurs in the microenvironments of Thymus. Both at the fetus stage and juvenile stage, thymus produces large number of new T-cells which leave thymus (after thymic education) to circulate and populate peripheral lymphoid tissues. While Thymic production of T-cells starts slowing down after puberty, T cell population remains same in the same range by division of mature T-cells in peripheral lymphoid tissues. This is unlike B-cells which are continuously produced by bone marrow. T-cells development requires production of two different T-cell lineages with α:β and γ:δ types of TcR. Down the line T cells with α:β type TcR divides into CD4 and CD8 T-cells. That occurs in immature T-cells after TcR arrangement is complete. Effecter T-cells A naïve T-cell, as we have learnt, require two signals to become activated. First signal is presence of a nonself origin peptide bound to a self MHC molecule. This itself is not sufficient. The second required signal is a costimulatory signal by an APC. Both these signals are possible when dendritic cells, macrophages and B-cells act as APC. 10
Physiologically, the largest role of this kind is played by mature dendritic cells. The activation of T-cells by APCs in general is called priming. Acquired immune response occur in the peripheral lymphoid tissues. Pathogens accumulate in such tissues and it is here that they prime T-cells. The development and organisation of peripheral lymphoid tissue is itself regulated by cytokines and chemokines. Curiously, these molecules belonging to TNF/TNFR family which are involved in inflammation and cell apoptosis are also essential for lymphoid developments. Lymph node development require lymphotoxin (LT) family members. Spleen has distorted architecture if TNF/TNFR family molecules are not present. Location of lymphocytes, macrophages and dendritic cells is regulated by chemokines. These are MIP-3β and secondary lymphoid chemokine (SLC) are required for T-cell localization at the right place. 11
Figure 9: Immature dendritic cells take up antigen in the tissues 12
Infections of the mucosal surfaces collect in Payer s patches of the gut or tonsils. Infection entering blood are trapped in spleen. Infecting pathogens in the peripheral site are trapped in the lymph node immediately at the downstream from the site of infection. All these lymphoid organs have antigen capturing cells which act as APC to T-cells. This process is supported by innate immunity. This support starts at the local site of infection. Cells with receptors recognise the nonself molecular patterns. Inflammation ensures that plasma enters the site of infection and later tissue fluids are drained into the lymph. Locally resident dendritic cells mature and take up both particulate and soluble antigens. This activation is triggered by receptor bound antigens and facilitated by cytokines produced during inflammation. Overall, innate immunity thus accelerates the process of antigens to the lymphoid tissue and their presentation to T-cells. Within the lymph nodes, dendritic cells are present mostly in T-cell areas, forming network with their tentacles among the T-cells. As a part of their differentiation, dendritic cells reaching lymph nodes can no longer capture antigens. They are just powerful APC for naïve T-cells in the lymph nodes. Macrophages have wider distribution in lymph nodes. They are concentrated in marginal sinus (where the lymph enters) and in the medullary cords (where the efferent lymph collects before mixing with the blood). Macrophages take up microbes and prevent them entering the blood stream. Thus, their role in lymph nodes is different from dendritic cells present there. Naïve T-cells circulate through the peripheral lymphoid organs. In lymph nodes their entry route is through specialized regions of vascular endothelium which is named as high endothelial venules (HEV). The exit is through medulla of the lymph nodes. Two possibilities arise for these naïve T-cells. First is that some of these are not clonally selected and leave the lymph nodes as the naïve T-cells only through the lymphatic to start another round of circulation. Nevertheless, their passage through the lymph node is not encounter free. They interact with self MHC: self peptide complexes on dendritic cells to reinforce the positive selection which they went through during thymic education. It is believed that this in fact is a signal for their survival. 13
