42.2D: Cytotoxic T Lymphocytes and Mucosal Surfaces - Biology

The lymphatic system houses large populations of immune cells which are released upon detection of a pathogen.

Learning Objectives

  • Describe the features of the lymphatic system as they relate to the immune response

Key Points

  • The lymphatic system contains lymph: a fluid that bathes tissues and organs and contains white blood cells (not red blood cells).
  • Once B and T cells mature, the majority of them enter the lymphatic system, where they are stored in lymph nodes until needed.
  • Lymph nodes also store dendritic cells and macrophages; as antigens are filtered through the lymphatic system, these cells collect them so as to present them to B and T cells.
  • The spleen, which is to blood what lymph nodes are to lymph, filters foreign substances and antibody -complexed pathogens from the blood.

Key Terms

  • lymph: a colorless, watery, bodily fluid carried by the lymphatic system, consisting mainly of white blood cells

Lymphatic system

Lymph, the watery fluid that bathes tissues and organs, contains protective white blood cells, but does not contain erythrocytes (red blood cells). Lymph moves about the body through the lymphatic system, which is made up of vessels, lymph ducts, lymph glands, and organs such as tonsils, adenoids, thymus, and spleen. Although the immune system is characterized by circulating cells throughout the body, the regulation, maturation, and intercommunication of immune factors occur at specific sites that are known as lymph nodes.

The blood circulates immune cells, proteins, and other factors through the body. Approximately 0.1 percent of all cells in the blood are leukocytes, which include monocytes (the precursor of macrophages) and lymphocytes. Most cells in the blood are red blood cells. Cells of the immune system can travel between the distinct lymphatic and blood circulatory systems, which are separated by interstitial space, by a process called extravasation (passing through to surrounding tissue).

Recall that cells of the immune system originate from stem cells in the bone marrow. B cell maturation occurs in the bone marrow, whereas progenitor cells migrate from the bone marrow and develop and mature into naïve T cells in the organ called the thymus. On maturation, T and B lymphocytes circulate to various destinations. Lymph nodes scattered throughout the body house large populations of T and B cells, dendritic cells, and macrophages. Lymph gathers antigens as it drains from tissues. These antigens are filtered through lymph nodes before the lymph is returned to circulation. Antigen-presenting cells (APCs) in the lymph nodes capture and process antigens, informing nearby lymphocytes about potential pathogens.

The spleen houses B and T cells, macrophages, dendritic cells, and NK cells. The spleen is also the site where APCs that have trapped foreign particles in the blood can communicate with lymphocytes. Antibodies are synthesized and secreted by activated plasma cells in the spleen, which filters foreign substances and antibody-complexed pathogens from the blood. Functionally, the spleen is to the blood as lymph nodes are to the lymph.

Mucosal Immune System

The mucosal immune system is tightly regulated to prevent inappropriate immune reactions to food antigens or the commensal flora, and is responsible for guarding a vast surface area against pathogenic entry. Antigens can gain access to the mucosal immune system by a number of different mechanisms including direct DC uptake and antibody-facilitated antigen uptake. The normal response to food antigens is an active tolerance response mediated by regulatory T cells, which are induced by IL-10, TGF-β and retinoic acid secreted by resident DCs and macrophages. These immune mechanisms are critical for the prevention of food- or flora-induced pathology.

The mucosal T cell integrin alpha M290 beta 7 recognizes a ligand on mucosal epithelial cell lines

The integrin alpha M290 beta 7 is expressed at high levels on mucosal T cells, particularly on those within the epithelium of the gut. We now report that a mouse T cell hybridoma, MTC-1, with similar surface expression of this molecule, adhered strongly to cells of the mouse rectal carcinoma line CMT93 and that adhesion was blocked completely by the monoclonal antibody (mAb) M290. Other mAb to the alpha M290 or beta 7 subunits had little or no inhibitory effect. M290 also inhibited adhesion of the hybridoma to cells of the mouse lung carcinomas CTM64/61 and KLN205 but had little or no effect on adhesion to seven other mouse epithelial cell lines or to the human colon carcinoma line, HT29. Intraepithelial lymphocytes (IEL) isolated from the small intestine of BALB/c mice displayed potent T cell receptor-dependent cytotoxic effector function against CMT93 in the presence of low concentrations of Phytolacca americana lectin. This cytotoxic activity also was inhibited by the M290 mAb. Treatment of CMT93 cells with tumor necrosis factor-alpha and interferon-gamma induced expression de novo of ICAM-1 and reduced the inhibitory effect of M290 in tests both for adhesion and cytotoxicity. In further experiments cytotoxic activity of IEL against the mastocytoma P815 was investigated. This target cell was considered not to possess a ligand for the integrin. In this case cytotoxic effector function was triggered by anti-CD3 mAb and, in contrast to results with CMT93, target cell lysis was increased in the presence of M290 and other antibodies to the integrin, suggesting a co-stimulatory effect. These results show that alpha M290 beta 7 recognizes a ligand on the surface of certain epithelial cell lines. Further, they provide the first clear indication that this integrin may play an important role in functional interactions between T cells and the mucosal epithelium.

