We are searching data for your request:
Upon completion, a link will appear to access the found materials.
Lymphocytes originate from a common progenitor in a process known as hematopoeisis.
- Examine dual lymphocyte development
- B cells and T cells are the major types of lymphocytes.
- B cells mature into B lymphocytes in the bone marrow, while T cells migrate to, and mature in, a distinct organ called the thymus.
- Following maturation, the lymphocytes enter the circulation and peripheral lymphoid organs (e.g. the spleen and lymph nodes) where they survey for invading pathogens and/or tumor cells.
- The lymphocytes involved in adaptive immunity (i.e. B and T cells) differentiate further after exposure to an antigen to form effector and memory lymphocytes.
- lymphocyte: A type of white blood cell or leukocyte that is divided into two principal groups and a null group: B-lymphocytes, which produce antibodies in the humoral immune response, T-lymphocytes, which participate in the cell-mediated immune response, and the null group, which contains natural killer cells, cytotoxic cells that participate in the innate immune response.
- leukocyte: A white blood cell.
- haematopoiesis: Hematopoeisis is the formation of blood cellular components from a common progenitor stem cell.
The cells of the adaptive immune system are a type of leukocyte, called a lymphocyte. The human body has about 2 trillion lymphocytes, constituting 20-40% of white blood cells (WBCs); their total mass is about the same as the brain or liver. The peripheral blood contains 20–50% of circulating lymphocytes; the rest move within the lymphatic system. B cells and T cells are the major types of lymphocytes.
B Cell and T Cell Differentiation
Mammalian stem cells differentiate into several kinds of blood cell within the bone marrow. This process is called haematopoiesis. During this process, all lymphocytes originate from a common lymphoid progenitor before differentiating into their distinct lymphocyte types. The differentiation of lymphocytes into distinguishable types follows various pathways in a hierarchical fashion as well as in a more plastic fashion. The formation of lymphocytes is known as lymphopoiesis. B cells mature into B lymphocytes in the bone marrow, while T cells migrate to, and mature in, a distinct organ called the thymus. Following maturation, the lymphocytes enter the circulation and peripheral lymphoid organs (e.g. the spleen and lymph nodes) where they survey for invading pathogens and/or tumor cells.
The lymphocytes involved in adaptive immunity (i.e. B and T cells) differentiate further after exposure to an antigen; they form effector and memory lymphocytes. Effector lymphocytes function to eliminate the antigen, either by releasing antibodies (in the case of B cells), cytotoxic granules (cytotoxic T cells) or by signaling to other cells of the immune system (helper T cells). Memory cells remain in the peripheral tissues and circulation for an extended time ready to respond to the same antigen upon future exposure.
CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome
The Cancer Immunoediting concept has provided critical insights suggesting dual functions of immune system during the cancer initiation and development. However, the dynamics and roles of CD4+ and CD8+ T cells in the pathogenesis of breast cancer remain unclear. Here we utilized two murine breast cancer models (4T1 and E0771) and demonstrated that both CD4+ and CD8+ T cells were increased and involved in immune responses, but with distinct dynamic trends in breast cancer development. In addition to cell number increases, CD4+ T cells changed their dominant subsets from Th1 in the early stages to Treg and Th17 cells in the late stages of the cancer progression. We also analyzed CD4+ and CD8+ T cell infiltration in primary breast cancer tissues from cancer patients. We observed that CD8+ T cells are the key effector cell population mediating effective anti-tumor immunity resulting in better clinical outcomes. In contrast, intra-tumoral CD4+ T cells have negative prognostic effects on breast cancer patient outcomes. These studies indicate that CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcomes, which provides new insights relevant for the development of effective cancer immunotherapeutic approaches.
Keywords: CD4+ T cells CD8+ T cells Th17 cells breast tumor microenvironment regulatory T cells.
Conflict of interest statement
The authors declare no financial or commercial conflict of interest.
Figure 1. Accumulation of both CD4 +…
Figure 1. Accumulation of both CD4 + and CD8 + T cells in the TILs…
Figure 2. Dynamics of tumor-infiltrating CD4 +…
Figure 2. Dynamics of tumor-infiltrating CD4 + T cell subsets in mouse breast cancer models
Figure 3. Distributions of CD4 + T…
Figure 3. Distributions of CD4 + T cell subsets in peripheral blood and draining lymph…
Figure 4. Accumulation of CD4 + and…
Figure 4. Accumulation of CD4 + and CD8 + T cells in clinical breast cancer…
Figure 5. Distinct roles of tumor-infiltrating CD4…
Figure 5. Distinct roles of tumor-infiltrating CD4 + and CD8 + T cells in predicting…
Dual G9A and EZH2 inhibition stimulates an anti-tumour immune response in ovarian high-grade serous carcinoma
Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumour immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells whilst suppressing tumour-promoting FoxP3 + CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53 -/- null ID8 ovarian tumours and resulted in tumour burden reduction.
These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumour growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.
The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice
In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted (hAd5[E1-, E2b-, E3-]) that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell-surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity – including mucosal immunity – against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.
Competing Interest Statement
All authors with an ImmunityBio affiliation have a role in the development of the vaccine described.
Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits
T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.
Copyright © 2016 Elsevier Inc. All rights reserved.
Figure 1. Design of Combinatorial Antigen Sensing…
Figure 1. Design of Combinatorial Antigen Sensing Circuits in T cells Using Sequentially Regulated SynNotch…
Figure 2. SynNotch Regulated CAR Expression –…
Figure 2. SynNotch Regulated CAR Expression – Combinatorial Antigen Requirement for Jurkat T cell Activation
6 hours. Subsequently, activation of the T cell by CAR activation (monitored by CD69 expression) then occurs with a lag of several more hours (t1/2
13 hrs). FACS histograms for CAR expression are shown in Figure S1B. (F) Timecourse of AND-gate T cell inactivation upon removal of synNotch ligand. Jurkat T cells expressing the AND-gate circuit were stimulated for 24 hours by plate-bound α-Myc antibody (synNotch receptor has extracellular Myc-tag). START indicates time at which cells were removed from the ligand, and the decay of GFP tagged CAR expression was monitored (t1/2
8 hrs). FACS histograms for CAR expression are shown in Figure S1C.
