T CELLS

• T cells have two major roles in the immune reaction. They are either regulatory cells ( helper cells) or as effector cells ( Cytotoxic).

• Regulator ( tell cells what to do by signals), effector ( perform the function itself).

• The regulatory role of CD4 helper cells is mediated by signal proteins (interleukins). For example, helper T cells make • (1) interleukin (IL)-2, which activates CD4 and CD8 cells• (2) IL-4, which help B cells make antibodies, especially IgE• (3) gamma interferon, which enhances killing by macrophages.

• The effector functions of T cells are carried out mainly by cytotoxic (CD8) T cells, which DIRECTLY kill cells ( virus infected cells, tumor cells , allografts).

• T cell progenitor cells differentiate from the outer layer of cortical epithelial thymus cells (nurse cells), T cell progenitors differentiate under the influence of Thymic hormones (Thymosins and thymopoietins) into T-cell subpopulations that are characterized by their surface proteins (CD3, CD4, and CD8).

• CD3 is present on the surface of ALL T cells, and is associated with antigen receptors (TCR).

• The CD3 is a complex of five transmembrane proteins, its main function is the transmission of signals from outside the membrane to within the cell ( hence the transmembrane part!)

CD4 & CD8 Types of T Cells

• As it is associated with the antigen receptor (TCR) the signal transmitted its that the TCR is occupied (ON). (One of the CD3 transmembrane proteins, the zeta chain, is linked to a tyrosine kinase called fyn, which is involved with signal transduction).

• Second messengers further transmit the signal (see later).

• CD4 is a single transmembrane protein, whereas CD8 is made up of two transmembrane proteins. (Lck kinase is a possible way for their signal transmission)

• T cells are subdivided into two major categories on the basis of whether they have CD4 or CD8 proteins on their surface, when they mature they have either one, but not both.

https://commons.wikimedia.org/wiki/File:TCR_complex.svg

• CD4 lymphocytes perform their regulatory functions in the following manner:

• (1) they help activate CD8 T-cells to become activated cytotoxic T cells

• (2) They help activate B cells to develop into antibody-producing plasma cells

• (3) they help macrophages effect delayed hypersensitivity (e.g., limit infection by Mycobacterium tuberculosis).

• The first two functions are carried out by two different subpopulations of CD4 cells.

• The first function is carried out by Th-1 CD4 cells help activate cytotoxic T cells by producing IL-2 and help initiate the delayed hypersensitivity response ( the third function) by producing primarily IL-2 and gamma interferon

• The second function is carried out by Th-2 cells, which help activate B-cells by producing primarily IL-4 and IL-5 .

• HOWEVER, the role of Th1 cells (e.g., gamma interferon) , also affects B cells to class switch from IgM to IgG by producing cytokines which produce two subclasses of IgG (namely IgG 1 and IgG 3) that are very effective in opsonisation of bacteria. (we will get to this during antibody lectures)

The human cell that produces the IL-4, which induces naïve helper T cells to become Th-2 cells, has not been identified.

Ingested bugs→IL-12 produced→IL-12induces naïve Th-0 cells to become Th-1 cells → gamma interferon and IL-2 (cell mediated immunity)

IL-4 induces naïve Th-0 cells to become Th-2 cells→ production of IL-4 and IL-5→ activation of B cells to become plasma cells (antibody immunity)

• There is a balance between Th1 cells and Th1 cells.

• This balance is is provided by the production of IL-12 from macrophages. IL-12 increases the number of Th-1 cells (cell mediated), enhancing host defenses against organisms that are controlled by a delayed hypersensitivity response.

• Moreover, Interferon from Th1 cells also inhibits the production of Th2 cells, tipping the scale further towards Th1 response). Th1 = IL-12 (stimulatory to Th1) + Interferon (inhibitory to Th2)

• IL-10 produced by Th-2 cells inhibits IL-12 production by macrophages and drives the system toward an antibody response and away from a cell-mediated response (towards Th2).

• CD4 cells make up about 65% of peripheral T cells and predominate in the thymic medulla, tonsils, and blood. Th2 = IL-4 (stimulatory to Th2) + IL10 (inhibitory to Th1)

IL-12 +-

+IL10 -

Remember this from lecture 1?

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Back to normal behavior

• To mount a protective immune response against a specific microbe requires that the appropriate subpopulation (i.e., either Th-1 or Th-2 cells) to play a dominant role in the response.

• For example, if an individual is infected with M. tuberculosis and Th-2 cells are the major responders, then humoral immunity will be stimulated rather than cell-mediated immunity.

• Humoral immunity is not protective against M. tuberculosis, and the patient will suffer severe tuberculosis (why?).

• Similarly, if an individual is infected with Streptococcus pneumoniae and Th-1 cells are the major responders, then humoral immunity will not be stimulated and the patient will have severe pneumococcal disease (remember this is a capsulated bacteria, you need antibodies to counter it).

• Precisely what component of a microbe activates either Th-1 or Th-2 cells is unknown.

• M. tuberculosis is a well studied and well known example of how the response is stimulated.