Vaccines for mucosal immunity to combat emerging infectious diseases

The mucosal immune system consists of molecules, cells, and organized lymphoid structures intended to provide immunity to pathogens that impinge upon mucosal surfaces. Mucosal infection by intracellular pathogens results in the induction of cell- mediated immunity, as manifested by CD4-positive (CD4 + ) T helper-type 1 cells, as well as CD8 + cytotoxic T-lymphocytes. These responses are normally accompanied by the synthesis of secretory immunoglobulin A (S-IgA) antibodies, which provide an important first line of defense against invasion of deeper tissues by these pathogens. New-generation live, attenuated viral vaccines, such as the cold-adapted, recombinant nasal influenza and oral rotavirus vaccines, optimize this form of mucosal immune protection. Despite these advances, new and reemerging infectious diseases are tipping the balance in favor of the parasite continued mucosal vaccine development will be needed to effectively combat these new threats.

BIO 140 - Human Biology I - Textbook

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Chapter 25

The Adaptive Immune Response: T lymphocytes and Their Functional Types

  • 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&mdashits ability to specifically recognize and make a response against a wide variety of pathogens&mdashis 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&rsquos 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&rsquos 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).

Figure 1: Notice the constant and variable regions of each chain, anchored by the transmembrane region.

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 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 2).

Figure 2: A typical protein antigen has multiple antigenic determinants, shown by the ability of T cells with three different specificities to bind to different parts of the same antigen.

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&rsquos 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 3).

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&rsquos 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 &ldquoprofessional&rdquo 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 1).

Macrophages stimulate T cells to release cytokines that enhance phagocytosis. Dendritic cells also kill pathogens by phagocytosis (see Figure 2), 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

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&rsquos own body is referred to as T cell tolerance . While thymocytes are in the cortex of the thymus, they are referred to as &ldquodouble negatives,&rdquo meaning that they do not bear the CD4 or CD8 molecules that you can use to follow their pathways of differentiation (Figure 4). 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 &ldquoself&rdquo 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: 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&rsquos 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 4). 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 5). 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 &ldquohard-wired&rdquo into its DNA, and all of its progeny will have identical DNA and T cell receptors, forming clones of the original T cell.

Figure 5: Stem cells differentiate into T cells with specific receptors, called clones. The clones with receptors specific for antigens on the pathogen are selected for and expanded.

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 5).

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 6).

Figure 6: (a) CD4 is associated with helper and regulatory T cells. An extracellular pathogen is processed and presented in the binding cleft of a class II MHC molecule, and this interaction is strengthened by the CD4 molecule. (b) CD8 is associated with cytotoxic T cells. An intracellular pathogen is presented by a class I MHC molecule, and CD8 interacts with it.

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 2).

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 2).

Table 2: Functions of T Cell Types and Their Cytokines

T cell Main target Function Pathogen Surface marker MHC Cytokines or mediators
Tc Infected cells Cytotoxicity Intracellular CD8 Class I Perforins, granzymes, and fas ligand
Th1 Macrophage Helper inducer Extracellular CD4 Class II Interferon-&gamma and TGF-&beta
Th2 B cell Helper inducer >Extracellular CD4 Class II IL-4, IL-6, IL-10, and others
Treg Th cell Suppressor None CD4, CD25 ? TGF-&beta 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.

10-15. Secretory IgA is the antibody isotype associated with the mucosal immune system

The dominant antibody isotype of the mucosal immune system is IgA. This class of antibody is found in humans in two isotypic forms, IgA1 and IgA2. The expression of IgA differs between the two main compartments in which it is found𠅋lood and mucosal secretions. In the blood, IgA is mainly found as a monomer and the ratio of IgA1 to IgA2 is approximately 4:1. In mucosal secretions, IgA is almost exclusively produced as a dimer and the ratio of IgA1 to IgA2 is approximately 3:2. A number of common intestinal pathogens possess proteolytic enzymes that can digest IgA1, whereas IgA2 is much more resistant to digestion. The higher proportion of plasma cells secreting IgA2 in the gut lamina propria may therefore be the consequence of selective pressure by pathogens against individuals with low IgA2 levels in the gut. The mechanism of isotype switching to IgA is discussed in Section 9-14.