Only one out of 10 4-10 6 naïve T-cells is likely to meet the antigen on the APC and get clonally selected. These proliferate, increasingly in number by 100-1000 fold. These cells remain in secondary lymphoid organs to complete their differentiation into armed effector T- cells which are antigen specific. This process takes place several days. Once this is over, the effector T-cells also leave the lymphoid organs, enter the blood circulation and like a targeted missile go back to the site of infection from where the antigen entered. The migration of T-cells and their activation to the effector cells involve inter-cellular interactions wherein cell adhesion molecules play an important role. Selectins play a critical role in all leukocytes reaching a particular tissue. Naïve T-cells express L-selectin which are responsible for their entering peripheral lymphoid tissue through their blood. L-selectin binds to vascular addressins which are present on the surface of vascular endothelial cells. Among these addressins are CD34 and GlyCAM-1 on HEV in lymph nodes. Another MADCAM-1 is present on endothelium cells of the mucosa and thus is responsible for the T-cell entry into the MALT. Next, integrins and some other proteins of Ig superfamily enable T-cells into crossing over the endothelial barrier and facilitate their further interactions. Integrins need signal through the presence of chemokines before interacting with T-cells. Chemokine called secondary lymphoid tissue chemokine (SLC) facilitates naïve T-cells migrating to the lymphoid tissues. This is present on high vascular endothelium, stromal cells, and dendritic cells in the lymphoid tissues and binds to CCR7 chemokine receptor on naïve T-cells. This increases integrin binding to T-cells and enables their entry into the lymphoid tissue. 14
Figure 13: Adhesion molecules involved in leukocyte interactions Members of the Ig superfamily act as adhesion molecules which are important for T-cell activation intracellular adhesion molecules (ICAMs): ICAM-1, ICAM-2 and ICAM-3 bind to T- cell integrin LFA-1. Figure 14: Cell-surface molecules of the immunoglobulin superfamily are important in the interactions of lymphocytes with antigen-presenting cells While passing through lymph nodes cortex, naïve T-cell bind through LFA-1, CD2 and ICAM- 3 to ICAM 1, ICAM 2, LFA-3 on APC such as dendritic cells (which also in addition have DC- SIGN. Experiments with genetically engineered mice indicate that there may be some redundancy involved for these cell adhesion molecules. Figure 15: Transient adhesive interactions between T cells and antigen-presenting cells are stabilized by specific antigen recognition. 15
The interaction between naïve T-cells and APC remain transient. In case T-cell recognizes antigenic peptide on APC, its TcR signals a conformational change in LFA-1. This firms up the T-cell-APC interactions as now LFA-1 binds to ICAMs on APC more strongly. This starts T-cell on its journey to become effective T-cell. In case, T-cell does not recognise the antigenic peptide, it disengages from APC and exits to recirculate. As pointed out earlier, this has much higher probability. Figure 16: Armed effector T cells can respond to their target cells without costimulation. T-cell differentiating into an effector T-cell secrets IL-2 after dual signal from APP interaction. This is kind of self-help as it also has IL-2 receptor. IL-2 binding triggers clonal expansion and differentiation. A key feature of the effector T-cells is that it can respond to the antigen without any costimulatory signal. Thus Tc will kill any virus infected cell of the host. This feature is important for both cytotoxic CD8T-cells as well as CD4 T-cells. For the former, as it can kill the virus infected cells. For CD4 T-cells, their function to activate B-cell and macrophages which have taken up antigens requires this feature as the both B-cells and macrophages have weak co-stimulatory signal. 16
Figure 17: The stages of activation of CD4 T cells. CD8 T-cells after thymic education are already committed naïve Tc cells. These become effector Tc cells after clonally selected through antigen encounter. CD4 T-cells after thymic education, however, still have to differentiate into either T h 1 or T h 2 cells. Both differ in the spectrum of cytokines which is secreted by them. This in turn shapes their functionality. In case of CD4 T-cells, this choice, that is, between T h 1 or T h 2 options is made during clonal expansion after antigen encounter. Some factors which determine this include cytokines secreted by the pathogens (IFN-γ, IL-12 and IL-4), the nature of the co-stimulator and nature of MHC: peptide complex. 17
Figure 18: The differentiation of naive CD4 T cells into different subclasses of armed effector T cells is influenced by cytokines elicited by the pathogen. The T h 1 vs T h 2 option is chosen by CD4 T-cells at the early phase of the antigen encounter in the peripheral lymphoid tissues. This decides whether the acquired immune response will be primarily through macrophage activation or T-cell B cell cooperation. In vitro experiments indicate that IL-12 and IFN-γ encourage differentiation into T h 1 cells. IFN-γ inhibits T h 2 proliferation. IL-12 (secreted by macrophages and dendritic cells), IFN-γ (secreted by NK and CD8 T-cells) are the two cytokines produced when viruses and some intracellular bacteria infect, these infections are tackled by T h 1 cells. CD4 T cells in the presence of IL4 and IL6 differentiate into T h 2 cells. IL-4 and IL-10 both inhibit differentiation into T h 1 cells. 18