Figure 3. SynNotch Regulated CAR Expression in…
Figure 3. SynNotch Regulated CAR Expression in Human Primary T cells – Combinatorial Antigen Control…
Figure 4. SynNotch Receptors Drive Tumor Localized…
Figure 4. SynNotch Receptors Drive Tumor Localized CAR Expression In vivo
Figure 5. Selective Combinatorial Antigen Tumor Killing…
Figure 5. Selective Combinatorial Antigen Tumor Killing In vivo by SynNotch Gated CAR Expression
Figure 6. SynNotch Receptors Control and Localize…
Figure 6. SynNotch Receptors Control and Localize CAR T cell Response for Precision Immunotherapy
Requirements of bioreactors for lymphocyte culture
Although every cell therapy process has unique elements, it is not practical to design specialized devices for each specific product. Instead, ACT products should be grouped on shared process characteristics, defining strategies and technologies that fit better for each category as a whole . In that regard, ACT can be performed using two general principles: autologous and allogenic. In the autologous setting, a batch is individually produced from a patient’s biopsy, isolating and culturing the cell population of interest. In the allogenic workflow, cell source is a universal donor platform with highly expandable cells that have similar scale requirements as the manufacturing of cell derived proteins and the cell product may target multiple patients . Process-wise, increasing vessel scale and ensuring culture performance (scale-up) is related to allogeneic approaches, while parallelizing several independent units (scale-out) is generally the goal in optimizing autologous therapy . An autologous batch size is not expected to exceed more than a few liters volume, because of the limited amount of starting material and the time sensitivity of the cells to retain their functionality. Thus, scaling up autologous is not useful and scaling out for multiple batches still requires a thorough assessment of technical capacities . This delicate setting for autologous cell therapy drives bioprocess development towards automation [11, 25], as the ideal autologous platform should compensate for the effects of varying culture conditions on CQA’s performance . The allogenic set up, on the other hand, requires appropriate inoculation levels with minimal seed adaptation to maximize the expansion outcome. Therefore, the possibility of having a set of vessels geometrically and dynamically comparable is highly relevant . In the same way, achieving consistent process reproducibility is necessary for a standardized and safe allogenic platform, thus, allogenic bioprocess development is mostly driven towards process control than workflow automation. To harness a bioreactor’s full potential, its design and application should be fitted to the challenges of cultivating lymphocytes and the supplements necessary for their growth. These are, in the view of the authors, and based on previous frameworks of requirements [14, 19, 25, 34, 35, 42], the main standards to be fulfilled by a culturing platform for ACT.
Suitable vessel size and scalability
Cell-based therapies often require the application of vast quantities of cells (10 8 –10 10 ) to patients therefore the space required for their growth is a practical limitation. Assuming a culture density of 10 6 to 10 7 cells/mL (a high value for ACT), it would demand a volume starting from few milliliters up to tens of liters during culture . The available bioreactor scale must be flexible enough to fully accommodate the range of cell growth across all feasible batches, and to compensate for the expected potential growth variability from the source . To achieve this, ultra-high cell density cultures or an industrial scale production that is able to maintain uniform culture conditions are required . Some current expansion processes include a preliminary stage where cells are activated and rapidly multiplied in static systems, generating enough cells for bioreactor inoculation. However, enough bioreactor space for the actual expansion is still necessary.
To avoid cross contamination (between different batches or patients) and microbiological contamination, closed systems (bags, expansion sets, flasks), incubators and hoods should be used . Bioreactors should guarantee sterility by keeping a closed system . Each manipulation step (e.g. inoculation, activation, transduction, media changes, stimulation, sampling, washing) creates a risk for error and contamination that may lead to a failed run . For that purpose, single-use, closed, disposable cell production “kits” may represent a desired design strategy for patient-specific cell therapy manufacturing protocols , particularly if such kits can be designed for simplicity .
Once the specific requirements for the cells being expanded have been defined, process parameters such as temperature, shear stress, dissolved oxygen (DO) and CO2 and environmental variables like osmolality and pH must be kept at optimal values . .Extensive, online process monitoring and integrated control is required for adaptation to process changes . DO and pH of the medium are typically held constant to provide a consistent environment supporting optimal cell expansion. DO and pH signals, are valuable for assessing the status of the expansion medium and cell proliferation, triggering a proportional feeding strategy , although this is a fairly limited approach. Some technologies that should be considered for ACT process monitoring and control are included in Table 1. The final goal of process monitoring should be to find descriptors that can give information about the influence of batch-to-batch or donor-to-donor variability on the expansion process . The best approach for process control development would be to use PAT data to facilitate process related decisions in real-time, or even predictively. This can include decision points for transduction, perfusion initiation, harvest point, or even quality control release based on minimum viability or endotoxin level. Ideally, such technologies would evolve to measure surface markers expression of key phenotypic markers.
Handling of shear stress
Ex vivo expansion of all immune cell types should avoid mechanical stress by chaotic, inhomogeneous medium dynamics . It has been long established that animal cells are sensitive to shear, which, above certain levels, compromises their viability. Besides the direct effect that mechanical forces can exert on a cell membrane’s integrity, animal cells are adapted to the environment of each tissue, evolving sensitive mechanisms for detecting shear changes. To develop an acceptable understanding of how these forces influence cell behavior, it is necessary to recreate similar level of shear forces than found in the body within a bioreactor, allowing for a detailed characterization and control of the mechanotransduction process  and the direct effects of shear on the cells. Importantly, agitation must be designed to manage not only shear exposure of cells, but also the efficiency of mass transfer, suspension of cells and avoidance of heterogeneities that may cause cell inconsistencies .
The designed bioreactor process should stay out of any artificial deleterious influences on cell integrity by passaging and reseeding the cells, as it may decrease total yield . Sampling and harvesting of cells, medium, or both should be also designed with simplicity in mind. Taking samples has certain drawbacks that need to be mitigated : to get a representative bioreactor sample, a significant volume should be drawn, which can impact on yield, especially if multiple small scale vessels are used for the cell expansion. Repeated sampling can also increase the risk of contaminating the bioreactor. Issues that need to be resolved in such cell therapy process development platforms include deciding on the amount of cells needed to reflect heterogeneity and the usage of live cell-based image analysis and “lab-on-chip” strategies .
Stimulation and supplementation
Media changes in bioreactors are usually done by nutrient addition, or by total or partial media replacement, or by perfusion. If a cell culture produces non-damaging levels of waste products, concentrated levels of nutrients can be added over time to feed the growing culture. Inevitably, waste metabolites such as lactate and ammonia start to accumulate, and either media replacement or perfusion is required. Perfusion, in which fresh media is gradually fed and old media is removed while the cells are retained, is the ideal way to intervene and still maintain a stable environment for cell therapy . It also should be noted that cell exhaustion can be induced by current activation methods, which generally also demand careful operator attention . Because of that, precise optimization of the feeding of nutrients and cell activators/stimulants is needed, being able to precisely supply them into the culturing medium, allowing for different feeding profiles.
Gas transfer happens passively in static systems, which limits oxygen availability in high volume vessels, as the diffusive flux of a gas is inversely proportional to the thickness of the liquid that needs to be permeated, according to Fick’s law and the McMurtrey model of oxygen diffusion . Oxygen transfer may be limited in non-perfused bioreactors because low agitation rates are required to minimize shear stress on the lymphocytes and headspace aeration is also generally preferred for the same reasons. This, on the long run, may hinder the final expansion output of the system . .Oxygen can be supplied to a bioreactor either via the headspace or via a sparger which disperses gas into the medium, however, sparging has been shown to be possibly detrimental for immune cell growth . The physiological oxygen concentration is usually lower than the atmospheric. Because of that, establishing culturing protocols that resembles in vivo oxygenation conditions may improve expansion yield and cell functionality . Similarly, the use of CO2 levels representative of the biological fluctuation threshold could also be beneficial of the process outcome. It must be noted that reduced oxygen tension results in reduced human T cell proliferation, increased intracellular oxidative damage and susceptibility to apoptosis upon activation, highlighting the importance of controlling oxygen levels in culture .