There are special mechanisms for the secretion of polymeric IgA and IgM antibody into the gut lumen (see Section 9-13). Polymeric IgA and IgM are synthesized throughout the gut by plasma cells located in the lamina propria and are transported into the gut by immature epithelial cells located at the base of the intestinal crypts. These express the polymeric immunoglobulin receptor on their basolateral surfaces. This receptor binds polymeric IgA or IgM and transports the antibody by transcytosis to the luminal surface of the gut. Upon reaching the luminal surface of the enterocyte, the antibody is released into the secretions by proteolytic cleavage of the extracellular domain of the polymeric IgA receptor. Secreted IgA and IgM bind to the mucus layer overlying the gut epithelium where they can bind to and neutralize gut pathogens and their toxic products (Fig. 10.20).

Figure 10.20

The major antibody isotype present in the lumen of the gut is secretory polymeric IgA. This is synthesized by plasma cells in the lamina propria and transported into the lumen of the gut through epithelial cells at the base of the crypts. Polymeric IgA (more. )


Intramuscular rAd immunization induces potent and durable mucosal CD8+ T-lymphocyte responses

We first studied the ability of intramuscularly administered rAd serotype 5 (rAd5) vectors to elicit mucosal cellular immune responses. C57BL/6 mice were immunized intramuscularly with 10 9 viral particles (VP) of rAd5 expressing SIV Gag (rAd5-Gag), and Gag-specific CD8+ T-lymphocyte responses specific for the dominant D b -restricted epitope AL11 (AAVKNWMTQTL) (30) were assessed by D b /AL11 tetramer binding assays over 24 weeks. We evaluated the magnitude and kinetics of AL11-specific CD8+ T-lymphocyte responses in multiple systemic and mucosal compartments, including peripheral blood, spleen, inguinal and mesenteric lymph nodes, Peyer's patches, vaginal mucosa, and the intraepithelial and lamina propria lymphocyte populations (IEL and LPL) of both the small and large intestines. To determine the effector or memory phenotype of AL11-specific CD8+ T-lymphocytes, multiparameter flow cytometry was utilized to assess CD44, CD62L and CD127 expression (37-39). Lymphocytes from systemic and mucosal compartments exhibited comparable viability as determined by vital dye exclusion and their ability to produce interferon-γ (IFN-γ) and interleukin-2 (IL-2) following stimulation with phytohemagglutinin and ionomycin (data not shown). Lymphocytes isolated from mucosal effector surfaces were also free of contamination from blood or inductive lymphoid tissue, as demonstrated by minimal numbers of naïve (CD44-CD62L+) and central memory (CD44+CD62L+) T-lymphocytes in the cell populations isolated from these compartments (data not shown).

After a single intramuscular immunization with 10 9 VP rAd5-Gag, high frequency AL11-specific CD8+ T-lymphocyte responses were observed in multiple systemic and mucosal compartments, as shown for a representative experiment ( Fig. 1A ) or in summary for 4-6 mice per time point ( Fig. 1B ). The kinetics of the responses were similar at all anatomic sites evaluated. At week 2 following vaccination, mean peak AL11-specific CD8+ T-lymphocyte responses in blood were 6.4% of total CD8+ T-lymphocytes, while mean peak responses in spleen were 4.0%. Mean peak responses in both systemic and mucosal lymphoid inductive sites were several-fold lower (inguinal lymph nodes 0.6%, mesenteric lymph nodes 0.7% and Peyer's patches 1.3%). Surprisingly, mean peak responses in small and large bowel lamina propria (6.1% and 4.0%, respectively) were comparable in magnitude to those seen in blood and spleen, although mean peak responses in the small and large bowel IEL compartment were several-fold lower (1.6% in each compartment). In the vaginal tract, mean peak responses were, remarkably, 33% of CD8+ T-lymphocytes. In all anatomic compartments, AL11-specific CD8+ T-lymphocyte responses exhibited considerable durability up to 24 weeks following a single immunization. A similar anatomic distribution of CD8+ T lymphocyte responses was observed after subcutaneous immunization (data not shown).

Intramuscular rAd immunization induces durable high frequency CD8+ T-lymphocyte memory in multiple mucosal compartments. C57BL/6 mice were immunized intramuscularly with 10 9 VP rAd5-Gag, and AL11-specific CD8+ T-lymphocyte responses were followed over a 24-week time course in multiple anatomic compartments using D b /AL11 tetramer binding assays, as shown for a representative experiment (A) and in summary for 4-6 mice/time point in (B). In (C), the memory phenotype (effector, black bars effector memory, white bars and central memory, gray bars) of the AL11-specific CD8+ T-lymphyocyte population was determined at weeks 2 and 24 post-immunization based on expression of CD62L and CD127. Error bars are +/− S.E.

