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7.4: The Adaptive Immune Response- T lymphocytes and Their Functional Types - Biology


Learning Objectives

By the end of this section, you will be able to:

  • Explain the advantages of the adaptive immune response over the innate immune response
  • List the various characteristics of an antigen
  • Describe the types of T cell antigen receptors
  • Outline the steps of T cell development
  • Describe the major T cell types and their functions

Innate immune responses (and early induced responses) are in many cases ineffective at completely controlling pathogen growth. However, they slow pathogen growth and allow time for the adaptive immune response to strengthen and either control or eliminate the pathogen. The innate immune system also sends signals to the cells of the adaptive immune system, guiding them in how to attack the pathogen. Thus, these are the two important arms of the immune response.

The Benefits of the Adaptive Immune Response

The specificity of the adaptive immune response—its ability to specifically recognize and make a response against a wide variety of pathogens—is its great strength. Antigens, the small chemical groups often associated with pathogens, are recognized by receptors on the surface of B and T lymphocytes. The adaptive immune response to these antigens is so versatile that it can respond to nearly any pathogen. This increase in specificity comes because the adaptive immune response has a unique way to develop as many as 1011, or 100 trillion, different receptors to recognize nearly every conceivable pathogen. How could so many different types of antibodies be encoded? And what about the many specificities of T cells? There is not nearly enough DNA in a cell to have a separate gene for each specificity. The mechanism was finally worked out in the 1970s and 1980s using the new tools of molecular genetics

Primary Disease and Immunological Memory

The immune system’s first exposure to a pathogen is called a primary adaptive response. Symptoms of a first infection, called primary disease, are always relatively severe because it takes time for an initial adaptive immune response to a pathogen to become effective.

Upon re-exposure to the same pathogen, a secondary adaptive immune response is generated, which is stronger and faster that the primary response. The secondary adaptive response often eliminates a pathogen before it can cause significant tissue damage or any symptoms. Without symptoms, there is no disease, and the individual is not even aware of the infection. This secondary response is the basis of immunological memory, which protects us from getting diseases repeatedly from the same pathogen. By this mechanism, an individual’s exposure to pathogens early in life spares the person from these diseases later in life.

Self Recognition

A third important feature of the adaptive immune response is its ability to distinguish between self-antigens, those that are normally present in the body, and foreign antigens, those that might be on a potential pathogen. As T and B cells mature, there are mechanisms in place that prevent them from recognizing self-antigen, preventing a damaging immune response against the body. These mechanisms are not 100 percent effective, however, and their breakdown leads to autoimmune diseases, which will be discussed later in this chapter.

T Cell-Mediated Immune Responses

The primary cells that control the adaptive immune response are the lymphocytes, the T and B cells. T cells are particularly important, as they not only control a multitude of immune responses directly, but also control B cell immune responses in many cases as well. Thus, many of the decisions about how to attack a pathogen are made at the T cell level, and knowledge of their functional types is crucial to understanding the functioning and regulation of adaptive immune responses as a whole.

T lymphocytes recognize antigens based on a two-chain protein receptor. The most common and important of these are the alpha-beta T cell receptors (Figure 1).

There are two chains in the T cell receptor, and each chain consists of two domains. The variable region domain is furthest away from the T cell membrane and is so named because its amino acid sequence varies between receptors. In contrast, the constant region domain has less variation. The differences in the amino acid sequences of the variable domains are the molecular basis of the diversity of antigens the receptor can recognize. Thus, the antigen-binding site of the receptor consists of the terminal ends of both receptor chains, and the amino acid sequences of those two areas combine to determine its antigenic specificity. Each T cell produces only one type of receptor and thus is specific for a single particular antigen.

Antigens

Antigens on pathogens are usually large and complex, and consist of many antigenic determinants. An antigenic determinant (epitope) is one of the small regions within an antigen to which a receptor can bind, and antigenic determinants are limited by the size of the receptor itself. They usually consist of six or fewer amino acid residues in a protein, or one or two sugar moieties in a carbohydrate antigen. Antigenic determinants on a carbohydrate antigen are usually less diverse than on a protein antigen. Carbohydrate antigens are found on bacterial cell walls and on red blood cells (the ABO blood group antigens). Protein antigens are complex because of the variety of three-dimensional shapes that proteins can assume, and are especially important for the immune responses to viruses and worm parasites. It is the interaction of the shape of the antigen and the complementary shape of the amino acids of the antigen-binding site that accounts for the chemical basis of specificity.

Antigen Processing and Presentation

Although Figure 2 shows T cell receptors interacting with antigenic determinants directly, the mechanism that T cells use to recognize antigens is, in reality, much more complex. T cells do not recognize free-floating or cell-bound antigens as they appear on the surface of the pathogen. They only recognize antigen on the surface of specialized cells called antigen-presenting cells. Antigens are internalized by these cells.

Antigen processing is a mechanism that enzymatically cleaves the antigen into smaller pieces. The antigen fragments are then brought to the cell’s surface and associated with a specialized type of antigen-presenting protein known as a major histocompatibility complex (MHC) molecule. The MHC is the cluster of genes that encode these antigen-presenting molecules. The association of the antigen fragments with an MHC molecule on the surface of a cell is known as antigen presentation and results in the recognition of antigen by a T cell. This association of antigen and MHC occurs inside the cell, and it is the complex of the two that is brought to the surface. The peptide-binding cleft is a small indentation at the end of the MHC molecule that is furthest away from the cell membrane; it is here that the processed fragment of antigen sits. MHC molecules are capable of presenting a variety of antigens, depending on the amino acid sequence, in their peptide-binding clefts. It is the combination of the MHC molecule and the fragment of the original peptide or carbohydrate that is actually physically recognized by the T cell receptor.

Two distinct types of MHC molecules, MHC class I and MHC class II, play roles in antigen presentation. Although produced from different genes, they both have similar functions. They bring processed antigen to the surface of the cell via a transport vesicle and present the antigen to the T cell and its receptor. Antigens from different classes of pathogens, however, use different MHC classes and take different routes through the cell to get to the surface for presentation. The basic mechanism, though, is the same. Antigens are processed by digestion, are brought into the endomembrane system of the cell, and then are expressed on the surface of the antigen-presenting cell for antigen recognition by a T cell. Intracellular antigens are typical of viruses, which replicate inside the cell, and certain other intracellular parasites and bacteria. These antigens are processed in the cytosol by an enzyme complex known as the proteasome and are then brought into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) system, where they interact with class I MHC molecules and are eventually transported to the cell surface by a transport vesicle.

Extracellular antigens, characteristic of many bacteria, parasites, and fungi that do not replicate inside the cell’s cytoplasm, are brought into the endomembrane system of the cell by receptor-mediated endocytosis. The resulting vesicle fuses with vesicles from the Golgi complex, which contain pre-formed MHC class II molecules. After fusion of these two vesicles and the association of antigen and MHC, the new vesicle makes its way to the cell surface.