There is no ideal bioreactor that suits all purposes for all cells, but it should be able to replicate in vitro many of the conditions experienced in vivo, therefore it should allow for experimental testing, mechanical conditioning and monitoring of living cells in dynamic conditions . In a close physiological remembrance, immune cells cultured in bioreactors often require APCs for stimulation, three-dimensional culturing, controlled cell-cell contact and undisturbed local microenvironments . These needs should be taken into consideration during the design of suitable devices, starting from the fact that hematopoietic cells do not require a surface to grow, being anchorage independent . It is true that cells can be adapted to a specific bioreactor design as a replacement to engineering the bioreactor itself, but it must be noted that this approach may not be available to most cell therapies, as cells may become senescent after a certain amount of doublings . It should also be noted that some cells may need to be in extensive contact with each other, such as TILs  and T cells [65, 66], some of them also tend to form aggregates that must be controlled for optimal growth , usually by mechanical disruption of the clusters.
Different reactor configurations may fulfill these requirements to a varying extent. Given this framework, in the next chapter we explore the currently available options and highlight the most relevant characteristics that stand out from comparison.
NK Cells in Health and Disease
To date, the diverse functions of NK cells in mammalian immunity is not fully understood. However, accumulating data collected from patients with rare disorders characterized by NK cell deficiency have shed light on their relevance to human health (187) and studies using genetically modified mouse models have generated intriguing ideas with regards to their pro-inflammatory and immunosuppressive functions (188). NK cells produce and respond to inflammatory stimuli and are most well known for their roles in anti-viral immunity and tumor immunosurveillance however, NK cells are also involved in a variety of autoimmune disorders as drivers of pathologic inflammation (189). Emerging evidence also demonstrates that NK cells can regulate anti-inflammatory programs, such as tissue repair (190, 191). Whether NK cells act as primary innate effectors or accessory cells as part of the adaptive immune response appears to be context-dependent, but their contribution as first-line responders and essential inflammatory mediators is well established. Importantly, how the crosstalk between NK cells and lymphocytes (αβ-TCR + T, γδ-TCR + T, NKT, and B cells), myeloid cells (monocytes, macrophages, and DCs), or non-immune cells (epithelial or endothelial cells) enumerate a productive immune response is far from fully understood.
NK Cell Functions During Viral and Bacterial Infections
Natural killer cells are critical for defense against a wide variety of pathogens. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns and are essential components of the NK cell-mediated innate immune response (192). Activation of NK cells through PRRs elicit the production of TNF and IFN-γ which contribute to antibacterial defense (192, 193). NK cells also contribute to antifungal immunity by direct and indirect mechanisms (194). First, NK cells can directly damage fungal membranes through the targeted release of cytotoxic granules containing the membrane disrupting protein, perforin (195). They can also facilitate the antifungal host response through direct phagocytosis as well as the production of inflammatory mediators (196). Specifically, the production of GM-CSF by NK cells is critical for controlling C. albicans infection by promoting the fungicidal activity of neutrophils (197). However, the direct contribution of NK cells to microbial immunity has best been described with regards to their discrete actions against intracellular pathogens.
Intracellular pathogens have evolved a variety of mechanisms to evade the host immune response including subversion of the MHC immunosurveillance system (198). MHC molecules are highly polymorphic within a population and are encoded by human leukocyte antigen (HLA) genes in humans and, H-2 in mice (199). MHC molecules can be divided into two major classes, MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I molecules bind, and present endogenous peptides to cytotoxic CD8 + T cells and subversion of this immunosurveillance mechanism results in an insufficient adaptive immune response (200). MHC-II is abundantly expressed on antigen-presenting cells (APCs) and facilitates the presentation of exogenous peptides to CD4 + helper T cells (201). Nearly all somatic cells express endogenous peptides on their surface in the context of MHC-I, and this allows the immune system to sample the intracellular environment (201). The peptide–MHC-I complex also defines the immunological “self” condition and maintenance of this system is essential for both immune tolerances as well as the rejection of “non-self” cells (Figure 8) and tissues that express distinct MHC-I haplotypes (202).
Figure 8. Mechanisms of target cell recognition by natural killer (NK) cells. NK cells lack clonotypic receptors and rely on germline-encoded activation and inhibitory receptors to recognize other cells around them. The following are some of the primary mechanisms by which NK cells perceive target cells. (A) “Immunological Self”: recognition of autologous MHC class I (MHC-I) (human leukocyte antigen (HLA)) or histocompatibility antigen-2 (H2, mouse) by inhibitory receptors [killer cell immunoglobulin-like receptor (KIR) or Ly49] let the NK cells know that they are interacting with normal cells and contain their activation. (B) “Missing-self”: recognition of target cells that either does not express MHC-I or reduce them below optimal levels can induce NK cell activation. (C) “Induced-self”: recognition of activating ligands that are expressed on target cells by the germline-encoded receptors such as NKG2D (H60, mouse MIC-A/B, human), Ly49H (murine cytomegalovirus-derived m157, mouse), NCR1 (a number of viral proteins) can overcome MHC-I-mediated inhibitory signaling resulting in NK cell activation. (D) “Non-self”: recognition of transplanted tissue by NK cells, where the donor tissue expresses either allogeneic or haploidentical MHC-I.
Natural killer cells possess unique mechanisms to contain intracellular pathogens including viruses and some species of bacteria by lysing infected cells, releasing them and exposing them to adaptive cell-mediated immunity (203, 204). NK cells also produce inflammatory cytokines, such as IFN-γ to contain viral or bacterial growth (205). For example, hemagglutinin, a sialic acid receptor expressed by the influenza virus, serves as an activating ligand for NCR1 (208, 209). The murine cytomegalovirus (MCMV)-encoded membrane glycoprotein, m157, is recognized by the Ly49H receptor expressed in C57BL/6-derived NK cells (210). NK cells from other mouse backgrounds, such as 129/SvJ and BALB/c, do not express Ly49H, or another resistance factor, which renders them susceptible to MCMV as they are unable to mount a specific NK cell-mediated immune response to the virus (211). NKG2D has also involved in NK cell-mediated anti-viral immunity as evidenced by multiple observations in which human and mouse CMV proteins downregulate cellular stress ligands that activate NK cells through this receptor (214).
Natural killer cells have the unique ability to identify infected cells without direct engagement of the MHC-I complex (12, 218). Therefore, intracellular pathogens that evade CD8 + T cells by interfering with MHC-I surface expression remain vulnerable to NK cell-mediated immunity (219). In terms of anti-viral immunity, NK cells and CD8 + T cells have long been considered to represent the innate and adaptive arms of the immune response, respectively (220). However, the separation of these cells with regards to their contributions to adaptive immunity has recently been reconsidered due to the discovery of NK cells that exhibit immunological memory (160, 221). Although they do not utilize clonotypic receptors, such as the TCR, a relatively small population of memory NK cells has been described as long-lived effectors capable of rapid recall responses (222).
The formation of memory NK cells has been extensively investigated in mice infected with MCMV and studies using this system have been critical in defining the molecules that mediate this phenomenon (222). A vaccination study using antigens from viruses including, influenza, vesicular stomatitis virus, and human immunodeficiency virus type 1 also showed memory-like NK cell responses in mice (226) and NK cells exhibited enhanced protection against secondary infections with vaccinia virus and herpes simplex virus type 2 (227, 228). Collectively, these studies provide compelling evidence demonstrating the functional relevance of NK cell memory as a universal anti-viral immune mechanism. Observations in humans have also suggested the ability of human NK cells to form memory (229, 230) however, the full contribution of memory NK cells to anti-viral immunity and potential implications this may have on vaccine development has yet to be determined.