Figure 19: The two subsets of CD4 T cells each produce cytokines that can negatively regulate the other subset. There is a regulation by some sort of mutual exclusion. If the initial response is through one kind of CD4 T-cells, that tends to continue. There is no change over to the another kind of CD4T-cells taking over This is believed to be operated again through cytokines. T h 2 product IL-10 inhibits differentiation to T h 1 cells. IFN-γ produced by T h 1cells inhibits transformation to T h 2 cells. However, this sharing of the burden is not absolute. In vivo, many times both T h 1 and T h 2 are involved. 19
Figure 20: The nature and amount of ligand presented to a CD4 T cell during primary stimulation can determine its functional phenotype 20
Both amount and nature of the peptide derived from the antigen also influences the choice between T h 1 and T h 2 cells. High density of the peptide on APC favours T h 1 production. Low density of this on an APC induces T h 2 response. Those peptides which have a higher binding constant with TcR leads to preference for T h 1 cells formation. Peptides with low affinity for TcR favours formulation of T h 2 cells. Immune system having these clear cut choices has physiological relevance. Antigens that provoke allergy, (as we will learn in hypersensitivity modules), are able to induce IgE response in small amounts. Switch over to IgE production is aided by IL-4 and inhibited by IFN-γ. In fact, allergic antigens do not seem to induce even innate immunity cells to secret any cytokine which will favour T h 1 cell formation. In spite of such clear cut signals, most protein antigens results in mixed responses in terms of T h 1 and T h 2 cells. This happens because any antigen will generate peptides with different sequences. Their binding constants with MHC class II molecules will also dictate their density on APC. T-cells which are naïve CD4, upon presentation of high density of peptides by APC form T h 1 cells that starts secreting IL-2, TNF-β and IFN-γ and efficiently activate macrophages. Figure 21: Protective immunity consists of preformed immune reactants and immunological memory 21
In the aftermath of a successful immune response, when the infection is completely over come, effector cells withdraw so that tissue starts repairing itself. The immune response, often manifested by inflammation causes dents in tissue integrity and then restoration of normalcy requires that cell immune responses are withdrawn from the site of infection. Removal of cells which are no longer required follow the same mechanism which multicellular organism follow for such cells: apoptosis. Macrophages take over in rapidly cleaning away the cell mass as a result of this apoptosis. Phosphotidyl serine, normally part of inner surface of plasma membrane is present on the outer surface in cells undergoing apoptosis. Macrophages recognizes this molecule. What is left after the infection is memory! Let us now look at whatever we understand about how some T-cells become memory T-cells. These insights first came from Tc cells which are transformed more swiftly upon the second encounter with the same antigen. Theis response also is faster kinetically. A Tc cell starts cell lysis of the target pathogen within 5 minutes. Memory cells The CD8 memory T-cells retain surface markers of effector CD8 T-cells like CD44. At the same time, there are other surface markers like CD69 which are not found on the memory cells. Also, these memory cells have new surface proteins (which were not present as effector T- cells), important among these is Bcl-2 which presumably is responsible for their longer half lives. Memory T-cells more readily secrete cytokines and more readily become effector T-cells upon the next encounter with the specific antigen. The information about memory CD4 T-cells rests more on circumstantial evidence. CD4T cells which have longer half life and require another exposure to act like effector CD4 T-cells are presumed to be memory CD4 T-cells. The restimulation sees changes in level of L-selectin, CD44 and CD45. Memory CD4 T cells do not express L selectin but have higher levels of CD44. This determine their migration to tissues (when they are likely to be needed to respond to the second infection) rather than secondary lymphoid organs. 22
The change in CD45 is of a different kind, alternative splicing of exons corresponding to CD45 extracellular domain to become an isoform which synergises with TcR in antigen recognition. There is evidence that both CD4 and CD8 T-cells have two kinds of memory cells. For example effector memory CD4 T-cell are more tailored to deal with inflammation. Central memory T-cells recirculate and take longer to become effector T-cells. So, there are Tc cells and T h cells. T h cells are either T h 1 or T h 2. Both CD4 and CD8 T-cells have a population of memory cells. The mechanism which operate for a T-cell to become memory T-cells are slowly becoming clearer. We have purposely laid less emphasis on function of Tc cells as we are going to look at them shortly as a separately module. One obviously important entity is TcR. This also deserves discussion separately. Summary Two main classes of T-cells:CD4 (T h ) cells and CD8 (T c ) cells. Two kinds of T h cells: T h 1 and T h 2. Superantigens Naïve t cells become either CD4 or CD8 T-cells and corresponding memory cells. Memory T cells: different from both effector and naïve T-cells 23