Professional Antigen-presenting Cells

Many cell types express class I molecules for the presentation of intracellular antigens. These MHC molecules may then stimulate a cytotoxic T cell immune response, eventually destroying the cell and the pathogen within. This is especially important when it comes to the most common class of intracellular pathogens, the virus. Viruses infect nearly every tissue of the body, so all these tissues must necessarily be able to express class I MHC or no T cell response can be made.

On the other hand, class II MHC molecules are expressed only on the cells of the immune system, specifically cells that affect other arms of the immune response. Thus, these cells are called “professional” antigen-presenting cells to distinguish them from those that bear class I MHC. The three types of professional antigen presenters are macrophages, dendritic cells, and B cells.

Macrophages stimulate T cells to release cytokines that enhance phagocytosis. Dendritic cells also kill pathogens by phagocytosis, but their major function is to bring antigens to regional draining lymph nodes. The lymph nodes are the locations in which most T cell responses against pathogens of the interstitial tissues are mounted. Macrophages are found in the skin and in the lining of mucosal surfaces, such as the nasopharynx, stomach, lungs, and intestines. B cells may also present antigens to T cells, which are necessary for certain types of antibody responses, to be covered later in this chapter.

Table 1. Classes of Antigen-presenting Cells
MHCCell typePhagocytic?Function
Class IManyNoStimulates cytotoxic T cell immune response
Class IIMacrophageYesStimulates phagocytosis and presentation at primary infection site
Class IIDendriticYes, in tissuesBrings antigens to regional lymph nodes
Class IIB cellYes, internalizes surface Ig and antigenStimulates antibody secretion by B cells

T Cell Development and Differentiation

The process of eliminating T cells that might attack the cells of one’s own body is referred to as T cell tolerance. While thymocytes are in the cortex of the thymus, they are referred to as “double negatives,” meaning that they do not bear the CD4 or CD8 molecules that you can use to follow their pathways of differentiation. In the cortex of the thymus, they are exposed to cortical epithelial cells. In a process known as positive selection, double-negative thymocytes bind to the MHC molecules they observe on the thymic epithelia, and the MHC molecules of “self” are selected. This mechanism kills many thymocytes during T cell differentiation. In fact, only two percent of the thymocytes that enter the thymus leave it as mature, functional T cells.

Figure 4. Click to view a larger image. Thymocytes enter the thymus and go through a series of developmental stages that ensures both function and tolerance before they leave and become functional components of the adaptive immune response.

Later, the cells become double positives that express both CD4 and CD8 markers and move from the cortex to the junction between the cortex and medulla. It is here that negative selection takes place. In negative selection, self-antigens are brought into the thymus from other parts of the body by professional antigen-presenting cells. The T cells that bind to these self-antigens are selected for negatively and are killed by apoptosis. In summary, the only T cells left are those that can bind to MHC molecules of the body with foreign antigens presented on their binding clefts, preventing an attack on one’s own body tissues, at least under normal circumstances. Tolerance can be broken, however, by the development of an autoimmune response, to be discussed later in this chapter.

The cells that leave the thymus become single positives, expressing either CD4 or CD8, but not both. The CD4+ T cells will bind to class II MHC and the CD8+ cells will bind to class I MHC. The discussion that follows explains the functions of these molecules and how they can be used to differentiate between the different T cell functional types.

Mechanisms of T Cell-mediated Immune Responses

Mature T cells become activated by recognizing processed foreign antigen in association with a self-MHC molecule and begin dividing rapidly by mitosis. This proliferation of T cells is called clonal expansion and is necessary to make the immune response strong enough to effectively control a pathogen. How does the body select only those T cells that are needed against a specific pathogen? Again, the specificity of a T cell is based on the amino acid sequence and the three-dimensional shape of the antigen-binding site formed by the variable regions of the two chains of the T cell receptor. Clonal selection is the process of antigen binding only to those T cells that have receptors specific to that antigen. Each T cell that is activated has a specific receptor “hard-wired” into its DNA, and all of its progeny will have identical DNA and T cell receptors, forming clones of the original T cell.

Clonal Selection and Expansion

The clonal selection theory was proposed by Frank Burnet in the 1950s. However, the term clonal selection is not a complete description of the theory, as clonal expansion goes hand in glove with the selection process. The main tenet of the theory is that a typical individual has a multitude (1011) of different types of T cell clones based on their receptors. In this use, a clone is a group of lymphocytes that share the same antigen receptor. Each clone is necessarily present in the body in low numbers. Otherwise, the body would not have room for lymphocytes with so many specificities.

Only those clones of lymphocytes whose receptors are activated by the antigen are stimulated to proliferate. Keep in mind that most antigens have multiple antigenic determinants, so a T cell response to a typical antigen involves a polyclonal response. A polyclonal response is the stimulation of multiple T cell clones. Once activated, the selected clones increase in number and make many copies of each cell type, each clone with its unique receptor. By the time this process is complete, the body will have large numbers of specific lymphocytes available to fight the infection.

The Cellular Basis of Immunological Memory

As already discussed, one of the major features of an adaptive immune response is the development of immunological memory.

During a primary adaptive immune response, both memory T cells and effector T cells are generated. Memory T cells are long-lived and can even persist for a lifetime. Memory cells are primed to act rapidly. Thus, any subsequent exposure to the pathogen will elicit a very rapid T cell response. This rapid, secondary adaptive response generates large numbers of effector T cells so fast that the pathogen is often overwhelmed before it can cause any symptoms of disease. This is what is meant by immunity to a disease. The same pattern of primary and secondary immune responses occurs in B cells and the antibody response, as will be discussed later in the chapter.

T Cell Types and their Functions

In the discussion of T cell development, you saw that mature T cells express either the CD4 marker or the CD8 marker, but not both. These markers are cell adhesion molecules that keep the T cell in close contact with the antigen-presenting cell by directly binding to the MHC molecule (to a different part of the molecule than does the antigen). Thus, T cells and antigen-presenting cells are held together in two ways: by CD4 or CD8 attaching to MHC and by the T cell receptor binding to antigen.

Although the correlation is not 100 percent, CD4-bearing T cells are associated with helper functions and CD8-bearing T cells are associated with cytotoxicity. These functional distinctions based on CD4 and CD8 markers are useful in defining the function of each type.

Helper T Cells and their Cytokines

Helper T cells (Th), bearing the CD4 molecule, function by secreting cytokines that act to enhance other immune responses. There are two classes of Th cells, and they act on different components of the immune response. These cells are not distinguished by their surface molecules but by the characteristic set of cytokines they .