At week 2, AL11-specific CD8+ T-lymphocytes from all anatomic sites exhibited predominantly an effector (CD62L− CD127 low) phenotype ( Fig. 1C ). However, by week 24, AL11-specific CD8+ T-lymphocyte responses transitioned to a memory phenotype, with effector memory lymphocytes (CD62L− CD127 high) present at all anatomic sites and central memory lymphocytes (CD62L+ CD127 high) accumulating preferentially at both mucosal and systemic lymphoid inductive sites ( Fig. 1C ). These data demonstrate the phenotypic changes of vaccine-elicited CD8+ T-lymphocytes over time in the development of systemic and mucosal CD8+ T-lymphocyte memory at both inductive and effector sites.

To assess the functionality of vaccine-elicited mucosal CD8+ T-lymphocyte responses, we performed intracellular cytokine staining (ICS) assays to evaluate antigen-specific IFN-γ and IL-2 production at week 2 following intramuscular vaccination with 10 9 VP rAd5-Gag. High-frequency IFN-γ+ CD8+ T-lymphocyte responses were observed in multiple systemic and mucosal compartments following stimulation with either pooled SIV Gag peptides or the immunodominant AL11 peptide, and the anatomic distribution of these responses was concordant with the tetramer binding assays, as shown in representative mice ( Fig. 2A ) and in summary for 6 mice per group ( Fig. 2B ). IL-2+ CD8+ T-lymphocyte responses were of lower magnitude as compared with IFN-γ responses, consistent with our ongoing studies of rAd5 vectors in rhesus monkeys (40), but the magnitude of IL-2 responses nevertheless remained comparable between spleen and small bowel lamina propria ( Fig. 2C ). The majority of IL-2-secreting cells also produced IFN-γ (data not shown). In contrast, little to no IL-4 and IL-10 was secreted by these cell populations (data not shown). Thus, intramuscular immunization with rAd5-Gag induced the accumulation of potent, durable and functional antigen-specific memory CD8+ T-lymphocytes in multiple mucosal compartments. The functionality of the mucosal AL11-specific CD8+ T-lymphocytes elicited by this regimen was further confirmed by their capacity to expand and to control an intranasal rVaccinia-Gag challenge (data not shown).

Intramuscular rAd immunization induces high frequency functional mucosal CD8+ T-lymphocyte responses but low frequency mucosal CD4+ T-lymphocyte responses. C57BL/6 mice were immunized intramuscularly with 10 9 VP rAd5-Gag, and CD8+ T-lymphocyte responses to pooled Gag peptides or to the AL11 epitope peptide were monitored at week 2 post-immunization by intracellular cytokine staining for IFN-γ, as shown for a representative experiment in (A) and in summary for 6 mice/group in (B), or for IL-2 (C). C57BL/6 mice (n=6/group) were also immunized intramuscularly with 10 9 VP of rAd5-Gag (D) or primed with 10 9 VP rAd5-Gag and boosted with 10 9 VP rAd5HVR48-Gag (E), and CD4+ T-cell responses were assessed by intracellular cytokine staining for IFN-γ after stimulation with pooled Gag peptides at week 2 post-immunization. Error bars are +/− S.E.

Although mucosal antigen-specific CD8+ T-lymphocytes may be desirable for a candidate HIV-1 vaccine, it is possible that vaccine-elicited mucosal CD4+ T-lymphocytes may serve as additional targets of HIV-1 infection and prove detrimental. We therefore evaluated the mucosal CD4+ T-lymphocyte responses elicited by intramuscular rAd5-Gag immunization. At week 2 following a single immunization with 10 9 VP rAd5-Gag, low-frequency (0.18%) IFN-γ-secreting CD4+ T-lymphocytes could be detected in spleen following stimulation with pooled SIV Gag peptides ( Fig. 2D ). However, IFN-γ secreting CD4+ T-lymphocytes in the small bowel mucosa were not significantly above background despite clearly detectable CD8+ T-lymphocyte responses in this anatomic compartment ( Fig. 2A-B ). Moreover, IL-2 and IL-4 secreting CD4+ T lymphocytes were not detected in both systemic and mucosal compartments (data not shown). To increase our capacity to detect mucosal CD4+ T-lymphocyte responses, C57BL/6 mice were primed intramuscularly with 10 9 VP rAd5-Gag and boosted with 10 9 VP of the heterologous hexon-chimeric vector rAd5HVR48-Gag (31). At week 2 following the boost immunization, increased Gag-specific CD4+ T-lymphocyte responses were observed in splenocytes, but only low responses were observed in the small bowel mucosa ( Fig. 2E ). Thus, while high frequency IFN-γ and low frequency IL-2 secreting mucosal Gag-specific CD8+ T-lymphocytes were induced following intramuscular rAd5 immunization, Gag-specific CD4+ T-lymphocyte responses were over 10-fold lower in magnitude and only marginally detectable in this model.