Natural killer cells also recognize bacteria and bacterial products either directly or from infected cells and professional APCs (Figure 9) (231). Recent work has shown that NK cells can directly release granzymes proteases to initiate disruption of electron transport, generate superoxide anion, and inactivate bacterial oxidative defenses causing the death of Listeria monocytogenes, Escherichia coli, and Mycobacteria tuberculosis (232). In addition, NK cells using Granzyme B mediated the killing of facultative anaerobic bacteria such as L. monocytogenes by cleaving essential proteins that are required for protein translation (aminoacyl tRNA synthetases and ribosomal proteins), folding (protein chaperones), and protein degradation (Clp system) (235). Indirect killing and containment of L. monocytogenes (236, 237), Staphylococcus aureus (238), Lactobacillus johnsonii (239), Mycobacterium tuberculosis (240), and Mycobacterium bovis bacille Calmette-Guérin (241) by NK cells have been described. Mechanisms by which NK cells mediate indirect clearance of bacteria are complex. Substantial evidence suggests that interleukins including IL-12 and IL-18 from monocytes and DCs play a central role (242). Role of other inflammatory cytokines such as IL-27 and its cooperation with IL-18, IL-6, and IL-12 during the clearance of bacterial infections have been identified however, the precise mechanisms by which NK cells evoke the anti-microbial responses are yet to be elucidated (245, 246).
Figure 9. Natural killer (NK) cells in health and disease. As the largest lymphocyte population representing innate immunity, NK cells perform diverse functions. Through their ability to mediate killing and to produce soluble factors, NK cells perform multitudes of immunological functions. Counter-clockwise: bidirectional interactions between NK cell and dendritic cells (DCs)/macrophages result in priming. Activated DCs and macrophages generate interleukin (IL)-15, IL-12, IL-18, IL-35, IFN-α, IFN-β, IL-27, IL-1β, and IL-23. These, in turn, activate NK cells to be primed, proliferate, and to produce inflammatory factors and chemokines such as interferon-gamma (IFN-γ), granulocyte/monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, CCL3, CCL4, and CCL5. In addition, IFN-γ from NK cells can increase the MHC class I expression and the transcription of genes encoding immuno-proteasomal subunits in these professional antigen-presenting cells and thereby augmenting T cell priming and activation. Similarly, virus-infected cells produce IFN-α, IFN-β, and IL-1β and present either “stress-induced” self-ligands or viral proteins on the cell surface that activate NK cells. A reduction in graft-versus-host disease (GvHD) is mediated through the production of IL-10 by the CD56 bright CD16 Neg NK cell subset and augmentation of GvT is potentiated via direct tumor killing by CD56 dim CD16 Pos NK cell subset. In addition, production of IL-22 by NK subsets may help the regeneration of epithelial cells in the mucosal tissues. Irrespective of these observations, the mechanisms by which NK cells are activated to respond during active GvHD/GvT is not fully understood. Genetic manipulation of NK cells has helped to improve the effector functionality and the longevity of human NK cells in vivo. Stable integration of gene encoding IL-15 into the genome of NK cells promotes sustained proliferation via an artificial autocrine loop. Similarly, integration of gene encoding IL-12 makes this cytokine abundantly available within the microenvironmental milieu and thereby augment the effector functions of NK cells, specifically, the production of IFN-γ. Augmented expression of NK cell activation receptors (NKRs) including NKG2D and NCR1 by genetic engineering increases the anti-tumor cytotoxicity of NK cells. Other studies have shown the expression of single chain variable fragment that forms the core ectodomain of chimeric antigen receptor (CAR) to augments the tumor-targeted killing of NK cells. These genetically modified NK cells provide exciting newer opportunities for cell-based therapies. The bidirectional interaction between NK and T cells results in the regulation of adaptive immunity. IL-2 produced by CD4 + Th1 cells play a vital role in the proliferation and expansion of NK cells. Although in vitro experiments consistently have provided support toward this notion, the in vivo evidence is far from convincing. However, the inflammatory factors produced by NK cells have a significant impact on both CD8 + and CD4 + T cells. Expression of “self” ligands for NKG2D by T cells results in the recognition and killing of T cells by NK cells during GvHD and anti-viral responses. In addition, a cleaved soluble form of these ligands (MIC-A/B) is present in the serum of cancer patients. This, in turn, plays an important role in containing the effector functions of T cells via direct binding to the NKG2D receptor expressed on T cells. NK cells recognize bacteria-infected cells (such as epithelial cells) either using toll-like receptors (TLR) or by activated through soluble factors including aryl hydrocarbon receptor (Ahr). This results in the production of IFN-γ and IL-22 that helps with the reduction in bacterial load and regeneration of epithelial cells, respectively. NK cells can also directly mediate the lysis of bacteria using granzymes and perforin.
Anti-Tumor Functions and the Clinical Utilization of NK Cells
The vital role of NK cells in tumor immunosurveillance was recognized soon after their initial characterization (247, 248). NK cells can detect changes in surface expression of self-MHC-I molecules on autologous cells which distinctively qualifies them to detect cells that have undergone malignant transformation (Figure 8) (218, 248). Genomic mutations that arise during the transformation process are reflected by a variety of phenotypic changes which alter the expression of cell surface molecules, including downregulation of the inhibitory “self” MHC-I (200, 249). The activity of NK cells against this “missing-self” condition has been well described (250, 251) and serves as a critical mechanism through which NK cells facilitate anti-tumor immunity. Transformed cells also express increased numbers of stress-induced molecules on their surface which can be recognized by specific NK cell receptors, such as NKG2D (120, 252). This concept, known as “induced self” (Figure 8) recognition (253, 254), explains why NK cell does not kill normal cells, such as erythrocytes, that do not express MHC-I on their surface but retain cytotoxic activity against MHC-I sufficient tumors (255). Elicitation of NK cell function is determined by the relative strength of activating and inhibitory receptor signaling and this concept, known as “altered balance,” ultimately controls NK cell activity under normal and disease conditions (256).
Decades of research in rodents have demonstrated the importance of NK cells in tumor clearance (14, 117, 247, 248). In humans, an 11-year follow-up study showed that low NK cell cytotoxic activity was correlated to an increased risk of cancer (257) and the presence of tumor-infiltrating NK cells is a positive prognostic marker for multiple malignancies including colorectal carcinoma (258), gastric carcinoma (259), and squamous cell lung cancer (260). Results from multiple studies demonstrate that NK cells have promise as a cancer immunotherapeutic for the treatment of hematological malignancies including acute myeloid leukemia and acute lymphoblastic leukemia (261). Allogenic NK cell therapy has proven effective in the clinic and, unlike T cell-based interventions, NK cell transfusion carries a relatively low risk of adverse off-tumor effects such as graft-versus-host disease (GvHD) (264).