  • Th1 cells are a type of helper T cell that secretes cytokines that regulate the immunological activity and development of a variety of cells, including macrophages and other types of T cells.
  • Th2 cells, on the other hand, are cytokine-secreting cells that act on B cells to drive their differentiation into plasma cells that make antibody. In fact, T cell help is required for antibody responses to most protein antigens, and these are called T cell-dependent antigens.

Cytotoxic T cells

Cytotoxic T cells (Tc) are T cells that kill target cells by inducing apoptosis using the same mechanism as NK cells. They either express Fas ligand, which binds to the fas molecule on the target cell, or act by using perforins and granzymes contained in their cytoplasmic granules. As was discussed earlier with NK cells, killing a virally infected cell before the virus can complete its replication cycle results in the production of no infectious particles. As more Tc cells are developed during an immune response, they overwhelm the ability of the virus to cause disease. In addition, each Tc cell can kill more than one target cell, making them especially effective. Tc cells are so important in the antiviral immune response that some speculate that this was the main reason the adaptive immune response evolved in the first place.

Regulatory T Cells

Regulatory T cells (Treg), or suppressor T cells, are the most recently discovered of the types listed here, so less is understood about them. In addition to CD4, they bear the molecules CD25 and FOXP3. Exactly how they function is still under investigation, but it is known that they suppress other T cell immune responses. This is an important feature of the immune response, because if clonal expansion during immune responses were allowed to continue uncontrolled, these responses could lead to autoimmune diseases and other medical issues.

Not only do T cells directly destroy pathogens, but they regulate nearly all other types of the adaptive immune response as well, as evidenced by the functions of the T cell types, their surface markers, the cells they work on, and the types of pathogens they work against.

Table 2. Functions of T Cell Types and Their Cytokines
T cellMain targetFunctionPathogenSurface markerMHCCytokines or mediators
TcInfected cellsCytotoxicityIntracellularCD8Class IPerforins, granzymes, and fas ligand
Th1MacrophageHelper inducerExtracellularCD4Class IIInterferon-γ and TGF-β
Th2B cellHelper inducerExtracellularCD4Class IIIL-4, IL-6, IL-10, and others
TregTh cellSuppressorNoneCD4, CD25?TGF-β and IL-10

Chapter Review

T cells recognize antigens with their antigen receptor, a complex of two protein chains on their surface. They do not recognize self-antigens, however, but only processed antigen presented on their surfaces in a binding groove of a major histocompatibility complex molecule. T cells develop in the thymus, where they learn to use self-MHC molecules to recognize only foreign antigens, thus making them tolerant to self-antigens. There are several functional types of T lymphocytes, the major ones being helper, regulatory, and cytotoxic T cells.

Self Check

Answer the question(s) below to see how well you understand the topics covered in the previous section.

Critical Thinking Questions

  1. Describe the processing and presentation of an intracellular antigen.
  2. Describe clonal selection and expansion.

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  1. The antigen is digested by the proteasome, brought into the endoplasmic reticulum by the TAP transporter system, where it binds to class I MHC molecules. These are taken to the cell surface by transport vesicles.
  2. Antigen-specific clones are stimulated as their antigen receptor binds to antigen. They are then activated and proliferate, expanding their numbers. The result is a large number of antigen-specific lymphocytes.

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Glossary

antigenic determinant: (also, epitope) one of the chemical groups recognized by a single type of lymphocyte antigen receptor

antigen presentation: binding of processed antigen to the protein-binding cleft of a major histocompatibility complex molecule

antigen processing: internalization and digestion of antigen in an antigen-presenting cell

antigen receptor: two-chain receptor by which lymphocytes recognize antigen

clone: group of lymphocytes sharing the same antigen receptor

clonal expansion: growth of a clone of selected lymphocytes

clonal selection: stimulating growth of lymphocytes that have specific receptors

constant region domain: part of a lymphocyte antigen receptor that does not vary much between different receptor types

cytotoxic T cells (Tc): T lymphocytes with the ability to induce apoptosis in target cells

effector T cells: immune cells with a direct, adverse effect on a pathogen

helper T cells (Th): T cells that secrete cytokines to enhance other immune responses, involved in activation of both B and T cell lymphocytes

immunological memory: ability of the adaptive immune response to mount a stronger and faster immune response upon re-exposure to a pathogen

major histocompatibility complex (MHC): gene cluster whose proteins present antigens to T cells

memory T cells: long-lived immune cell reserved for future exposure to an pathogen

MHC class I: found on most cells of the body, it binds to the CD8 molecule on T cells

MHC class II: found on macrophages, dendritic cells, and B cells, it binds to CD4 molecules on T cells

negative selection: selection against thymocytes in the thymus that react with self-antigen

polyclonal response: response by multiple clones to a complex antigen with many determinants

primary adaptive response: immune system’s response to the first exposure to a pathogen

positive selection: selection of thymocytes within the thymus that interact with self, but not non-self, MHC molecules

regulatory T cells (Treg): (also, suppressor T cells) class of CD4 T cells that regulates other T cell responses

secondary adaptive response: immune response observed upon re-exposure to a pathogen, which is stronger and faster than a primary response

T cell tolerance: process during T cell differentiation where most T cells that recognize antigens from one’s own body are destroyed

Th1 cells: cells that secrete cytokines that enhance the activity of macrophages and other cells

Th2 cells: cells that secrete cytokines that induce B cells to differentiate into antibody-secreting plasma cells

variable region domain: part of a lymphocyte antigen receptor that varies considerably between different receptor types


Revealing the role of CD4(+) T cells in viral immunity

Protective immunity to chronic and acute viral infection relies on both the innate and adaptive immune response. Although neutralizing antibody production by B cells and cytotoxic activity of CD8(+) T cells are well-accepted components of the adaptive immune response to viruses, identification of the specific role of CD4(+) T cells in protection has been more challenging to establish. Delineating the contribution of CD4(+) T cells has been complicated by their functional heterogeneity, breadth in antigen specificity, transient appearance in circulation, and sequestration in tissue sites of infection. In this minireview, we discuss recent progress in identifying the multiple roles of CD4(+) T cells in orchestrating and mediating the immune responses against viral pathogens. We highlight several recent reports, including one published in this issue, that have employed comprehensive and sophisticated approaches to provide new evidence for CD4(+) T cells as direct effectors in antiviral immunity.


21.3 The Adaptive Immune Response: T lymphocytes and Their Functional Types

Innate immune responses (and early induced responses) are in many cases ineffective at completely controlling pathogen growth. However, they slow pathogen growth and allow time for the adaptive immune response to strengthen and either control or eliminate the pathogen. The innate immune system also sends signals to the cells of the adaptive immune system, guiding them in how to attack the pathogen. Thus, these are the two important arms of the immune response.