• It was found that a specific lipoprotein on the surface of that bacterium interacts with a specific Toll-like receptor (TLR) present on the surface of the macrophage, the interaction of the lipoprotein and the macrophage’s TLR induces the production of IL-12. ( so this is before the bacteria enters cells, it is now detected before entry by macrophages)

• (remember Toll like receptors, part of innate immunity, how cells such as macrophages and dendritic cell use pathogen associated patterns to detect microbes and engulf them)

• IL-12 is the stimulatory signal that drives the differentiation of undifferentiated (naïve) helper T cells to go down the Th-1 type of differentiation which drives a cell-mediated (delayed hypersensitivity) response against the organism/which is here the correct response.

• Another subpopulation of CD4 cells that differentiate into yet another subpopulation of immune responders (called Th-17), they have been shown to have a significant role in the mucosal immunity of the gastrointestinal (GI) tract (this will include the mucosa of the mouth!).

• The reason these cells are different , is that they are producing IL-17instead of gamma interferon (that is usually produced by Th1 cells, or IL-4 from Th-2 cells)

• The function of IL-17 is as a signal that recruits neutrophils to the site of bacterial infections.

• What do neutrophils do? How?

• how we found the significance of these cells was that HIVSELECTIVLEY targets these cells, which creates an almost total loss of function of Th-17 cells.

• It was shown that those patients have a high rate of blood infection caused by colonic (gut) bacteria such as Escherichia coli and Klebsiella ( the gut is not protecting them from the penetration of these bacteria into the blood). This is how we discovered this subpopulation of CD4 cells.

• In a similar fashion, IL-17 was found to also contribute to our immunity against some fungal infections (chronic mucocutaneouscandidiasis).

CD8 cells:

• As was discussed before, CD8 lymphocytes are cellular responders (effectors), which respond as cytotoxic cells against other cells (virus-infected cell, tumor and allograft cells).

• The way they kill these cells is by either release perforins ( the bullets) that poke holes in the membranes of the target cells, or by forcing these cell to undergo apoptosis ( programmed cell death).

• CD8 cells constitute about 35% of all peripheral T-cells, that are usually found mainly in the bone marrow and gut lymphoid tissue.

• To activate helper T cells, an antigen presenting cell must posses a MHC-II complex, which carries (as a complex) an antigen, which the T-cell receptor is able to recognize. ( antigen presenting cells are usually macrophages and dendritic cells, but there are others).

• The activation of cytotoxic T cells on the other hand requires antigen presentation from cells on MHC-I, that is complexed with an antigen recognizable by the cytotoxic CD8 cell TCR.

• APCs have both class I and class II proteins on their surface. • The first step in the activation of helper T cells is the uptake of the foreign

protein-antigen, microbe- by the APC. • This microbe or protein is then digested within the APC into smaller parts –

processing into small peptides for example-, and these unique small peptides are then complexed with MHC-II proteins and presented on the outside of the cell for a CD4 cell(using its TCR) to recognize it.

Activation of T Cells

• T cells are usually harbored in lymph nodes, while at the same time the antigen is being encountered in mucosal surfaces where APC are present under these epithelial surfaces.

• how do these APCs recruit the T cells from such distance?

• An APC that it is carrying an antigen on its surface will produce a specific receptor on its surface (which indicates it is currently presenting an antigen), this receptor is for(reactive to) the chemokine CCR7.

• The chemokine signal of CCR7 is being produced all the time ( like a radio wave, seen as a gradient of CCR7, highest in the lymph node and gets lower and lower away from the lymph node) from T cells in the lymph node at all times, once the correct radio (the receptor on the APC) is produced it will start to migrate to the NEAREST signal -lymph node- going from low concentrations of CCR7 ( away) to higher concentrations gradually (towards the NEAREST lymph node).

• As for the activation of cytotoxic T cells, this happens when the APC itself is infected with a virus, the virus within the cell is now producing foreign proteins which are presented on the surface with an MHC-I protein which activates the CD8 cells to perform cytotoxic function.

• The same happens when a piece of a dying infected cells is presented to CD8 cells on MHC-I proteins (also viral antigens are being presented on MHC-I protein).

• A Non APC cell will also present viral antigens on its surface on a MHC-I complex ( all nucleated cells have MHC-I)

• To easily remember this, know the rule of eight: CD4 cells interact with class II (4 × 2 = 8), and CD8 cells interact with class I (8 × 1 = 8).

The T cell Receptor

• Polymorphism of MHC proteins is needed in order to enable them to bind many different types of antigens to be recognized by TCR ( if all MHC proteins were exactly alike, they will only bind a small portion of antigen peptides).

• This polymorphism is achieved by having many different alleles(gene copies) within the class I and class II MHC genes.

• MHC-I and MHC-II proteins can only present peptides and they are unable to present any other type of molecule.

• As mentioned previously, MHC proteins present peptides from foreign and self sources, and to direct the immune response towards responding only to MHC proteins that are presenting foreign proteins, the T cells must be correctly trained in the thymus ( negative and positive selection) as to only release the ones that recognize non self MHC complexes and delete the ones that recognize self proteins.

• ( meaning we are not deleting cells that present self peptides on their MHC proteins, but deleting the cells-T cells- that would incorrectly respond to this self signal).

Costimulation Is Required to Activate T Cells we need two signals• The first signal is the initial activation of the T cell by the interaction

between the MHC complex protein with an antigen that is specific and interacts with that TCR. ( is the TCR then unique to each antigen?)

• It is an important observation that the CD4 protein on the surface of these T cells functions in stabilizing the connection between TCR and MHC protein on the surface of the APC.