We also assessed Gag-specific antibodies in serum, rectal washes and vaginal washes by ELISA in similarly vaccinated mice. Gag-specific IgG was detected in serum following immunization with 10 10 VP rAd5HVR48-Gag, but no Gag-specific IgG or IgA was detected in rectal or vaginal washes (data not shown).

Systemic CD8+ T-lymphocytes rapidly traffic to mucosal surfaces after intramuscular rAd vaccination

The comparable magnitude and kinetics of CD8+ T-lymphocyte responses in systemic and mucosal compartments following intramuscular rAd vaccination suggested a coordinated cellular immune response that bridged anatomic sites, contrasting with the anatomically skewed cellular immune responses reported in prior studies of several different vaccine modalities (6-10). One possibility was that the rAd vectors directly distributed to mucosal sites and primed local responses simultaneously in multiple anatomic compartments. However, GLP-grade rAd biodistribution studies in rabbits supporting regulatory submissions to the FDA showed no evidence of direct vector trafficking to mucosal lymphoid inductive sites following intramuscular immunization utilizing an ultrasensitive and validated quantitative PCR-based assay (D.H.B., unpublished data). These data strongly suggest that T-lymphocyte priming was restricted to systemic inductive sites. We therefore hypothesized that systemic CD8+ T-lymphocytes may have acquired the capacity to migrate to mucosal surfaces and to persist at those sites following intramuscular rAd vaccination.

To explore this possibility, we performed adoptive transfer studies to evaluate the trafficking of systemic CD8+ T-lymphocytes activated by rAd immunization. C57BL/6 mice were primed intramuscularly with 10 9 VP of the rare serotype vector rAd26-Gag (32), and boosted 6 weeks later with 10 9 VP rAd5HVR48-Gag to generate high frequencies of AL11-specific CD8+ T-lymphocytes (10% of CD8+ T-lymphocytes in spleen). On day 10 after the boost immunization, systemic CD8+ T-lymphocytes were purified from splenocytes by negative selection using immunomagnetic beads. CD8+ T-lymphocytes were then transferred intravenously to naïve recipient mice, and the anatomic distribution and phenotype of the transferred AL11-specific CD8+ T-lymphocytes were determined 14 days later. AL11-specific CD8+ T-lymphocytes from spleen rapidly migrated from the blood to all anatomic sites examined and established a tissue distribution pattern that recapitulated that seen after direct immunization, as shown for a representative experiment ( Fig. 3A ) and in summary for 5 mice ( Fig. 3B ). Moreover, the anatomic distribution of effector and memory phenotypes of the transferred AL11-specific CD8+ T-lymphocytes ( Fig. 3C ) proved comparable with that seen after active immunization ( Fig. 1C ), with central memory cells accumulating at systemic and mucosal inductive sites but largely excluded from mucosal effector surfaces. Importantly, transferred AL11-specific CD8+ T-lymphocytes that trafficked to the gastrointestinal tract also markedly increased expression of integrins and chemokine receptors critical for intestinal homing. 㬧 integrin, CCR9 and CD103 (integrin αIEL) (1, 41) were dramatically upregulated on AL11-specific CD8+ T-lymphocytes migrating to gastrointestinal LPL and IEL compartments despite being expressed at very low levels on donor lymphocytes prior to adoptive transfer ( Fig. 3D ). These findings suggest that vaccine-activated, systemic CD8+ T-lymphocytes exhibited substantial phenotypic plasticity as well as the capacity to traffic widely to mucosal tissues.

Systemic CD8+ T-lymphocytes upregulate mucosal homing markers and migrate to multiple mucosal compartments after adoptive transfer. Donor C57BL/6 mice were primed intramuscularly at week 0 with 10 9 VP rAd26-Gag and boosted at week 6 with 10 9 VP rAd5HVR48-Gag. On day 10 following the boost immunization, CD8+ T-lymphocytes were purified from splenocytes by negative immunomagnetic selection, and 2휐 7 purified lymphocytes were injected intravenously into naïve recipient mice (n=5/experiment). The tissue distribution of transferred AL11-specific CD8+ T-lymphocytes was determined at week 2 after adoptive transfer, as shown for a representative experiment (A) and in summary for 5 mice/group in (B). The effector and memory phenotypes (C) and the pattern of mucosal homing marker expression (D) were determined for AL11-specific CD8+ T-lymphocytes both prior to transfer and at week 2 post-transfer.