The Benefits of the Adaptive Immune Response

The specificity of the adaptive immune response—its ability to specifically recognize and make a response against a wide variety of pathogens—is its great strength. Antigens, the small chemical groups often associated with pathogens, are recognized by receptors on the surface of B and T lymphocytes. The adaptive immune response to these antigens is so versatile that it can respond to nearly any pathogen. This increase in specificity comes because the adaptive immune response has a unique way to develop as many as 10 11 , or 100 trillion, different receptors to recognize nearly every conceivable pathogen. How could so many different types of antibodies be encoded? And what about the many specificities of T cells? There is not nearly enough DNA in a cell to have a separate gene for each specificity. The mechanism was finally worked out in the 1970s and 1980s using the new tools of molecular genetics

Primary Disease and Immunological Memory

The immune system’s first exposure to a pathogen is called a primary adaptive response . Symptoms of a first infection, called primary disease, are always relatively severe because it takes time for an initial adaptive immune response to a pathogen to become effective.

Upon re-exposure to the same pathogen, a secondary adaptive immune response is generated, which is stronger and faster that the primary response. The secondary adaptive response often eliminates a pathogen before it can cause significant tissue damage or any symptoms. Without symptoms, there is no disease, and the individual is not even aware of the infection. This secondary response is the basis of immunological memory , which protects us from getting diseases repeatedly from the same pathogen. By this mechanism, an individual’s exposure to pathogens early in life spares the person from these diseases later in life.

Self Recognition

A third important feature of the adaptive immune response is its ability to distinguish between self-antigens, those that are normally present in the body, and foreign antigens, those that might be on a potential pathogen. As T and B cells mature, there are mechanisms in place that prevent them from recognizing self-antigen, preventing a damaging immune response against the body. These mechanisms are not 100 percent effective, however, and their breakdown leads to autoimmune diseases, which will be discussed later in this chapter.

T Cell-Mediated Immune Responses

The primary cells that control the adaptive immune response are the lymphocytes, the T and B cells. T cells are particularly important, as they not only control a multitude of immune responses directly, but also control B cell immune responses in many cases as well. Thus, many of the decisions about how to attack a pathogen are made at the T cell level, and knowledge of their functional types is crucial to understanding the functioning and regulation of adaptive immune responses as a whole.

T lymphocytes recognize antigens based on a two-chain protein receptor. The most common and important of these are the alpha-beta T cell receptors (Figure 21.15).

There are two chains in the T cell receptor, and each chain consists of two domains. The variable region domain is furthest away from the T cell membrane and is so named because its amino acid sequence varies between receptors. In contrast, the constant region domain has less variation. The differences in the amino acid sequences of the variable domains are the molecular basis of the diversity of antigens the receptor can recognize. Thus, the antigen-binding site of the receptor consists of the terminal ends of both receptor chains, and the amino acid sequences of those two areas combine to determine its antigenic specificity. Each T cell produces only one type of receptor and thus is specific for a single particular antigen.

Antigens

Antigens on pathogens are usually large and complex, and consist of many antigenic determinants. An antigenic determinant (epitope) is one of the small regions within an antigen to which a receptor can bind, and antigenic determinants are limited by the size of the receptor itself. They usually consist of six or fewer amino acid residues in a protein, or one or two sugar moieties in a carbohydrate antigen. Antigenic determinants on a carbohydrate antigen are usually less diverse than on a protein antigen. Carbohydrate antigens are found on bacterial cell walls and on red blood cells (the ABO blood group antigens). Protein antigens are complex because of the variety of three-dimensional shapes that proteins can assume, and are especially important for the immune responses to viruses and worm parasites. It is the interaction of the shape of the antigen and the complementary shape of the amino acids of the antigen-binding site that accounts for the chemical basis of specificity (Figure 21.16).

Antigen Processing and Presentation

Although Figure 21.16 shows T cell receptors interacting with antigenic determinants directly, the mechanism that T cells use to recognize antigens is, in reality, much more complex. T cells do not recognize free-floating or cell-bound antigens as they appear on the surface of the pathogen. They only recognize antigen on the surface of specialized cells called antigen-presenting cells. Antigens are internalized by these cells. Antigen processing is a mechanism that enzymatically cleaves the antigen into smaller pieces. The antigen fragments are then brought to the cell’s surface and associated with a specialized type of antigen-presenting protein known as a major histocompatibility complex (MHC) molecule. The MHC is the cluster of genes that encode these antigen-presenting molecules. The association of the antigen fragments with an MHC molecule on the surface of a cell is known as antigen presentation and results in the recognition of antigen by a T cell. This association of antigen and MHC occurs inside the cell, and it is the complex of the two that is brought to the surface. The peptide-binding cleft is a small indentation at the end of the MHC molecule that is furthest away from the cell membrane it is here that the processed fragment of antigen sits. MHC molecules are capable of presenting a variety of antigens, depending on the amino acid sequence, in their peptide-binding clefts. It is the combination of the MHC molecule and the fragment of the original peptide or carbohydrate that is actually physically recognized by the T cell receptor (Figure 21.17).

Two distinct types of MHC molecules, MHC class I and MHC class II , play roles in antigen presentation. Although produced from different genes, they both have similar functions. They bring processed antigen to the surface of the cell via a transport vesicle and present the antigen to the T cell and its receptor. Antigens from different classes of pathogens, however, use different MHC classes and take different routes through the cell to get to the surface for presentation. The basic mechanism, though, is the same. Antigens are processed by digestion, are brought into the endomembrane system of the cell, and then are expressed on the surface of the antigen-presenting cell for antigen recognition by a T cell. Intracellular antigens are typical of viruses, which replicate inside the cell, and certain other intracellular parasites and bacteria. These antigens are processed in the cytosol by an enzyme complex known as the proteasome and are then brought into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) system, where they interact with class I MHC molecules and are eventually transported to the cell surface by a transport vesicle.

Extracellular antigens, characteristic of many bacteria, parasites, and fungi that do not replicate inside the cell’s cytoplasm, are brought into the endomembrane system of the cell by receptor-mediated endocytosis. The resulting vesicle fuses with vesicles from the Golgi complex, which contain pre-formed MHC class II molecules. After fusion of these two vesicles and the association of antigen and MHC, the new vesicle makes its way to the cell surface.

Professional Antigen-presenting Cells

Many cell types express class I molecules for the presentation of intracellular antigens. These MHC molecules may then stimulate a cytotoxic T cell immune response, eventually destroying the cell and the pathogen within. This is especially important when it comes to the most common class of intracellular pathogens, the virus. Viruses infect nearly every tissue of the body, so all these tissues must necessarily be able to express class I MHC or no T cell response can be made.

On the other hand, class II MHC molecules are expressed only on the cells of the immune system, specifically cells that affect other arms of the immune response. Thus, these cells are called “professional” antigen-presenting cells to distinguish them from those that bear class I MHC. The three types of professional antigen presenters are macrophages, dendritic cells, and B cells (Table 21.4).