• Other proteins also serve to stabilize the contact between the two cells (APC and T cell)• lymphocyte function-associated antigen 1 [LFA-1] protein 2 on CD4 or CD8 T

cells binds with intracellular adhesion molecule 1 [ICAM-1] protein on the surface of APCs this is how the connection is established, which is then further strengthened by CD4

• A second signal, costimulatory signal is also required

• B7 protein on the APC must interact with CD28 protein on the helper T cell to proceed with the costimulatory signal.

• If the secondary co stimulation occurs, only then that IL-2 is produced by the helper T cell

• The production of IL-2 is the vital step that produces activated helper T cells that are able to regulate the immune response in Th-1 or Th-2 manner and produce memory cells.

• If this costimulatory signal does not occur a state of Anergy occurs (unresponsiveness). This unresponsiveness will only be specific to that epitope ( they will still respond to other epitopes).

• Production of the costimulatory protein depends on activation of the Toll-like receptor on the APC surface.

• This is yet another defense mechanism against an immune response towards self proteins, there is no B7 production for self proteins however, foreign antigens are able to induce the production of B7.

MHC TCR/BCR

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Activation of T cells.

• On the left: APC presenting antigen with MHC-II → TCR specifically recognizes antigen→CD4 helper cell is activated and produces IL-2 which will only occur if the B7 protein binds the CD28 protein on the T cell. CD4 protein helps stabilize the connection between the cells.

• On the Right: A virus-infected cell uses its MHC-I to present viral antigen--> viral antigen recognized by TCR (it is specific to that antigen)→IL-2 produced by the helper T-cell activates this CD8 cell to kill the viral infected cell.→ CD8 protein helps stabilize the interaction between the two cells.

• Class II MHC protein consists of two polypeptides, both of which are encoded by genes in the human leukocyte antigen (HLA) locus, whereas Class I MHC protein has only one of its polypeptide encoded by the HLA locus and β2 - microglobulin (β2 MG), which is encoded somewhere else.

• To turn off these helper T- Cells, a different protein called cytotoxic T lymphocyte antigen-4 (CTLA-4) appears on the T-cell surface and binds to B7 and displaces the bound CD28.

• Now the co stimulatory signal is no longer working and thus CTLA-4 inhibits T-cell activation (IL-2 is now not produced due to the lack of the costimulatory signal).

• This is a regulatory mechanism to control the T cells and create a balance between on and off status.

• If this OFF switch is not present (mutant T cells that do not have CTLA-4) cannot be deactivated and cause autoimmune reactions.

• The use of CTLA-4 protein is shown to reduce the rejection of organ transplants in experimental animals ( remember it is a cellular response).

Inhibition of activated helper T cells.

• Agonists of CTLA4 are used coupled with Ig to reduce immunity and are in trials to treat immune disorders such as Rheumatoid arthritis and renal transplants in specific patients (with EBV virus).

• Ig Fc fragment provides resistance against degradation, resulting in increased plasma levels of CTLA-4 for a longer duration than CTLA-4 alone

• Antagonists of CTLA4 (enhancers of cellular immunity) are used to increase immunity, this is in trials to be used as a potential therapy to reduce the tolerance of immune system to tumor cells and thus help mount a response against them

• Antagonists to CTLA-4 can be in the form of antibodies, that would inactivate CTLA4 and thus improve the immune response against some human cancer cells and cause the cancer to regress.

• So the antibody is an inhibitor of an inhibitor of the immune response.

• Another inhibitory protein on the surface of T cells has also been described (PD-1 (programmed cell death-1)).

• When PD-1 interacts with its ligand (PDL-1) on the surface of APCs, such as dendritic cells and macrophages, the immune response is inhibited-similar to CTLA-4. Similarly, antibodies against PD-1 enhance the immune response and are effective against some cancers, as shown in recent trials.

Activation of T cells.

• On the left: APC presenting antigen with MHC-II → TCR specifically recognizes antigen→CD4 helper cell is activated and produces IL-2 which will only occur if the B7 protein binds the CD28 protein on the T cell. CD4 protein helps stabilize the connection between the cells.

• On the Right: A virus-infected cell uses its MHC-I to present viral antigen--> viral antigen recognized by TCR (it is specific to that antigen)→IL-2 produced by the helper T-cell activates this CD8 cell to kill the viral infected cell.→ CD8 protein helps stabilize the interaction between the two cells.

• Class II MHC protein consists of two polypeptides, both of which are encoded by genes in the human leukocyte antigen (HLA) locus, whereas Class I MHC protein has only one of its polypeptide encoded by the HLA locus and β2 - microglobulin (β2 MG), which is encoded somewhere else.

• To turn off these helper T- Cells, a different protein called cytotoxic T lymphocyte antigen-4 (CTLA-4) appears on the T-cell surface and binds to B7 and displaces the bound CD28.

• Now the co stimulatory signal is no longer working and thus CTLA-4 inhibits T-cell activation (IL-2 is now not produced due to the lack of the costimulatory signal).

• This is a regulatory mechanism to control the T cells and create a balance between on and off status.

• If this OFF switch is not present (mutant T cells that do not have CTLA-4) cannot be deactivated and cause autoimmune reactions.

• The use of CTLA-4 protein is shown to reduce the rejection of organ transplants in experimental animals ( remember it is a cellular response).

Inhibition of activated helper T cells.

• Agonists of CTLA4 are used coupled with Ig to reduce immunity and are in trials to treat immune disorders such as Rheumatoid arthritis and renal transplants in specific patients (with EBV virus).