Autologous NK cells may be inhibited by “self” MHC-I, thus limiting GvT effects in the absence of exogenous cytokines or antibodies (265, 266). Therefore, allogeneic NK cells along with hematopoietic stem cell transplant has been explored as a potential treatment for patients with high-risk solid tumors (263, 267, 268). Using non-myeloablative conditioning regimens to provide potent immune suppression without toxicity, the burden of cure then relies on the ability of transplanted donor cells to provide a GvT effect. Precedence in using low-intensity conditioning before transplanting allogeneic stem cells has been reported in Ewing sarcoma (269), osteosarcoma (272, 273), germ cell tumors (274), rhabdomyosarcoma (275), neuroblastoma (278), Wilms tumor (281), and CNS tumors (282), suggesting that alloreactive donor NK cells infiltrate heterogeneous solid tumors and cross the blood𠄻rain barrier. A sizeable reduction in tumor burden has been observed (269). Using HLA-haploidentical family donors (parents and siblings), matched by only one HLA haplotype to the patient, have not only shown favorable outcomes in patients with solid tumors (263, 267, 283) but are also readily available and highly motivated donor sources. Thus, using HLA-haploidentical donors to augment GvT may be an effective strategy in patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) for treatment of solid tumors (263, 284).
Regulatory Functions of NK Cells
Most functions of NK cells are analogous to either CD8 + T or Th1 cells, including the production of pro-inflammatory cytokines (IFN-γ, TNF-α, and GM-CSF) and mediating cytotoxicity against infected or tumor cells (95). However, in addition to these, recent reports suggest NK cells also play regulatory functions (285, 286). NK cells mediate regulatory functions of other cell types including myeloid [DC (246, 287), monocytes (291), and macrophages (246, 294)] or lymphoid [T (297, 298) and B (299) cells] via cytokines production or through direct cellll contact in a receptor–ligand interaction-dependent manner. As part of the innate immune responses, effector functions of NK cells during the early phase is expected to dictate the threshold, direction, and the outcome of an immune response. These NK cell-mediated regulatory functions are predicted to occur during viral, bacterial, or protozoan infections, anti-tumor immune responses, unexpected immuno-pathological outcomes such as GvHD, and autoimmune diseases (302). Few of the examples are described below. A unique innate immunoregulatory function for the smaller CD56 bright subset of human NK cells (CD56 bright CD16 dim NKG2A + KIR − ) was proposed due to their inherent ability to produce significant amounts of IL-10, and IL-13 along with IFN-γ, TNF-α, and GM-CSF compared to that of the more substantial CD56 dim CD16 + subset (58). An Il-27-stimulated CD56 bright CD16 dim NKG2A + KIR − subset was able to suppress the proliferation of autologous CD4 + T cells in patients with multiple sclerosis through a cytotoxic mechanism involving perforin (303) or by the release of Granzymes (304, 305). Importantly, CD56 bright CD16 dim NKG2A + KIR − subset through their ability to produce adenosine and by the restricted expression of the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (CD203a/PC-1) and the nucleotide-metabolizing ectoenzyme CD38 (an NAD + nucleosidase) was able to inhibit the proliferation of autologous CD4 + T cells (306).
Regulatory role of NK cells during GvHD is highly controversial (307). GvHD is one of the major complications and limiting factor in allogeneic HCT (308). Studies in both mouse and human lead to either suppressing or promoting rejection of HCT by NK cells. Furthermore, persistence or expansion of NK cells following HCT resulted in rejection and severe GvHD (309) while allograft-derived donor NK cells helped the engraftment of HCT by suppressing GvHD (310). Mechanistically, NK cells can help to contain GvHD through distinct mechanisms including the killing of professional APCs and thereby controlling the proliferation and expansion of graft-specific T cell (314, 315). In addition, NK cells were able to directly lyse graft-specific T cells following the expression of activating ligands of NKG2D on these T cell (316, 317). Expression of both mouse (316, 318) (H60a, H60b, H60c, Rae-1, and Mult-1) and human (319) (MIC-A, MIC-B, and ULBPs) activating ligands of NKG2D on stimulated T cells has been reported in a number of models. Also, shedding of these murine and human activating ligands has been demonstrated to employ a critical negative regulatory function on both T (322) and NK (325, 326) cells. These findings provide an exciting new avenue in understanding an inherent regulatory interaction between NK cell and APCs or T cells and thereby potential clinical utilization. Irrespective of the recent advances, the precise functions and associated mechanisms by which NK cells contribute to an immune-suppressive or immune-sufficient tumor microenvironment is far from fully defined. Similarly, the complex interplay of cytokines and ILs that are derived from and regulating the functions of NK and professional APCs during viral or bacterial infections is yet to be fully appreciated. Furthermore, defining the interactions between conventional NK cells (ILC1) and ILC2 or ILC3 can help to formulate better immunotherapeutic approaches to infections associated with mucosal tissues.
NK Cells and CAR Therapy
Recent efforts to improve the clinical efficacy of NK cell immunotherapy has led to the development of genetically engineered NK cells that express a chimeric antigen receptor (CAR). Primary NK cells and NK cell lines can be engineered to express CARs which redirect the anti-tumor specificity of NK cells on an antigen-dependent basis (327). Through the manipulation of signaling motifs critical for lymphocyte activation, CARs are also designed to utilize specific intracellular signaling molecules which can further refine NK cell function and optimize their therapeutic potential (328, 329). Interestingly, the use of a clonal cell line derived from a human NK cell leukemia, known as NK-92, has been genetically modified to express fully functional CARs and these cells have shown great promise with regards to their safety and efficacy in recent clinical trials (327, 330, 331). Moreover, the use of irradiated cell lines may provide a fast and affordable off-the-shelf option for a personalized cellular immunotherapy treatment (332, 333) and are quickly rising to the forefront of cell-based cancer immunotherapies (Figure 9).
Future perspectives and outstanding questions
Collectively, TLS and the associated T and B lymphocytes might serve as biomarkers useful to select patients who might better respond to immunotherapy. However, there are still many questions that remain to be answered before they can be incorporated into clinical practice as prognostic tools. 155
Do immature and mature TLS differentially impact a patient’s prognosis?
It is still unclear whether the degree of TLS maturation impacts a patient’s prognosis or treatment efficacy. Indeed, whether immature and disorganized TLS with sparse cellular aggregates and no evidence of effective conventional adaptive immunity convey similar prognostic value as mature and structurally well-defined TLS harboring follicles and germinal centers remains unclear. Recently, Li and colleagues (2020) endeavored to examine this issue in oral squamous cell carcinoma. They found that the presence of TLS was associated with increased 5 years overall- and relapse-free survival, and importantly, both immature and mature TLS conveyed equally positive outcomes. 58 In contrast, Posch et al. (2018) delineated that TLS in colorectal tumors exhibited different degrees of maturation which were associated with differential prognostic values. 47 In particular, mature TLS containing germinal centers had a more positive prognostic outcome compared with immature TLS. 47 Thus, evaluation of TLS maturation status in every tumor type would bring TLS into focus as an accurate prognostic tool for cancer treatment.
Can patient survival and response to treatment be predicted based on prospective evaluation of TLS ?
In most cases, the presence of TLS in tumors and their correlation with patient outcomes have been evaluated retrospectively. Given the consistent positive correlation of TLS with the anti-tumor immune response, prognosis and immunotherapeutic responses, prospective studies are warranted to determine the utility of measuring TLS presence, composition and density as prognostic tools or predictive markers of therapy efficacy.
Does TLS composition differently impact patient prognosis?