Macrophages stimulate T cells to release cytokines that enhance phagocytosis. Dendritic cells also kill pathogens by phagocytosis (see Figure 21.17), but their major function is to bring antigens to regional draining lymph nodes. The lymph nodes are the locations in which most T cell responses against pathogens of the interstitial tissues are mounted. Macrophages are found in the skin and in the lining of mucosal surfaces, such as the nasopharynx, stomach, lungs, and intestines. B cells may also present antigens to T cells, which are necessary for certain types of antibody responses, to be covered later in this chapter.

MHC Cell type Phagocytic? Function
Class I Many No Stimulates cytotoxic T cell immune response
Class II Macrophage Yes Stimulates phagocytosis and presentation at primary infection site
Class II Dendritic Yes, in tissues Brings antigens to regional lymph nodes
Class II B cell Yes, internalizes surface Ig and antigen Stimulates antibody secretion by B cells

T Cell Development and Differentiation

The process of eliminating T cells that might attack the cells of one’s own body is referred to as T cell tolerance . While thymocytes are in the cortex of the thymus, they are referred to as “double negatives,” meaning that they do not bear the CD4 or CD8 molecules that you can use to follow their pathways of differentiation (Figure 21.18). In the cortex of the thymus, they are exposed to cortical epithelial cells. In a process known as positive selection , double-negative thymocytes bind to the MHC molecules they observe on the thymic epithelia, and the MHC molecules of “self” are selected. This mechanism kills many thymocytes during T cell differentiation. In fact, only two percent of the thymocytes that enter the thymus leave it as mature, functional T cells.

Later, the cells become double positives that express both CD4 and CD8 markers and move from the cortex to the junction between the cortex and medulla. It is here that negative selection takes place. In negative selection , self-antigens are brought into the thymus from other parts of the body by professional antigen-presenting cells. The T cells that bind to these self-antigens are selected for negatively and are killed by apoptosis. In summary, the only T cells left are those that can bind to MHC molecules of the body with foreign antigens presented on their binding clefts, preventing an attack on one’s own body tissues, at least under normal circumstances. Tolerance can be broken, however, by the development of an autoimmune response, to be discussed later in this chapter.

The cells that leave the thymus become single positives, expressing either CD4 or CD8, but not both (see Figure 21.18). The CD4 + T cells will bind to class II MHC and the CD8 + cells will bind to class I MHC. The discussion that follows explains the functions of these molecules and how they can be used to differentiate between the different T cell functional types.

Mechanisms of T Cell-mediated Immune Responses

Mature T cells become activated by recognizing processed foreign antigen in association with a self-MHC molecule and begin dividing rapidly by mitosis. This proliferation of T cells is called clonal expansion and is necessary to make the immune response strong enough to effectively control a pathogen. How does the body select only those T cells that are needed against a specific pathogen? Again, the specificity of a T cell is based on the amino acid sequence and the three-dimensional shape of the antigen-binding site formed by the variable regions of the two chains of the T cell receptor (Figure 21.19). Clonal selection is the process of antigen binding only to those T cells that have receptors specific to that antigen. Each T cell that is activated has a specific receptor “hard-wired” into its DNA, and all of its progeny will have identical DNA and T cell receptors, forming clones of the original T cell.

Clonal Selection and Expansion

The clonal selection theory was proposed by Frank Burnet in the 1950s. However, the term clonal selection is not a complete description of the theory, as clonal expansion goes hand in glove with the selection process. The main tenet of the theory is that a typical individual has a multitude (10 11 ) of different types of T cell clones based on their receptors. In this use, a clone is a group of lymphocytes that share the same antigen receptor . Each clone is necessarily present in the body in low numbers. Otherwise, the body would not have room for lymphocytes with so many specificities.

Only those clones of lymphocytes whose receptors are activated by the antigen are stimulated to proliferate. Keep in mind that most antigens have multiple antigenic determinants, so a T cell response to a typical antigen involves a polyclonal response. A polyclonal response is the stimulation of multiple T cell clones. Once activated, the selected clones increase in number and make many copies of each cell type, each clone with its unique receptor. By the time this process is complete, the body will have large numbers of specific lymphocytes available to fight the infection (see Figure 21.19).

The Cellular Basis of Immunological Memory

As already discussed, one of the major features of an adaptive immune response is the development of immunological memory.

During a primary adaptive immune response, both memory T cells and effector T cells are generated. Memory T cells are long-lived and can even persist for a lifetime. Memory cells are primed to act rapidly. Thus, any subsequent exposure to the pathogen will elicit a very rapid T cell response. This rapid, secondary adaptive response generates large numbers of effector T cells so fast that the pathogen is often overwhelmed before it can cause any symptoms of disease. This is what is meant by immunity to a disease. The same pattern of primary and secondary immune responses occurs in B cells and the antibody response, as will be discussed later in the chapter.

T Cell Types and their Functions

In the discussion of T cell development, you saw that mature T cells express either the CD4 marker or the CD8 marker, but not both. These markers are cell adhesion molecules that keep the T cell in close contact with the antigen-presenting cell by directly binding to the MHC molecule (to a different part of the molecule than does the antigen). Thus, T cells and antigen-presenting cells are held together in two ways: by CD4 or CD8 attaching to MHC and by the T cell receptor binding to antigen (Figure 21.20).

Although the correlation is not 100 percent, CD4-bearing T cells are associated with helper functions and CD8-bearing T cells are associated with cytotoxicity. These functional distinctions based on CD4 and CD8 markers are useful in defining the function of each type.

Helper T Cells and their Cytokines

Helper T cells (Th) , bearing the CD4 molecule, function by secreting cytokines that act to enhance other immune responses. There are two classes of Th cells, and they act on different components of the immune response. These cells are not distinguished by their surface molecules but by the characteristic set of cytokines they secrete (Table 21.5).

Th1 cells are a type of helper T cell that secretes cytokines that regulate the immunological activity and development of a variety of cells, including macrophages and other types of T cells.

Th2 cells , on the other hand, are cytokine-secreting cells that act on B cells to drive their differentiation into plasma cells that make antibody. In fact, T cell help is required for antibody responses to most protein antigens, and these are called T cell-dependent antigens.

Cytotoxic T cells

Cytotoxic T cells (Tc) are T cells that kill target cells by inducing apoptosis using the same mechanism as NK cells. They either express Fas ligand, which binds to the fas molecule on the target cell, or act by using perforins and granzymes contained in their cytoplasmic granules. As was discussed earlier with NK cells, killing a virally infected cell before the virus can complete its replication cycle results in the production of no infectious particles. As more Tc cells are developed during an immune response, they overwhelm the ability of the virus to cause disease. In addition, each Tc cell can kill more than one target cell, making them especially effective. Tc cells are so important in the antiviral immune response that some speculate that this was the main reason the adaptive immune response evolved in the first place.