• Ig Fc fragment provides resistance against degradation, resulting in increased plasma levels of CTLA-4 for a longer duration than CTLA-4 alone

• Antagonists of CTLA4 (enhancers of cellular immunity) are used to increase immunity, this is in trials to be used as a potential therapy to reduce the tolerance of immune system to tumor cells and thus help mount a response against them

• Antagonists to CTLA-4 can be in the form of antibodies, that would inactivate CTLA4 and thus improve the immune response against some human cancer cells and cause the cancer to regress.

• So the antibody is an inhibitor of an inhibitor of the immune response.

• Another inhibitory protein on the surface of T cells has also been described (PD-1 (programmed cell death-1)).

• When PD-1 interacts with its ligand (PDL-1) on the surface of APCs, such as dendritic cells and macrophages, the immune response is inhibited-similar to CTLA-4. Similarly, antibodies against PD-1 enhance the immune response and are effective against some cancers, as shown in recent trials.

Immunology Lecture 6

T Cells Recognize Only Peptides

• T cells only recognize antigens that are polypeptides and only when those are presented on MHC proteins.

• The restriction of CD4 cells and CD8 cells to bind MHC-II or MHC-I is due to the fact that binding sites on the TCR only recognize the appropriate MHC protein (CD4 can only bind MHC-II, and CD4 will stabilize that connection, whereas a CD8 cells can do so but only with MHC-I)

• Further more, the CD8 and CD4 proteins which stabilize the connection, further aid in this restriction.

• MHC-I are usually used to present intracellulary produced antigens (viral proteins for example), MHC-II proteins usually present antigens of extracellular origin (bacterial proteins) - two distinct pathways with distinct organelles used for each.

• This is why when I give a vaccine composed of dead viral cells, it will not cause a CD8 (cytotoxic response) and cause an antibody response As these viruses will not be presented on MHC-I cells, because they did not replicate and infect cells.

• They will instead be presented on MHC-II and go through the B cell humoral response.

• You will have ready made antibodies that will trap the virus before entry to cells (or when they burst open cells) -so next time you meet the virus, you have antibodies to intercept before it enters your cells-.

• .

• This restriction, is due to the distinction in the acquisition and PROCESSINGof these proteins through different organelles and pathways.

• 1) An endogenous protein (viral protein – cancer protein) is processed in different compartments within the cytoplasm than those derived from extracellular sources.

• The association of endogenous foreign proteins with MHC-I complex happens due this difference in processing, where these proteins are cleaved by a proteasome and then the peptides are chaperoned by a “TAP transporter” that transports these proteins to the Rough ER where it is linked with MHC-I.

• The endogenous protein-MHC-I complex now travels to Golgi and then to the cell surface where it is presented → in short, MHC-I presented proteins go through the typical endogenous protein manufacturing process and end up on MHC-I

• 2)The other route is for extracellular proteins is through the cleavage of these proteins in an endosome (as opposed to rough ER), this is where the peptide fragments are linked with the MHC-II protein into a complex.

• From the endosome the peptide-MHC-II complex migrates to the cell surface.

• Major question: why don’t the intracellular proteins –some of those get degraded, get presented on MHC-II? →There is a protection mechanism placed here that (mostly) prevents endogenously produced proteins from being linked with an MHC-II protein ( and hence the restriction mechanism would be bypassed).

• This occurs due to the presence of an invariant chain attached to MHC-II proteins when they are not inside the endosome ( sort of like a lock mechanism), no proteins will be able to attach to MHC-II outside the endosome (while it is being sent from endosome to the membrane), and no endogenously produced proteins enter the endosome, effectively creating the restriction mechanism. ( the lock mechanism is only removed for MHC-II proteins within the endosome only).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026165/figure/F2/

*some crossing between

the two pathways DOES

occur:

MHC-I can bind peptides

derived from exogenous

proteins internalized by

endocytosis or

phagocytosis, a

phenomenon called

cross-presentation.

Specific subsets of

dendritic cells (DCs) are

particularly adept at

mediating this process,

which is critically

important for the

initiation of a primary

response by naïve

CD8+ T cells

This is actually beneficial

which would allow

cytotoxic response

against certain cells that

have taken up certain

toxic material (toxins)

• As for B cells the story is quite different, these cells interact with their surface immunogloblulin (IgM and IgD)(not a TCR).

• Since antigen presentation with MHC-II is not needed to activate B cells ( not always).

Remember the T cell independent antigen loop and the ability of B cells to present antigens (which are not always peptides), which ultimately activate themselves indirectly by presenting the antigen to a CD4 cell.

• In contrast to MHC-II antigen presentation, where it can only present peptides, the IgM and IgD antigen receptors on the surface of the B cell can recognize non peptides as antigens (polysaccharides, nucleic acids, and small molecules -drugs such as penicillin-).

• As mentioned in previous lectures, this is how haptens (non peptides) can bypass the peptide requirement and act as an immunogen ( and then satisfy the size requirement using the carrier protein).

• Then to be associated with MHC-II antigen presentation, the carrier protein’s peptides are instead used in this case, bypassing a third requirement (peptide presenting to CD4 on MHC-II) to activate CD4 helper T cells.