Given these observations, we hypothesized that intramuscular vaccination altered the migration patterns of antigen-specific CD8+ T-lymphocytes and conferred mucosal homing capacity to these cells, which typically have more restricted trafficking patterns. To compare directly the tissue migration patterns of quiescent and vaccine-activated systemic CD8+ T-lymphocytes, we performed adoptive transfer experiments utilizing congenic mice. Systemic CD8+ T-lymphocytes were purified from splenocytes of naïve CD45.1+ mice (B6.SJL) and transferred intravenously to naïve CD45.2-congenic recipients (C57BL/6). As expected, transferred naïve CD8+ T-lymphocytes migrated rapidly to the spleen and lymph nodes in recipient mice ( Fig. 4A ). However, in the absence of immunization, trafficking of adoptively transferred CD8+ T-lymphocytes to mucosal effector sites was highly restricted at day 14 post-transfer, as demonstrated by the low proportion of CD45.1+ to CD45.2+ CD8+ T-lymphocytes in the gastrointestinal IEL and LPL compartments ( Fig. 4A ). In contrast, intramuscular immunization of the recipient mice with 10 9 VP rAd5-Gag on day 2 post-transfer generated AL11-specific CD45.1+ CD8+ T-lymphocytes that efficiently migrated to gastrointestinal mucosa, as shown by the comparable proportions of these cells relative to AL11-specific CD45.2+ CD8+ T-lymphocytes across both systemic and mucosal compartments on day 14 ( Fig. 4B ). These findings demonstrate that vaccine-activated, but not quiescent, peripheral CD8+ T-lymphocytes have the capacity to migrate rapidly and extensively to multiple mucosal tissues. Moreover, the congenic adoptive transfer system allowed for a comparison of the relative magnitudes of mucosal immune responses generated by adoptively transferred and native CD8+ T-lymphocytes within the same mouse. The comparable responses in multiple anatomic compartments ( Fig. 4B ) suggest that trafficking of systemic lymphocytes to mucosal sites accounted for the vast majority of antigen-specific mucosal CD8+ T-lymphocytes. Thus, trafficking of systemic lymphocytes, rather than direct local priming at mucosal sites, appears to be the predominant mechanism for generating widespread mucosal immunity following systemic vaccination with rAd vectors.

Vaccination facilitates trafficking of systemic CD8+ T-lymphocytes to mucosal surfaces. 10 7 purified systemic CD8+ T-lymphocytes were isolated from splenocytes of naïve CD45.1+ donors and adoptively transferred into naïve congenic CD45.2+ recipients. The ratio of transferred/native CD8+ T-lymphocytes was determined on day 12 post-transfer for the total CD8+ T-lymphocyte population at each anatomic site in the absence of immunization (A) or for the responding AL11-specific CD8+ T-lymphocyte population at each anatomic site on day 14 post-immunization with rAd5-Gag (B). In each case, the top panel demonstrates the gating strategy used for analysis, the middle panel shows a representative experiment, and the bottom panel shows the ratio of transferred/native CD8+ T-lymphocytes at each anatomic site averaged for 4 mice/group. (C) 10 7 purified systemic CD4+ T-lymphocytes were isolated from splenocytes of naïve CD45.1+ donors and adoptively transferred into naïve congenic CD45.2+ recipients. The ratio of transferred/native CD4+ T-lymphocytes was determined on day 14 post-transfer at each anatomic site in the absence of immunization (black bars) or following intramuscular immunization with rAd5-Gag on day 2 (white bars). Error bars are +/− S.E.

We also assessed the capacity of CD4+ T-lymphocytes to traffic to mucosal sites utilizing the same adoptive transfer and immunization protocol. Naïve CD4+ T-lymphocytes exhibited limited capacity to migrate to mucosal sites ( Fig. 4C, black bars ), comparable with the restricted trafficking observed with naïve CD8+ T-lymphocytes ( Fig. 4A ). Vaccination did not detectably increase the capacity of total CD4+ T-lymphocytes to migrate to mucosal surfaces ( Fig. 4C, white bars ). However, we were unable to study the trafficking of antigen-specific CD4+ T-lymphocytes to mucosal surfaces as a result of the low magnitude of Gag-specific CD4+ T-lymphocyte responses in this experimental model ( Fig. 2D-E ).