Regulatory T Cells

Regulatory T cells (Treg) , or suppressor T cells, are the most recently discovered of the types listed here, so less is understood about them. In addition to CD4, they bear the molecules CD25 and FOXP3. Exactly how they function is still under investigation, but it is known that they suppress other T cell immune responses. This is an important feature of the immune response, because if clonal expansion during immune responses were allowed to continue uncontrolled, these responses could lead to autoimmune diseases and other medical issues.

Not only do T cells directly destroy pathogens, but they regulate nearly all other types of the adaptive immune response as well, as evidenced by the functions of the T cell types, their surface markers, the cells they work on, and the types of pathogens they work against (see Table 21.5).


Molecular mechanisms of autophagy

Macroautophagy is an evolutionarily conserved nutrient recycling pathway that delivers cytoplasmic components to the lytic compartment of a cell. This process can be divided into nonselective/bulk autophagy and selective autophagy 1 . In bulk autophagy, the cargo is randomly selected and sequestered in a cup-shaped structure called a phagophore. The expansion and closure of a phagophore results in the engulfment of part of the cytoplasm within a double-membrane structure called an autophagosome, which subsequently fuses with a lysosome where lumenal components are degraded the resulting breakdown products are then released back into the cytosol for reuse. This process occurs at a low basal level in virtually all cells. However, autophagy is upregulated in response to metabolic stress. Autophagy, therefore, helps maintain metabolic homeostasis by generating macromolecules that fuel anabolism and enable cell survival 2,3 .

Macroautophagy is the best-characterized type of autophagy and involves five major steps: initiation, nucleation, expansion, fusion, and degradation-recycling 4 . The initiation of the double-membrane phagophore begins with the phosphorylation of various components of the PIK3C3/VPS34 kinase complex by the ULK kinase complex comprised of ULK1/ULK2, RB1CC1/FIP200, ATG13, and ATG101. This phosphorylation results in the activation of the lipid kinase PIK3C3/VPS34 and the production of local pools of phosphatidylinositol-3-phosphate (PtdIns3P) needed for the nucleation of the phagophore 5 . The ATG9 trafficking system, composed of ATG2, WDR45/WIPI4, and the transmembrane protein ATG9A in conjunction with lipids channeled from the ER, supplies membrane precursors to meet the high demand for the membrane required for autophagosome biogenesis 6,7,8,9 . Following phagophore nucleation, two essential ubiquitin-like conjugation systems are activated for membrane expansion and fusion. The two systems function to covalently conjugate Atg8-family proteins (i.e., the MAP1LC3/LC3 and GABARAP subfamilies) to the expanding phagophore. First, the E1-like enzyme ATG7 and the E2-like enzyme ATG10 conjugate the ubiquitin-like protein ATG12 to ATG5. This complex subsequently binds to ATG16L1 and acts as an E3 enzyme for the conjugation of Atg8-family proteins to the membrane-resident lipid phosphatidylethanolamine as facilitated by the E1 enzyme ATG7 and E2 enzyme ATG3. Lipidation of Atg8-family proteins drives phagophore expansion and facilitates the recognition of specific cargo via interaction with receptors 10,11 . Upon completion of phagophore expansion and closure, the resulting double-membraned autophagosome topologically separates the autophagic cargo from the cytoplasm. Following the dissociation of the autophagic machinery from its surface, the outer membrane of the autophagosome fuses with a lysosome to form an autolysosome. Subsequently, the inner membrane and its enclosed contents are exposed to lysosomal resident hydrolases and degraded to generate simple metabolites, which are released into the cytoplasm via transporters, and subsequently reused.


Single-Cell Resolution of T Cell Immune Responses

Single antigen-specific B or T lymphocytes are the smallest functional units, into which an adaptive immune response can be dissected. Today, novel high-throughput technologies are providing researches with increasingly complex information on the diverse phenotypic signatures of individual lymphocytes. With a focus on T cells, we summarize here, how computational approaches are becoming increasingly important to identify the relevant developmental boundaries and connections between these high-dimensional lymphocyte states. We then describe how these insights may be further expanded by novel experimental approaches that allow to map the fate of individual T cells and their progeny in vivo and in vitro. Finally, we highlight how these experiments have uncovered a probabilistic regulatory structure of T cell immune responses and briefly discuss, how two distinct theoretical frameworks used to describe this structure may be merged to best capture single T cell behavior in computational terms.

Keywords: Continuous imaging Genetic barcoding Limiting dilution Mass cytometry Reporter constructs Single-T-cell adoptive transfer Single-T-cell fate mapping Single-cell RNA sequencing Trajectory inference.


Charting the B-cell surface

Up until approximately 1980, the molecular architecture of the B-cell surface was known to consist of membrane-bound Ig, complement component receptors, and Fc receptors beyond that, the molecular constitution of the cell surface was completely uncharacterized. That all changed with the advent of monoclonal antibody (mAb) technology, a classic example of how the field of B-cell immunology has contributed important experimental tools that influence diverse areas of biology. 50 The authors' involvement in characterizing B-lineage cell surface molecules with mAbs began as a postdoctoral fellow (T.W.L.) with John Kersey, and a graduate student (T.F.T.) with Max Cooper. In parallel, with Lee Nadler and the team of Ed Clark and Jeff Ledbetter, most of these B cell–restricted cell surface molecules were identified. Fittingly, the first B cell–specific molecule described was termed B1 by Nadler and colleagues (Stashenko et al 51 ) and is now known as CD20.

Over the past 25 years, approximately 10 B cell–specific cell surface molecules have been identified by mAbs, with non–B-cell expression identified for some following their original characterization (Table 1). To bring a common nomenclature to the field, these mAbs were given WHO-sanctioned designations as “clusters of differentiation” (CD) by a series of international workshops on human leukocyte differentiation antigens. The first workshop held in Paris in 1982 was organized by Alain Bernard, Laurence Boumsell, Jean Dausset, César Milstein, and Stuart Schlossman. The CD nomenclature facilitated the classification of mAbs generated by different laboratories around the world against leukocyte cell surface epitopes. The mAbs were assigned a CD number once 2 independent mAbs were shown to bind to the same molecule. Since then, CD numbers have been assigned to more than 320 unique clusters and subclusters of mAbs. The CD designation is now universally embraced as a label for the target molecule rather than just a grouping of mAbs with common reactivity.