• Now at this point the helper T cell will produce the appropriate cytokines (lymphokines) to activate a the B cell to start producing antibodies against this hapten complex

• Signal transduction:• Antigen-MHC complex on the APC interacts with the TCR on the surface,

the effect o this interaction is produced by a signal sent from the TCR to the inside of the cell (nucleus).

• The signal is transmitted by the CD3 protein (remember CD3 is a part of the TCR) complex through certain pathways that eventually lead to an influx of Calcium into the cell (the stimulus to transduce the signal).

• The influx of Calcium causes (activates) a specific protein (Calcineurin) (a serine phosphatase enzyme) to exert its enzymatic function in the nucleus (switch on the genes for IL-2 and IL-2 receptor)

• Now that we know what is calcineurin and how it functions, we can remove MHC-II cell mediated immunity by blocking calcineurin ( a drug called cyclosporine does this), this is useful in organ transplantation (cell immunity is responsible for organ rejection*)

• →but why does block only cell mediated and not AB mediated?.

IL-2

• IL-2 is produced by CD4 cells which is activated by APC presenting on MHC-II

• IL-4 from unknown source is what activates Th2 response!

• Signal transduction pathways involved in T-cell anergy.

• Stimulatory signals delivered by the engagement of the T-cell receptor (TCR; signal 1) and co-stimulatory molecules (CD28; signal 2), both work to induce different signaling pathways that result in the activation of multiple transcription factors at the gene level

• The positive signals include:

• 1- phospholipase C1 (PLC1), which induces the Ca2+ influx,

• 2- nuclear factor of activated T cells (NFAT)- nuclear factor-B (NF-B) pathway

• 3- protein kinase C (PKC), which regulate the nuclear 4- activator protein 1 (AP1) pathways. (controlled by signal 2 -costimulation)

• In the nucleus, NFAT + AP1 + other transcription factors = induce a program of gene expression that leads to interleukin-2 (IL-2) production and activation.

• TCR engagement in the absence of co-stimulation (signal 1 without signal 2) results in the induction of NFAT proteins without concomitant AP1 activation. In the absence of cooperative binding to AP1 ,NFAT alone will regulate the transcription of a distinct set of genes that will produce anergy response NO IL-2, such as Casitas B-lineage lymphoma B (CBL-B). Anergy-associated factors inhibit T-cell function at different levels leading to T-cell unresponsiveness (NO IL-2 PRODUCTION).

1

2

3

signal1

signal2

• It is in this step (IL-2 production) that clonal proliferation of helper T cells happens for that specific antigen- meaning if the CD4 cell reaches the IL-2 production level, it will also proliferate to make clones for itself:

• IL-2 (also known as T-cell growth factor), stimulates the helper T cell to multiply into a clone of antigen-specific helper T cells ( it also stimulates CD8 cells).

• The majority of these helper T cells will carry out their effector and regulatory functions.

• From these clones of cells a few are kept away as memory Cells for rapid function in subsequent exposures to this antigen.

• Cytotoxic T cells and B cells clones made for a specific antigen also form memory cells.

• Activated CD4-positive T cells also produce another lymphokine called gamma interferon, which enhances the ability of APCs by making them produce more MHC-II proteins (and thus present more antigen).

• Gamma interferon also enhances the microbiocidal activity of macrophages.

• It is important to know that activation of T cells is not all or none. There is a grey area of partial activation that may occur.

• Full activation has the full complement of lymphokines released, partial activation leads to a release of a few of those lymphokines which would lead to a weaker response.

• This is dependent on the epitope that was used in the activation of the T cell, which would result in a different transduction pathways being used for transduction of the signal ( and activation of the proper lymphokine producing genes)

• The explanation for this is as follow ( which explains why some people can clear certain infections more efficiently than others…genetics! Perhaps even random events)

• As our cells have three genes for the class I locus (A, B, and C) and three genes at the class II locus (DP, DQ, and DR) from each parent ( for a total of six possible copies of each gene in Class I or 6 copies making Class II proteins).

• as each gene copy has multiple alleles for each locus, each MHC protein is now able to present peptides with different amino acid sequence

Memory T Cells

• With these cells we are able to mount a quicker, effective response to antigens –YEARS– after the initial exposure to an antigen.

• This memory response to a specific antigen is due to several features:

• (1) in the first exposure to an antigen, there are very few cells responding to this stimulus ( they have to start from zero cells), for activation of CD4 cells and so on, second exposure will find many cells already produced and ready ( don’t need to start from zero at this point)

• So a T cell has to reach activation (production of IL-2) by finding proper MHC-II and TCR interaction, then reach activation, at this point some of the cells are kept as memory cells.

• (2) cells ( as opposed to antibodies) can live for many years and replicate themselves ( maintenance) whereas antibodies cannot.

• (3) a memory cell has a special ability which allows it to have a much stronger (almost exaggerated) response to a smaller amount of antigen, and would require less co stimulation, this heightened response is because these cells produce higher amount of interleukins than regular naïve cells , this is the basis how your memory cells will produce a fast response against a certain antigen.

The T-Cell Receptor

• From the previous/next figure we find that the the TCR is composed of composed of 2 polypeptide chains (alpha and beta), which are associated with CD3 proteins ( the T cell receptor and CD3 protein work together in receiving the antigen- mostly TCR and transduction of the signal , which is done mainly by CD3 alongside TCR transmembrane apparatus).