Heterologous prime-boost regimens with rare serotype and hexon-chimeric rAd vectors elicit potent anamnestic mucosal cellular immune responses

The capacity of antigen-specific CD8+ T-lymphocytes to traffic from systemic to mucosal compartments after a single immunization raised the possibility that mucosal cellular immune responses may increase further following heterologous boost immunizations. However, the anatomic distribution of recall responses has previously been reported to be biased by the site of initial antigen exposure (1, 2). Therefore, we investigated whether repeated intramuscular administration of rAd vectors in heterologous prime-boost regimens would augment mucosal responses or, alternatively, would bias recall responses away from mucosal surfaces. The utility of homologous rAd prime-boost regimens is limited by the inability of homologous vector readministration to boost responses efficiently, as a result of the generation of potent vector-specific neutralizing antibodies by the priming immunization (30, 42, 43). Therefore, we utilized serologically distinct rare serotype and hexon-chimeric rAd vectors for these studies.

Naïve C57BL/6 mice or mice previously primed with 10 9 VP rAd26-Gag were boosted intramuscularly with 10 9 VP rAd5HVR48-Gag, and CD8+ T-lymphocyte responses were examined in multiple anatomic compartments. As compared with naïve mice, mice previously primed with rAd26-Gag exhibited substantially higher peak frequencies of AL11-specific CD8+ T-lymphocytes following rAd5HVR48-Gag immunization in both systemic and mucosal compartments ( Fig. 5A ), and the magnitude of the boost effect was comparable at systemic and mucosal sites. After boosting, frequencies of AL11-specific CD8+ T-lymphocytes approached 20% in the small bowel lamina propria and exceeded 60% in the vaginal tract, and these responses persisted for over 12 weeks. Thus, rather than directing CD8+ T-lymphocyte responses away from mucosal surfaces, boosting with a heterologous vector resulted in potent and persistent secondary recall responses in multiple mucosal compartments.

Heterologous rAd prime-boost regimens are significantly superior to homologous regimens in inducing mucosal CD8+ T-lymphocyte recall responses. (A) To compare primary and recall responses, AL11-specific CD8+ T-lymphocyte responses at week 2 and week 12 following immunization with 10 9 VP rAd5HVR48-Gag were evaluated in previously naïve C57BL/6 mice (white bars) or in mice primed 6 weeks earlier with 10 9 VP rAd26-Gag (black bars) (n=4/group at each time point). (B) C57BL/6 mice (n=4/group at each time point) were primed intramuscularly at week 0 with 10 9 VP rAd26-Gag and boosted at week 8 with either 10 9 VP rAd26-Gag (homologous vector dashed lines) or 10 9 VP rAd5HVR48-Gag (heterologous vector solid lines). AL11-specific CD8+ T-lymphocyte responses were assessed at multiple time points following the priming and boosting immunizations. Means +/− S.E. for each group are shown. Asterisks denote two-sided t-tests. ILN, inguinal lymph nodes MLN, mesenteric lymph nodes PP, Peyer's patches SB, small bowel LB, large bowel IEL, intraepithelial lymphocytes LPL, lamina propria lymphocytes VT, vaginal tract.

We next directly compared the magnitude and kinetics of mucosal CD8+ T-lymphocyte responses elicited by systemic heterologous versus homologous rAd prime-boost regimens. C57BL/6 mice were primed intramuscularly at week 0 with rAd26-Gag and were boosted intramuscularly at week 8 with the homologous rAd26-Gag vector or the heterologous rAd5HVR48-Gag vector. Systemic and mucosal AL11-specific CD8+ T-lymphocyte responses were assessed for 12 weeks following boost immunization. Homologous boosting with rAd26-Gag resulted in little to no increase in CD8+ T-lymphocyte responses as expected ( Fig. 5B ). In contrast, heterologous boosting with rAd5HVR48-Gag generated significantly enhanced peak and memory CD8+ T-lymphocyte responses in both systemic and mucosal compartments (p=0.005 for spleen, p=0.001 for small bowel LPL, p=0.005 for large bowel LPL, and p=0.02 for vaginal tract lymphocytes comparing heterologous versus homologous responses at week 10 using two-tailed t-tests). These data demonstrate that heterologous rAd prime-boost regimens were significantly superior to homologous rAd regimens for generating potent and durable cellular immune memory in the gastrointestinal and vaginal tracts.