While the presence of TLS often positively impacts clinical outcomes, TLS composition itself might dictate treatment efficacy, tumor recurrence and patient survival. Indeed, Yamaguchi and colleagues 156 classified TLS into five categories based on their immune cell composition and found that TLS enriched in helper T cells were associated with disease relapse in advanced colorectal cancer. Another example is the diversity found among ASC where IgA producing cells are almost exclusively associated with a poor prognosis, while IgG + secreting cells frequently correlated with increased patient survival. Thus, better understanding of the composition of the immune infiltrate and function of TLS-forming cells, such as the isotype of ASC may be important.
Therapeutic intervention – can we specifically induce or enhance TLS formation in tumors?
Strategies augmenting de novo TLS formation in patient tumors could potentiate antitumor treatments leading to an increase in therapy response rates and patient progression- and overall-survival. While a number of preclinical studies have demonstrated the potential value of such treatments, additional studies and clinical trials are necessary to determine the therapeutic value conveyed by combining TLS- or B lymphocyte-specific targeting with current immunotherapeutic treatments.
The development of new technologies that enable the interrogation of more than 50 cellular markers simultaneously allows the precise characterization of the cellular composition, function and localization within tumors. 157 Similar approaches could be used to investigate in detail TLS composition and function. Recently, Schurch and colleagues 160 elegantly identified nine conserved distinct components characteristic of colorectal cancer immune microenvironment – described as llular neighbourhoods’ – which differentially impact a patient’s survival. The expansion of our capabilities to study a large number of parameters simultaneously might increase our understanding of the tumor microenvironment. This is likely to shed light on the cellular and molecular events associated with TLS formation and intratumoral T and B lymphocytes function associated with successful anti-tumor immunity and therapy responses.
In 1982, Nobel laureate James P. Allison first discovered the T-cell receptor.  Then, Tak Wah Mak  and Mark M. Davis  identified the cDNA clones encoding the human and mouse TCR respectively in 1984. These findings allowed the entity and structure of the elusive TCR, known before as the "Holy Grail of Immunology", to be revealed. This allowed scientists from around the world to carry out studies on the TCR, leading to important studies in the fields of CAR-T, cancer immunotherapy and checkpoint inhibition.
The TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (α) and beta (β) chains expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as α:β (or αβ) T cells, though a minority of T cells express an alternate receptor, formed by variable gamma (γ) and delta (δ) chains, referred as γδ T cells. 
Each chain is composed of two extracellular domains: Variable (V) region and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel β-sheets. The Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide/MHC complex.
The variable domain of both the TCR α-chain and β-chain each have three hypervariable or complementarity-determining regions (CDRs). There is also an additional area of hypervariability on the β-chain (HV4) that does not normally contact antigen and, therefore, is not considered a CDR. [ citation needed ]
The residues in these variable domains are located in two regions of the TCR, at the interface of the α- and β-chains and in the β-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex.  CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the β-chain interacts with the C-terminal part of the peptide.
CDR2 is thought to recognize the MHC. CDR4 of the β-chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens.
The constant domain of the TCR consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which form a link between the two chains.
The TCR is a member of the immunoglobulin superfamily, a large group of proteins involved in binding, recognition, and adhesion the family is named after antibodies (also called immunoglobulins). The TCR is similar to a half-antibody consisting of a single heavy and single light chain, except the heavy chain is without its crystallisable fraction (Fc). The two subunits of TCR are twisted together. Whereas the antibody uses its Fc region to bind to Fc Receptors on leukocytes, TCR is already docked onto the cell membrane. However, it is not able to mediate signal transduction itself due to its short cytoplasmic tail, so TCR still requires CD3 and zeta to carry out the signal transduction in its place [ citation needed ] , just as antibodies require binding to FcRs to initiate signal transduction. In this way the MHC-TCR-CD3 interaction for T cells is functionally similar to the antigen(Ag)-immunoglobulin(Ig)-FcR interaction for myeloid leukocytes, and Ag-Ig-CD79 interaction for B cells.
The generation of TCR diversity is similar to that for antibodies and B-cell antigen receptors. It arises mainly from genetic recombination of the DNA-encoded segments in individual somatic T cells by somatic V(D)J recombination using RAG1 and RAG2 recombinases. Unlike immunoglobulins, however, TCR genes do not undergo somatic hypermutation, and T cells do not express activation-induced cytidine deaminase(AID). The recombination process that creates diversity in BCR (antibodies) and TCR is unique to lymphocytes (T and B cells) during the early stages of their development in primary lymphoid organs (thymus for T cells, bone marrow for B cells).
Each recombined TCR possess unique antigen specificity, determined by the structure of the antigen-binding site formed by the α and β chains in case of αβ T cells or γ and δ chains on case of γδ T cells. 
- The TCR alpha chain is generated by VJ recombination, whereas the beta chain is generated by VDJ recombination (both involving a random joining of gene segments to generate the complete TCR chain).
- Likewise, generation of the TCR gamma chain involves VJ recombination, whereas generation of the TCR delta chain occurs by VDJ recombination.
The intersection of these specific regions (V and J for the alpha or gamma chain V, D, and J for the beta or delta chain) corresponds to the CDR3 region that is important for peptide/MHC recognition (see above).
It is the unique combination of the segments at this region, along with palindromic and random nucleotide additions (respectively termed "P-" and "N-"), which accounts for the even greater diversity of T-cell receptor specificity for processed antigenic peptides.
Later during development, individual CDR loops of TCR can be re-edited in the periphery outside thymus by reactivation of recombinases using a process termed TCR revision (editing) and change its antigenic specificity.
In the plasma membrane the TCR receptor chains α and β associate with six additional adaptor proteins to form an octameric complex. The complex contains both α and β chains, forming the ligand-binding site, and the signaling modules CD3δ, CD3γ, CD3ε and CD3ζ in the stoichiometry TCR α β - CD3εγ - CD3εδ - CD3ζζ. Charged residues in the transmembrane domain of each subunit form polar interactions allowing a correct and stable assembly of the complex.  The cytoplasmic tail of the TCR is extremely short, hence the CD3 adaptor proteins contain the signalling motifs needed for propagating the signal from the triggered TCR into the cell. The signalling motifs involved in TCR signalling are tyrosine residues in the cytoplasmic tail of these adaptor proteins that can be phosphorylated in the event of TCR-pMHC binding. The tyrosine residues reside in a specific amino acid sequence of the signature Yxx(L/I)x6-8Yxx(L/I), where Y, L, I indicate tyrosine, leucine and isoleucine residues, x denotes any amino acids, the subscript 6-8 indicates a sequence of 6 to 8 amino acids in length. This motif is very common in activator receptors of the non-catalytic tyrosine-phosphorylated receptor (NTR) family and is referred to as immunoreceptor tyrosine-based activation motif (ITAM).  CD3δ, CD3γ and CD3ε each contain a single ITAM, while CD3ζ contains three ITAMs. In total the TCR complex contains 10 ITAMs.  Phosphorylated ITAMs act as binding site for SH2-domains of additionally recruited proteins.