• In order for the TCR to detect many different antigens, it thus has a structure that resembles immunoglobulin heavy chains in a few ways which would allow it to bind to different antigens:

• (1) the genes that code for TCR chains are made by the rearrangement of multiple regions of DNA (which creates variability)

• (2) the gene loci have many segments -V (variable), D (diversity), J (joining), and C (constant) , all of which work together to provide high variability and diversity ( at this level of diversity more than a 100 million different receptor proteins can be made)

• (3) the variable regions have hypervariable domains (even more diversity at the end of the protein)

• (4) the two genes (RAG-1 and RAG-2) that encode the recombinase enzymes that catalyze these gene rearrangements are similar in T cells and B cells.

• variable (V), diversity (D), joining (J) gene segments, and constant (C) genes.

• The TCRα locus (chromosome 14) consists of 70–80 Vα gene segments, each preceded by an exon encoding the leader sequence (L) in white. A cluster of 61 Jα gene segments is located a considerable distance from the Vα gene segments. The Jα gene segments are followed by a single C gene, which contains separate exons for the constant and hinge domains and a single exon encoding the transmembrane and cytoplasmic regions. The TCRβ locus (chromosome 7) has a different organization, with a cluster of 52 functional Vβ gene segments located distantly from two separate clusters each containing a single D gene segment, together with six or seven J gene segments and a single C gene. Each TCRβ C gene has separate exons encoding the constant domain, the hinge, the transmembrane region, and the cytoplasmic region (not shown). The TCRα locus is interrupted between the J and V gene segments by another T-cell receptor locus—the TCRδ locus

https://www.ncbi.nlm.nih.gov/books/NBK27145/bin/CH4F11.jpg

https://images.nature.com/full/nature-assets/gene/journal/v17/n3/images/gene20169f1.jpg

https://www.ncbi.nlm.nih.gov/books/NBK27145/bin/CH4F12.jpg

• From the previous figures, we can deduce that each T cell has a unique TCR on its surface, with hundreds of million of different T cells (with different specific receptors) in each human being.

• Each unique T cell, once activated, will proliferate (clonal expansion), to yield a large number of cells that are able to deal with the specific antigen they are unique to.

• Antibodies have a similar genetic rearrangement that yields a high number of hypervariable antibodies.

• However, the TCR (even though it is uniquely specific to an antigen like an antibody is), it differs from the antibody in two ways:

• (1) it has two chains rather than four (2) it recognizes antigen only in conjunction with MHC proteins, whereas immunoglobulins recognize free antigens.

• This is where the B cell (T cell independent activation) is appreciated, B cells don’t use TCR but use an immunoglobulin (IgM or IgD) so they bypass the need of MHC.

• Also TCR proteins are always anchored into the outer membrane of T cells, There is no circulating form as there is with certain antibodies (monomeric IgM is in the B cell membrane, but pentameric IgM circulates in the plasma)

Effect of Super antigens on T Cells• Certain proteins, particularly staphylococcal enterotoxins and toxic shock

syndrome toxin (TSST), act as “superantigens” (next figure).

• In contrast to the typical antigen, which activates one (or a few) helper T cell, superantigens are “super” because they activate a large number of helper T cells.

• For example, toxic shock syndrome toxin binds directly to class II MHC proteins without internal processing of the toxin. This complex interacts with the variable

portion of the beta chain (Vβ) of the TCR of many different T cells.

MEANING THESE ANTIGENS HAVE REGIONS THAT ARE DETECTABLE BY MANY DIFFERENT TCRs (overcomes their specificity)- which will produce a heightened exaggerated response.

• This activates the T cells, causing the release of IL-2 from the T cells and IL-1 and tumor necrosis factor (TNF) from macrophages. These interleukins account for many of the findings seen in toxin-mediated staphylococcal diseases.

• Certain viral proteins (e.g., those of mouse mammary tumor virus [a retrovirus]) also possess superantigen activity

Top: the “regular” antigen processing by an APC is shown, the epitope is then presented on the MHC-II complex and signal transduction and activation of few helper T cell ensues.

Bottom: the superantigen, is DIRECTLY WITHOUT PROCESSING (so it is FAST acting) is bound to MHC-II protein complex ( using the Vβ portion of the T cell receptor).

Because it bypasses the antigen-specific site, superantigen can activate many helper T cells.

Features of T Cells

• T cells make up about 65% to 80% of the recirculating pool of small lymphocytes, the rest are B cells.

• Within lymph nodes, T cells are found in the inner and subcortical regions, whereas B cells are located primarily in the germinal centers of the lymph node.

• T cells have a very long half life, they can survive for months or even years.

• These cells can be stimulated to proliferate by using mitogens ( mitosis generating molecules).

• mitogens for T cells include: phytohemagglutinin or concanavalin A [endotoxin, a lipopolysaccharide found on the surface of gram-negative bacteria, is a mitogen for B cells but not T cells]).

• T cells are detected in blood by using sheep blood, this is because all T cells have receptors on their surface that reacts with sheep RBCs, and form a unique structure seen by the eye “rosettes”.

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Effector Functions of T Cells- recap

• The four types of T cells (Th-1, Th-2, and Th-17 types of CD4 cells, and CD8 cells) mediate different aspects of our host defenses.

• Th-1 cells mediate delayed hypersensitivity reactions against intracellular organisms.

• Th-2 cells mediate protection against helminths (worms).

• Th-17 cells protect against the spread of bacterial infections by recruiting neutrophils to the site of infection.