Cell surface CD molecules that are preferentially expressed by B cells

Name . Original names . Cellular reactivity . Structure .
CD19 B4 Pan-B cell, FDCs? Ig superfamily
CD20 B1 Mature B cells MS4A family
CD21 B2, HB-5 Mature B cells, FDCs Complement receptor family
CD22 BL-CAM, Lyb-8 Mature B cells Ig superfamily
CD23 FcϵRII Activated B cells, FDCs, others C-type lectin
CD24 BA-1, HB6 Pan-B cell, granulocytes, epithelial cells GPI anchored
CD40 Bp50 B cells, epithelial cells, FDCs, others TNF receptor
CD72 Lyb-2 Pan-B cell C-type lectin
CD79a,b Igα,β Surface Ig + B cells Ig superfamily
Name . Original names . Cellular reactivity . Structure .
CD19 B4 Pan-B cell, FDCs? Ig superfamily
CD20 B1 Mature B cells MS4A family
CD21 B2, HB-5 Mature B cells, FDCs Complement receptor family
CD22 BL-CAM, Lyb-8 Mature B cells Ig superfamily
CD23 FcϵRII Activated B cells, FDCs, others C-type lectin
CD24 BA-1, HB6 Pan-B cell, granulocytes, epithelial cells GPI anchored
CD40 Bp50 B cells, epithelial cells, FDCs, others TNF receptor
CD72 Lyb-2 Pan-B cell C-type lectin
CD79a,b Igα,β Surface Ig + B cells Ig superfamily

FDCs indicates follicular dendritic cells.

Following their identification using mAbs, the structures of B cell–restricted target molecules have been determined and gene-targeted mice lacking expression of these CDs have been generated. Most of these target molecules regulate B-cell development and function, facilitate communication with the extracellular environment, or provide a cellular context in which to interpret BCR signals. Critical to the latter function are CD79a (Igα) and CD79b (Igβ), which are noncovalently associated components of the multiprotein cell surface BCR complex first described by John Cambier (Campbell et al 52 ). CD79a and CD79b cytoplasmic domains contain highly conserved motifs for tyrosine phosphorylation and Src family kinase docking that are essential for initiating BCR signaling and B-cell activation. 53

Studies from many laboratories evaluating murine and human B-lineage cells allow us to draw general conclusions regarding the function of specific cell surface molecules. CD19 is expressed by essentially all B-lineage cells and regulates intracellular signal transduction by amplifying Src-family kinase activity. CD20 is a mature B cell–specific molecule that functions as a membrane-embedded Ca 2+ channel. Importantly, ritixumab is a chimeric CD20 mAb, which was the first mAb approved by the Food and Drug Administration (FDA) for clinical use in cancer therapy (eg, follicular lymphoma). CD21 is the C3d and Epstein-Barr virus receptor that interacts with CD19 to generate transmembrane signals and inform the B cell of inflammatory responses within microenvironments. CD22 functions as a mammalian lectin for α2,6-linked sialic acid that regulates follicular B-cell survival and negatively regulates signaling. CD23 is a low-affinity receptor for IgE expressed on activated B cells that influences IgE production. CD24 was among the first pan-B-cell molecules to be identified, but this unique GPI-anchored glycoprotein's function remains unknown. CD40 serves as a critical survival factor for GC B cells and is the ligand for CD154 expressed by T cells. CD72 functions as a negative regulator of signal transduction and as the B-cell ligand for Semaphorin 4D (CD100). There may be other unidentified molecules preferentially expressed by B cells, but the cell surface landscape is likely dominated by molecules shared with multiple leukocyte lineages. For example, David Pisetsky demonstrated in 1991 that B cells proliferate in response to bacterial DNA stimulation (Messina et al 54 ), indicating the existence of B-cell surface receptors for DNA. These are now known to represent Toll-like receptors that are expressed by multiple leukocyte lineages.


The Cellular Basis of Immunological Memory

As already discussed, one of the major features of an adaptive immune response is the development of immunological memory.

During a primary adaptive immune response, both memory T cells and effector T cells are generated. Memory T cells are long-lived and can even persist for a lifetime. Memory cells are primed to act rapidly. Thus, any subsequent exposure to the pathogen will elicit a very rapid T cell response. This rapid, secondary adaptive response generates large numbers of effector T cells so fast that the pathogen is often overwhelmed before it can cause any symptoms of disease. This is what is meant by immunity to a disease. The same pattern of primary and secondary immune responses occurs in B cells and the antibody response, as will be discussed later in the chapter.


Materials and Methods

Ethics statement

All animal work was approved by the UCT Health Sciences Animal Ethics Committee (Project licence 012/054) to be in accordance with guidelines laid down by the South African Bureau of Standards. Research at Imperial College was approved and in accordance with regulations of the Home Office (PPL70/6957).

M3R −/− mice were backcrossed to BALB/c background for 10 generations for this study. Mice were bred under specific pathogen-free conditions and used aged 6–8 weeks. Protocols for all experiments were reviewed and approved by the UCT and ICL Animal Ethics committees.

Parasite and bacterial infection

For primary parasite infection, mice were infected subcutaneously with 500 N. brasiliensis infective larvae. To enumerate adult worms, mice were killed at various times post-infection (p.i.), intestines opened longitudinally, incubated in 10 ml saline for 3 hrs at 37°C and parasites counted under a dissecting microscope. For secondary infections, mice were treated at day 9 p.i. with ivermectin via drinking water to eliminate parasites, rested for 28 days and re-infected with 500 L3. Larvae were recovered from lungs by finely slicing the tissues, placing them in 5 ml saline for 3 hrs, and parasites subsequently enumerated.

Salmonella enterica serovar Typhimurium aroA − (SL3261) is an attenuated strain of STm SL1344 (44) and was maintained and used to infect mice intraperitoneally with an infectious dose of 5 × 10 5 CFU as described previously [40]. Tissue bacterial burdens were determined by direct culturing.

In vitro CD4 T cell differentiation into Th1 or Th2 phenotype

CD4 T cells were isolated from mesenteric lymph nodes of naïve mice using flow cytometry (>99% purity). Sorted CD4 T cells were plated at 1 × 10 5 cells/well on plates coated with 10 μg ml −1 anti-CD3 (BD Bioscience) and 5 μg ml −1 anti-CD28 (BD Bioscience). Th1 polarization conditions: 5 ng ml −1 rIL-12 (BD Bioscience) and 50 μg ml −1 anti-IL-4 (homemade Clone: 11B11) Th2 polarization: 50 ng ml −1 rIL-4 (BD Bioscience) and 50 μg ml −1 anti-IFN-γ. Cells were cultured in a final volume of 100 μl for 72 hrs, then transferred to fresh round-bottom 96 well plates and resuspended in appropriate antibody cocktails with the addition of 20 U ml −1 IL-2 (BD Bioscience) and cultured for another 48 hrs. Finally, cells were plated at 2 × 10 5 cells/well and incubated in 96 well plates coated with 20 μg ml −1 anti-CD3 (BD Bioscience). Supernatants were harvested after 48 hrs restimulation and used for ELISA.