Each T cell expresses clonal TCRs which recognize a specific peptide loaded on a MHC molecule (pMHC), either on MHC class II on the surface of antigen-presenting cells or MHC class I on any other cell type.  A unique feature of T cells is their ability to discriminate between peptides derived from healthy, endogenous cells and peptides from foreign or abnormal (e.g. infected or cancerous) cells in the body.  Antigen presenting cells do not discriminate between self and foreign peptides and typically express a large number of self derived pMHC on their cell surface and only a few copies of any foreign pMHC. For example, it has been shown that cells infected with HIV have only 8-46 HIV specific pMHCs next to 100000 total pMHC per cell.  
Because T cells undergo positive selection in the thymus there is a non-negligible affinity between self pMHC and the TCR, nevertheless, the T-cell receptor signalling should not be activated by self pMHC such that endogenous, healthy cells are ignored by T cells. However, when these very same cells contain even minute quantities of pathogen derived pMHC, T cells must get activated and initiate immune responses. The ability of T cells to ignore healthy cells but respond when these same cells express a small number of foreign pMHC is known as antigen discrimination.  
To do so, T cells have a very high degree of antigen specificity, despite the fact that the affinity to the peptide/MHC ligand is rather low in comparison to other receptor types.  The affinity, given as the dissociation constant (Kd), between a TCR and a pMHC was determined by surface plasmon resonance (SPR) to be in the range of 1-100 μM, with an association rate (kon) of 1000 -10000 M −1 s −1 and a dissociation rate (koff) of 0.01 -0.1 s −1 .  In comparison, cytokines have an affinity of KD = 10-600 pM to their receptor.  It has been shown that even a single amino acid change in the presented peptide that affects the affinity of the pMHC to the TCR reduces the T cell response and cannot be compensated by a higher pMHC concentration.  A negative correlation between the dissociation rate of the pMHC-TCR complex and the strength of the T cell response has been observed.  That means, pMHC that bind the TCR for a longer time initiate a stronger activation of the T cell. Furthermore, T cells are very sensitive. Interaction with a single pMHC is enough to trigger activation.  Also, the decision whether a T cell response to an antigen is made quickly. T cells rapidly scan pMHC on an antigen presenting cell to increase the chance of finding a specific pMHC. On average, T cell encounter 20 APCs per hour. 
Different models for the molecular mechanisms that underlie this highly specific and highly sensitive process of antigen discrimination have been proposed. The occupational model simply suggests that the TCR response is proportional to the number of pMHC bound to the receptor. Given this model, a shorter lifetime of a peptide can be compensated by higher concentration such that the maximum response of the T cell stays the same. However, this cannot be seen in experiments and the model has been widely rejected.  The most accepted view is that the TCR engages in kinetic proofreading. The kinetic proofreading model proposes that a signal is not directly produced upon binding but a series of intermediate steps insure a time delay between binding and signal output. Such intermediate "proofreading" steps can be multiple rounds of tyrosine phosphorylation. These steps require energy and therefore do not happen spontaneously, only when the receptor is bound to its ligand. This way only ligands with high affinity that bind the TCR for a long enough time can initiate a signal. All intermediate steps are reversible, such that upon ligand dissociation the receptor reverts to its original unphosphorylated state before a new ligand binds.  This model predicts that maximum response of T cells decreases for pMHC with shorter lifetime. Experiments have confirmed this model.  However, the basic kinetic proofreading model has a trade-off between sensitivity and specificity. Increasing the number of proofreading steps increases the specificity but lowers the sensitivity of the receptor. The model is therefore not sufficient to explain the high sensitivity and specificity of TCRs that have been observed. (Altan Bonnet2005) Multiple models that extend the kinetic proofreading model have been proposed, but evidence for the models is still controversial.   
The antigen sensitivity is higher in antigen-experienced T cells than in naive T cells. Naive T cells pass through the process of functional avidity maturation with no change in affinity. It is based on the fact that effector and memory (antigen-experienced) T cell are less dependent on costimulatory signals and higher antigen concentration than naive T cell. 
The essential function of the TCR complex is to identify specific bound antigen derived from a potentially harmful pathogen and elicit a distinct and critical response. At the same time it has to ignore any self-antigen and tolerate harmless antigens such as food antigens. The signal transduction mechanism by which a T cell elicits this response upon contact with its unique antigen is termed T-cell activation. Upon binding to pMHC, the TCR initiates a signalling cascade, involving transcription factor activation and cytoskeletal remodelling resulting in T cell activation. Active T cells secrete cytokines, undergo rapid proliferation, have cytotoxic activity and differentiate into effector and memory cells. When the TCR is triggered, T cells form an immunological synapse allowing them to stay in contact with the antigen presenting cell for several hours.  On a population level, T cell activation depends on the strength of TCR stimulation, the dose–response curve of ligand to cytokine production is sigmoidal. However, T cell activation on a single cell level can be characterised by a digital switch-like response, meaning the T cell is fully activated if the stimulus is higher than a given threshold, otherwise the T cell stay in its non-activated state. There is no intermediate activation state. The robust sigmoid dose-response curve on population level results from individual T cells having slightly different thresholds. 
T cells need three signals to become fully activated. Signal 1 is provided by the T-cell receptor when recognising a specific antigen on a MHC molecule. Signal 2 comes from co-stimulatory receptors such as CD28, presented on the surface of other immune cells. It is expressed only when an infection was detected by the innate immune system, it is a "Danger indicating signal". This two-signal system makes sure that T cells only respond to harmful pathogens and not to self-antigens. An additional third signal is provided by cytokines, which regulate the differentiation of T cells into different subsets of effector T cells.  There are myriad molecules involved in the complex biochemical process (called trans-membrane signaling) by which T-cell activation occurs. Below, the signalling cascade is described in detail.
Receptor activation Edit
The initial triggering follows the mechanism common for all NTR receptor family members. Once the TCR binds a specific pMHC, the tyrosine residues of the Immunoreceptor tyrosine-based activation motifs (ITAMs) in its CD3 adaptor proteins are phosphorylated. The residues serve as docking sites for downstream signalling molecules, which can propagate the signal.   Phosphorylation of ITAMs is mediated by the Src kinase Lck. Lck is anchored to the plasma membrane by associating with the co-receptor CD4 or CD8, depending on the T cell subtype. CD4 is expressed on helper T cells and regulatory T cells, and is specific for MHC class II. CD8, on the other hand, specific for MHC class I, is expressed on cytotoxic T cells. Binding of the co-receptor to the MHC bring Lck in close proximity to the CD3 ITAMs. It has been shown that 40% of Lck is active even before the TCR binds pMHC and therefore has the ability to constantly phosphorylate the TCR.  Tonic TCR signalling is avoided by the presence of phosphatase CD45 that removes phosphorylation from tyrosine residues and inhibits signal initiation. Upon binding the balance of kinase activity to phosphatase activity is perturbed, leading to a surplus of phosphorylation and initiation of the signal. How such perturbation is accomplished by TCR binding is still debated. Mechanisms involving conformational change of TCR, TCR aggregation and kinetic segregation have been suggested.  Tyrosine kinase Fyn might be involved in ITAM phosphorylation but is not essential for TCR signalling.  