• CD8 cells protect against viral infection by killing virus-infected cells.

Th-1 Cells• Th-1 cells and macrophages are the main effectors of cell mediated ( also

called delayed hypersensitivity reactions, type IV hypersensitivity reaction).

• This type of reaction is aimed to protect against intracellular pathogens ( fungi Histoplasma and Coccidioides) and (bacteria such as M. Tuberculosis), the main signaling molecule in this type of reaction is GAMMA INTERFERON, with other signals that help recruit or exclude macrophages (macrophage activation factor and macrophage migration inhibition factor (MIF)) also play a role.

• The actual function of destroying these intracellular pathogens is performed by macrophages, however Th-1 cells direct macrophages (recruit them to the site and tell them what to look for) by the production of interleukins.

• If one has a diminished delayed hyprsensitivity reaction, they will of course be unable to clear these pathogens.

• The well studied case of M. tuberculosis, a specific lipoprotein of the bacterium is picked up and then stimulates a specific Toll-like receptor on the macrophage, which signals the cell to synthesize IL-12 (indicating that a foreign object was encountered).

• IL-12 is an inducer of the correct type of response to this lipoprotein ( cellular Th1 response), thus IL-12 induces naïve helper T cells to differentiate into the Th-1 type of helper CD4 T cells to start the delayed hypersensitivity response, at this point Th-1 cells produce gamma interferon, which activates macrophages, thereby enhancing their ability to kill M. tuberculosis.

• This IL-12–gamma interferon axis is very important in the ability of our host defenses to control infections by intracellular pathogens, such as M. tuberculosis and Listeria monocytogenes.

• recap: Macrophage found a foreign protein→ IL-12→ activate Th1 CD4 cells →G-INF →activate macrophages

http://jcytol.org/articles/2015/32/3/images/JCytol_2015_32_3_188_168855_f1.jpg

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Histoplasmosis coccidosis TB

Th-2 Cells

• Th-2 CD4 cells along with eosinophils constitute the main effectors of reactions that are protective against helminths (worms) such as Schistosoma and Strongyloides (remember IgE and eosinophils).

• The most important interleukins for these reactions are IL-4 (why? Because we need Th2 response to produce IL-4),

• IL-4 increases the production of IgE, and IL-5, which activates eosinophils.• IgE binds to the surface of the worm. Eosinophils then bind to the heavy

chain of IgE and secrete their enzymes that destroy the worm. • Th-17 Cells • Th-17 cells protect against the spread of bacterial infections at mucosal

surfaces by producing IL-17. →IL-17 attracts neutrophils to the site of infection whereupon the bacteria are ingested and destroyed.

CD8 Cells

• CD8 cells mediate the cytotoxic response that is concerned primarily with destroying virus-infected cells and tumor cells but also play an important role in graft rejection(MHC-I).

• In response to virus-infected cells, the CD8 lymphocytes must recognize both viral antigens and class I molecules on the surface of infected cells.

• To kill the virus-infected cell, the cytotoxic T cell must be activated by IL-2 produced by a helper (CD4-positive) T cell, whereas a NK cell doesn’t.

• To become activated to produce IL-2, helper T cells recognize viral antigens bound to class II molecules on an APC (e.g., a dendritic cell or macrophage).

• The activated helper T cell, once it has recognized the MHC-II with viral epitope, will secrete IL-2 that will stimulate the CD8 cell that is specific to that virus- which then will form a clone of that now activated cytotoxic T cells.

• Activated cytotoxic T cells kill virus-infected cells primarily by inserting perforins and degradative enzymes called granzymes into the infected cell.

• 1- Perforins form a channel through the membrane, the cell contents are lost, and the cell dies.

• 2- Granzymes are proteases (enzymes that degrade proteins) and work against the proteins in the cell membrane, which also leads to the loss of cell contents.

• 3- Granzymes also activate caspases (a type of protease) that initiate apoptosis, resulting in cell death.

• After killing the virus-infected cell, the cytotoxic T cell itself is not damaged and can continue to kill other cells infected with the same virus.

• Cytotoxic T cells have no effect on free virus, only on virus-infected cells.

• The main Apoptotic mechanisn, by which cytotoxic T cells kill target cells is activation of apoptosis through the the Fas-Fas ligand (FasL) interaction.

• This apoptosis mechanism happens when Fas (which is a protein displayed on the surface of many cells) and cytotoxic TCR recognizes an epitope on the surface of a target cell (MHC-I), FasL (Fas Ligand) is induced in the cytotoxic T cell.

• Once FasL and Fas interact, apoptosis (death) of the target cell occurs.

• NK cells can also kill target cells by Fas-FasL–induced apoptosis.

• To eliminate virus infected cell, an antibody mediated mechanism is employed as well (In addition to direct killing by cytotoxic T cells) this is done by a combination of IgG and phagocytic cells.

• In this antibody-dependent cellular cytotoxicity (ADCC), the antibody directed at the virus, is bound to the surface of the infected cell. The bound Antibody is then recognized by phagocytic cells (macrophages or NK cells) by an IgG receptor on their surface → the infected cell is killed.

• The ADCC process is the mechanism of killing helminths (worms).

• However, in this case, ADCC is mediated by IgE, and eosinophils (not phagocytes) are the effector (does the function, in this case killing) cells.

• The mechanism is quite similar, IgE binds to surface proteins on the worm, and eosinophils have a receptor on their surface for the epsilon heavy chain (IgE).