Cytokine ELISA

Cytokine ELISAs were performed as previously described [41] using coating and biotinylated detection antibodies from R&D, with the exception of homemade coating antibodies for IL-4 (11B11) and IFN-γ (ANK18KL6). Streptavidin-conjugated HRP was used for detection with a commercially available substrate solution. MLN and lung cells were plated at 1 × 10 6 cells per well in 48 well plates pre-coated with 20 ug ml −1 anti-CD3 and restimulated for 72 hrs. Homogenates of lung and intestinal sections were prepared using a Polytron homogenizer and all samples standardized to 5 mg ml −1 protein prior to ELISA.

Calcium mobilisation assays

Changes in the cytosolic concentration of free Ca 2+ were measured using the calcium indicator Fura-4-AM. Cellular suspensions of MLN were stained with anti-CD3 and anti-CD4 antibodies and then washed and resuspended to a concentration of 0.5 × 10 7 cells/ml in Ca 2+ flux buffer (Hank’s balanced salt solution (HBSS) containing 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA), and labelled with 5 μM Fura-4-AM for 30 min at 37°C in the dark. Labelled cells were washed in Ca 2+ flux buffer. Changes in Ca 2+ in CD3 + CD4 + cells following stimulation with 10 μM ionomycin in the presence or absence of 10 μM EGTA were monitored by flow cytometry.

Cholinergic stimulation

Cells were stimulated with 0.1 μg ml −1 (sub-optimal) anti-CD3, 10 μM ACh + 10 μM BW284C51, a specific inhibitor of acetylcholinesterases used to increase the half-life of acetylcholine (ACh), 10 μM Oxotremorine M (Oxo M), 10 μM muscarine or buffer controls for 48 hrs. The muscarinic receptor antagonist atropine (AT) was used at a concentration of 100 μM, and the M3R-selective antagonist J104129 [42] at a concentration of 40 nM. Supernatants were analysed for cytokines as described.

Measurement of intestinal contraction

Jejunum sections (1 cm) were obtained and hooked onto a force transducer, placed in PBS maintained at 37°C in an organ bath, and stimulated with ACh from 10 −9 M to 10 −3 M. In between stimulations, the intestinal segment was allowed to return to baseline contraction (at least 5 min). All measurements were recorded using the Powerlab acquisition unit and analysed using the Chart5 program. The amplitude was measured as the difference between the peak and trough of the contraction and reported in millinewtons (mN).

Flow cytometry

Single cell suspensions were prepared and 1 × 10 6 cells incubated in PBS + 0.1% BSA, 1% normal rat serum and appropriate antibody cocktails. Cell populations were determined and acquired on a BD FACS Fortessa (Becton Dickinson). Cell populations were identified by the following antibody staining strategies: CD4 T cells: CD3 + CD4 + CD8 T cells: CD3 + CD8 + B cells: CD19 + B220 + Macrophages: CD11b + F4/80 + Dendritic cells: CD11c + . CD4 T cells populations were additionally stratified into naïve (CD44 lo CD62L hi ) and activated (CD44 hi CD62L lo ) T cell populations and stained for Ox40 (CD134). Alternatively activated macrophages were characterised by staining for YM1 and RELMα. Intra-cellular cytokine staining was carried on MLN, spleen or lung cells. Cells were re-suspended in complete media (IMDM (GIBCO/Invitrogen Carlsbad, CA), 10% FCS, P/S) at 2.5×10 7 /ml and stimulated with either10 μg/ml PMA/ionomycin or antigen and GolgiStop (as per manufacturer’s protocol BD Pharmingen) at 37°C for 4 hours. After re-stimulation, cells were surface stained for CD3, CD4 then fixed and permeabilized with Cytofix/

Cytoperm Plus (as per manufacturer’s instructions BD Pharmingen). Intracellular staining was performed by staining cells with either IL-13, IFN-γ or appropriately labeled isotype control. All analyses were performed with FlowJo software.

Adoptive transfer of CD4 T cells

CD4 T cells were purified from MLNs by positive selection using CD4 MACS beads (L3T4, MACS Miltenyi) according to the manufacturer’s protocol. Cells were further purified by flow cytometry to obtain purities above 95%, and 5 × 10 5 purified CD4 T cells from infected or naive animals transferred into naive WT or M3R −/− mice intravenously. Recipient mice were infected 24 hrs later with 500 L3 and killed 5 days post infection.

CDNA synthesis and RT-PCR

RNA was extracted using the Qiagen RNeasy Mini kit as per manufacturer’s protocols. RNA was converted to cDNA using random primers and Superscript II. The following primer pairs were used M1R: 5’-GGACAACAACACCAGAGGAGA-3’ 5’-GAGGTCACTTTAGGGTAGGG-3’ M2R: 5’-TGAAAACACGGTTTCCACTTC-3’, 5’-GATGGAGGAGGCTTCTTTTTG-3’ M3R: 5’-TTTACATGCCTGTCACCATCA-3’, 5’-ACAGCCACCATACTTCCTCCT-3’ M4R: 5’-TGCCTCTGTCATGAACCTTCT-3’, 5’-TGGTTATCAGGCACTGTCCTC-3’ M5R: 5’-CTCTGCTGGCAGTACTTGGTC-3’, 5’-GTGAGCCGGTTTTCTCTTCTT-3’ β-actin: 5’-TGGAATCCTGTGGCATCCATGAAAC-3’, 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’ IL-13: 5’-CTCCCTCTGACCCTTAAGGAG-3’ 5’-GAAGGGGCCGTGGCGAAACAG-3’

Histology

Lung and intestinal sections were fixed with 4% formalin in PBS solution, embedded in wax and cut into sections, then stained with Periodic Acid Schiff (PAS) stain to distinguish mucus-producing goblet cells. The histological mucus index (HMI) was used to quantify pulmonary goblet cell hyperplasia in individual mice, as described before (49).

Statistics

Values are expressed as mean ± standard deviation and significant differences were determined using either Mann-Whitney U test or ANOVA (GraphPad Prism4).


Medical Definition of T cell

T cell: A type of white blood cell that is of key importance to the immune system and is at the core of adaptive immunity, the system that tailors the body's immune response to specific pathogens. The T cells are like soldiers who search out and destroy the targeted invaders.

Immature T cells (termed T-stem cells) migrate to the thymus gland in the neck, where they mature and differentiate into various types of mature T cells and become active in the immune system in response to a hormone called thymosin and other factors. T-cells that are potentially activated against the body's own tissues are normally killed or changed ("down-regulated") during this maturational process.

There are several different types of mature T cells. Not all of their functions are known. T cells can produce substances called cytokines such as the interleukins which further stimulate the immune response. T-cell activation is measured as a way to assess the health of patients with HIV/AIDS and less frequently in other disorders.

T cell are also known as T lymphocytes. The "T" stands for "thymus" -- the organ in which these cells mature. As opposed to B cells which mature in the bone marrow.


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