Proximal TCR signaling Edit
Phosphorylated ITAMs in the cytoplasmic tails of CD3 recruit protein tyrosine kinase Zap70 that can bind to the phosphorylated tyrosine residues with its SH2 domain. This brings Zap70 into close proximity to Lck which results to its phosphorylation and activation by Lck.  Lck phosphorylates a number of different proteins in the TCR pathway.  Once activated, Zap70 is able to phosphorylate multiple tyrosine residues of the transmembrane protein LAT. LAT is a scaffold protein associated with the membrane. It itself does not have any catalytic activity but it provides binding sites for signalling molecules via phosphorylated tyrosine residues. LAT associates with another scaffolding protein Slp-76 via the Grap2 adaptor protein, which provides additional binding sites. Together LAT and Slp-76 provide a platform for the recruitment of many downstream signalling molecules. By bringing these signalling molecules into close proximity, they can then be activated by Lck, Zap70 and others kinases. Therefore, the LAT/Slp76 complex act as a highly cooperative signalosome. 
Molecules that bind the LAT/Slp76 complex include: Phospholipase Cγ1 (PLCγ1), SOS via a Grb2 adaptor, Itk, Vav, Nck1 and Fyb. 
Signal transduction to the nucleus Edit
PLCγ is a very important enzyme in the pathway as it generates second messenger molecules. It is activated by the tyrosine kinase Itk which is recruited to the cell membrane by binding to Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 is produced by the action of Phosphoinositide 3-kinase(PI-3K), which phosphorylates Phosphatidylinositol 4,5-bisphosphate (PIP2) to produce PIP3. It is not known that PI-3K is activated by the T cell receptor itself, but there is evidence that CD28, a co-stimulatory receptor providing the second signal, is able to activate PI-3K. The interaction between PLCγ, Itk and PI-3K could be the point in the pathway where the first and the second signal are integrated. Only if both signals are present, PLCγ is activated.  Once PLCγ is activated by phosphorylation, it hydrolyses PIP2 into two secondary messenger molecules, namely the membrane-bound diacyl glycerol(DAG) and the soluble inositol 1,4,5-trisphosphate (IP3). 
These second messenger molecules amplify the TCR signal and distribute the prior localised activation to the entire cell and activate protein cascades that finally lead to the activation of transcription factors. Transcription factors involved in T cell signalling pathway are the NFAT, NF-κB and AP1, a heterodimer of proteins Fos and Jun. All three transcription factors are needed to activate the transcription of interleukin-2(IL2) gene. 
NFAT activation depends on calcium signaling. IP3 produced by PLC-γ is no longer bound to the membrane and diffuses rapidly in the cell. Binding of IP3 to calcium channel receptors on the endoplasmic reticulum (ER) induces the release of calcium (Ca 2+ ) into the cytosol. The resulting low Ca 2+ concentration in the ER causes STIM1 clustering on the ER membrane, which in turn leads to activation of cell membrane CRAC channels that allows additional calcium to flow into the cytosol from the extracellular space. Therefore, levels of Ca 2+ are strongly increased in the T cell. This cytosolic calcium binds calmodulin, inducing a conformational change of the protein such that it can then bind and activate calcineurin. Calcineurin, in turn, dephosphorylates NFAT. In its deactivated state, NFAT cannot enter the nucleus as its nuclear localisation sequence (NLS) cannot be recognised by nuclear transporters due to phosphorylation by GSK-3. When dephosphorylated by Calcineurin translocation of NFAT into the nucleus is possible.  Additionally, there is evidence that PI-3K via signal molecules recruits the protein kinase AKT to the cell membrane. AKT is able to deactivate GSK3 and thereby inhibiting the phosphorylation of NFAT, which could contribute to NFAT activation. 
NF-κB activation is initiated by DAG, the second, membrane bound product of PLCγ hydrolysation of PIP2. DAG binds and recruits Protein kinase C θ (PKCθ) to the membrane where it can activated the membrane bound scaffold protein CARMA1. CARMA1 then undergoes a conformational change which allow it to oligomerise and bind the adapter proteins BCL10, CARD domain and MALT1. This multisubunit complex binds the Ubiquitin ligase TRAF6. Ubiquitination of TRAF6 serves as scaffold to recruit NEMO, IκB kinase (IKK) and TAK1.  TAK 1 phosphorylates IKK, which in turn phosphorylates the NF-κB inhibitor I-κB, leading to the ubiquitination and subsequent degradation of I-κB. I-κB blocks the NLS of NF-κB therefore preventing its translocation to the nucleus. Once I-κB is degraded, it cannot bind to NF-κB and the NLS of NF-κB becomes accessible for nuclear translocation. 
Activation of AP1 involves three MAPK signalling pathways. These pathway use a phosphorylation cascade of three successive acting protein kinases to transmit a signal. The three MAPK pathways in T cells involve kinases of different specificities belonging to each of the MAP3K, MAP2K, MAPK families. Initial activation is done by the GTPase Ras or Rac which phosphorylate the MAP3K.  A cascade involving the enzymes Raf, MEK1, ERK results in the phosphorylation of Jun, conformational change allows Jun to bind to Fos and hence AP-1 to form. AP-1 then acts as transcription factor. Raf is activated via the second messenger DAG, SOS, and Ras. DAG recruits among other proteins the RAS guanyl nucleotide-releasing protein (RasGRP), a guanine nucleotide exchange factor (GEF), to the membrane. RasGRP activates the small GTPase Ras by exchanging Guanosine diphosphate (GDP) bound to Ras against Guanosine triphosphate (GTP). Ras can also be activated by the guanine nucleotide exchange factor SOS which binds to the LAT signalosom. Ras then initiates the MAPK cascade.  The second MAPK cascade with MEKK1, JNKK, JNK induces protein expression of Jun. Another cascade, also involving MEKK1 as MAPK3, but then activating MKK3 /6 and p38 induces Fos transcription. Activation of MEKK1, additionally to being activated by Ras, involves Slp-76 recruiting the GEF Vav to the LAT signalosom, which then activates the GTPase Rac. Rac and Ras activate MEKK1 and thereby initiate the MAPK cascade. 
Regenerative capacity and the developing immune system
Many components of the vertebrate immune system have evolved with dual, interrelated functions of both protecting injured tissues from infection and providing for tissue maintenance and repair of injuries. The capacity for organ regeneration, prominent among invertebrates and certain phylogenically primitive vertebrates, is poorly developed in mammals. We have proposed that evolution of the mammalian immune system has produced inflammatory cellular interactions at sites of injury which have optimized tissue defense and facilitated tissue repair, but that these improvements included concomitant loss of regenerative capacity. This chapter briefly reviews work in two regenerating systems: scar-free repair of fetal mammalian skin and regeneration of amputated limbs in larval frogs. In both organs the potential to regenerate anatomically and functionally complete new structures is lost gradually during ontogeny and this loss coincides with development of an immune system producing an inflammatory response in injured tissues. Failure of organ regeneration has long been associated with scarring or fibrosis and this phenomenon is a direct result of inflammatory interactions of immune cells and fibroblasts at sites of injury. Several aspects of immunity related to repair are reviewed, including the importance of antigen-presenting cells and lymphocytes, relevant cytokines and growth factors released by these and other cells, immune functions of extracellular matrix components, and immunological functions of fibroblasts. Skin repair in various transgenic mouse models has been especially informative. Further study of immune mechanisms associated with the loss of regenerative capacity in the skin and amphibian limb will be useful for efforts to promote mammalian organ regeneration.