• The actual killing is mediated by granules inside the eosinophils that are released after they are activated by IgE, the major basic protein in these granules damages the surface of the worm. worm.

• In tumors, new antigens (not self) will be displayed on their MHC-I surface protein → CD8 cells will recognize this (and will stimulated to proliferate by IL-2) and target these tumor cells for destruction (either direct or by inducing apoptosis)

• IL-2 produced means that this clone of CD8 cell will proliferate, which will mean that this CD8 clonal expansion is now able to kill this type of tumor cells (this phenomenon is called immune surveillance).

• In allografts, cytotoxic (CD8) cells recognize the class I MHC molecules on the surface of the foreign cells (and depending on how similar it is to the self, they will either reject or accept).

• But what activates these CD8 cells in the allograft rejection? They need IL-2, produced by helper (CD4), they will recognize the foreign class II molecules on certain cells in the graft (APCs from the allograft, macrophages and lymphocytes).

• The activated helper cells secrete IL-2, which stimulates the cytotoxic cell to form a clone of cells that will function to kill the allograft cells (this means now the body has memory against this allograft and there is very little that we can do to help, this also means that if we aim to reduce allograft rejection we must work on having the CD4 cells not activating clones of CD8 cells that target the graft.

• Older slides below/retract

Th-2 Cells

• Th-2 CD4 cells along with eosinophils constitute the main effectors of reactions that are protective against helminths (worms) such as Schistosoma and Strongyloides (remember IgE and eosinophils).

• The most important interleukins for these reactions are IL-4 (why? Because we need Th2 response to produce IL-4),

• IL-4 increases the production of IgE, and IL-5, which activates eosinophils.• IgE binds to the surface of the worm. Eosinophils then bind to the heavy

chain of IgE and secrete their enzymes that destroy the worm. • Th-17 Cells • Th-17 cells protect against the spread of bacterial infections at mucosal

surfaces by producing IL-17. IL-17 attracts neutrophils to the site of infection whereupon the bacteria are ingested and destroyed.

CD8 Cells

• CD8 cells mediate the cytotoxic response that is concerned primarily with destroying virus-infected cells and tumor cells but also play an important role in graft rejection.

• In response to virus-infected cells, the CD8 lymphocytes must recognize both viral antigens and class I molecules on the surface of infected cells.

• To kill the virus-infected cell, the cytotoxic T cell must be activated by IL-2 produced by a helper (CD4-positive) T cell, whereas a NK cell doesnt.

• To become activated to produce IL-2, helper T cells recognize viral antigens bound to class II molecules on an APC (e.g., a dendritic cell or macrophage).

• The activated helper T cell, once it has recognized the MHC-II with viral epitope, will secrete IL-2 that will stimulate the CD8 cell that is specific to that virus- which then will form a clone of that now activated cytotoxic T cells.

• Activated cytotoxic T cells kill virus-infected cells primarily by inserting perforins and degradative enzymes called granzymes into the infected cell.

• Perforins form a channel through the membrane, the cell contents are lost, and the cell dies.

• Granzymes are proteases (enzymes that degrade proteins) and work against the proteins in the cell membrane, which also leads to the loss of cell contents.

• Granzymes also activate caspases (a type of protease) that initiate apoptosis, resulting in cell death.

• After killing the virus-infected cell, the cytotoxic T cell itself is not damaged and can continue to kill other cells infected with the same virus.

• Cytotoxic T cells have no effect on free virus, only on virus-infected cells.

• Another mechanism by which cytotoxic T cells kill target cells is the Fas-Fas ligand (FasL) interaction which is an activator for apoptosis.

• This apoptosis mechanism happens when Fas (which is a protein displayed on the surface of many cells) and cytotoxic TCR recognizes an epitope on the surface of a target cell (MHC-I), FasL (Fas Ligand) is induced in the cytotoxic T cell.

• Once FasL and Fas interact, apoptosis (death) of the target cell occurs.

• NK cells can also kill target cells by Fas-FasL–induced apoptosis.

• In addition to direct killing by cytotoxic T cells, virus-infected cells can be destroyed by a combination of IgG and phagocytic cells.

• In this process, called antibody-dependent cellular cytotoxicity (ADCC), antibody bound to the surface of the infected cell is recognized by IgG receptors on the surface of phagocytic cells (e.g., macrophages or NK cells), and the infected cell is killed.

• The ADCC process can also kill helminths (worms).

• IgE mediated ADCC has IgE as the antibody involved, and eosinophils are the effector cells. IgE binds to surface proteins on the worm, and the surface of eosinophils displays receptors for the epsilon heavy chain.

• This leads to the release of the major basic protein located in the granules of the eosinophils which damages the surface of the worm.

• Many tumor cells develop new antigens on their surface.

• These antigens bound to class I proteins are recognized by cytotoxic T cells, which are stimulated to proliferate by IL-2.

• The resultant clone of cytotoxic T cells can kill the tumor cells, a phenomenon called immune surveillance.

• In response to allografts, cytotoxic (CD8) cells recognize the class I MHC molecules on the surface of the foreign cells.

• Helper (CD4) cells recognize the foreign class II molecules on certain cells in the graft (e.g., macrophages and lymphocytes).

• The activated helper cells secrete IL-2, which stimulates the cytotoxic cell to form a clone of cells. These cytotoxic cells kill the cells in the allograft.