Intramuscular rAd immunization induces high frequency, durable mucosal CD8+ T-lymphocyte memory in rhesus monkeys

We next investigated whether our findings of robust mucosal cellular immunity in intramuscularly rAd vaccinated mice would translate into nonhuman primates. We immunized three rhesus monkeys (two that expressed the MHC class I allele MamuA*01 and one that did not express this allele) intramuscularly with 10 11 VP of rAd5HVR48-Gag. Mamu-A*01-restricted CD8+ T-lymphocyte responses to the highly immunodominant Gag epitope CM9 (CTPYDINQM) (44) were assessed by multiparameter tetramer binding assays for up to 1 year following immunization in multiple systemic and mucosal compartments. CD8+ T-lymphocyte responses were observed in duodenal mucosa as well as in blood and lymph nodes in the Mamu-A*01-positive animals at weeks 4 and 32 after vaccination, and the magnitude of mucosal responses proved comparable with the magnitude of systemic responses ( Fig. 6 ). Moreover, CM9-specific memory CD8+ T-lymphocytes persisted for at least 52 weeks following vaccination. At this late time point, CM9-specific CD8+ T-lymphocytes were still detected in duodenal mucosa, colorectal mucosa, bronchoalveolar lavage and vaginal mucosa. Importantly, the magnitude of these long-term mucosal responses proved comparable with those found in blood and lymph nodes, except for responses in vaginal mucosa that were approximately 5-fold higher in magnitude than responses in blood, consistent with our mouse studies (Figs. ​ (Figs.1, 1 , ​ ,2). 2 ). At all anatomic sites, CM9-specific CD8+ T-lymphocytes were predominantly of a CD28+CD95+ memory phenotype (45). These data demonstrate that a single intramuscular rAd vaccination generated potent and durable CD8+ T-lymphocyte memory that persisted for over one year in multiple mucosal tissues in nonhuman primates.

Mucosal cellular immune memory in rhesus monkeys after intramuscular rAd vaccination. Two rhesus monkeys expressing the MHC class I allele Mamu-A*01 (animals 184-03, gray bars, and 210-03, black bars) and one monkey negative for this allele (153-03, white bars) were vaccinated intramuscularly with a single injection of 10 11 VP rAd5HVR48-Gag. Gag CM9-specific CD8+ T-lymphocyte responses were evaluated in systemic and mucosal compartments at weeks 4, 32, and 52 following vaccination. The memory phenotype of the responding lymphocytes was determined by CD28 and CD95 expression.

Chapter 2 - The Role of T Lymphocytes in Mucosal Protection and Injury

This chapter describes the fundamental properties of T lymphocytes and the factors which govern their secretion of cytokines in various inflammatory conditions. It discusses the mechanism of recruitment of T lymphocytes to sites of inflammation, particularly mucosal surfaces. The chapter shows the way in which the secretion of various patterns of cytokines can influence the course and nature of the ensuing inflammatory response. In some cases, these responses serve to protect the host against infection and ensure subsequent healing and repair. In other cases, however, these responses may be chronic and result in long-term damage to host tissues. T lymphocytes play a vital role in the immune mechanisms which lead to the damage as well as the protection of mucosal surfaces. Through their specific antigen receptors, T lymphocytes are responsible for the initiation and orchestration of immune responses. The particular patterns of cytokines secreted by activated T lymphocytes determine the nature of the ensuing inflammatory response, and in particular, decide whether cell-mediated reactions or humoral reactions predominate. T lymphocytes also play a role in the genesis of immunological tolerance, which may abrogate unwanted inflammatory responses in mucosal surfaces exposed to a wide variety of foreign antigens.

42.2D: Cytotoxic T Lymphocytes and Mucosal Surfaces - Biology

The innate and adaptive immune responses discussed thus far comprise the systemic immune system (affecting the whole body), which is distinct from the mucosal immune system. Mucosa are another name for mucous membranes. Mucosal immunity is formed by mucosa-associated lymphoid tissue, or MALT, which functions independently of the systemic immune system it has its own innate and adaptive components. MALT is a collection of lymphatic tissue that combines with epithelial tissue lining the mucosa throughout the body. This tissue functions as the immune barrier and immune response in areas of the body in direct contact to the external environment. The systemic and mucosal immune systems use many of the same cell types. Foreign particles that make their way to MALT are taken up by absorptive epithelial cells called M cells and delivered to APCs (antigen-presenting cells) located directly below the mucosal tissue. M cells are located in the Peyer’s patch, which is a lymphoid nodule. APCs of the mucosal immune system are primarily dendritic cells, with B cells and macrophages playing minor roles. Processed antigens displayed on APCs are detected by T cells in the MALT and at various mucosal induction sites, such as the tonsils, adenoids, appendix, or the mesenteric lymph nodes of the intestine. Activated T cells then migrate through the lymphatic system and into the circulatory system to mucosal sites of infection.

MALT tissue: The topology and function of intestinal MALT is shown. Pathogens are taken up by M cells in the intestinal epithelium and excreted into a pocket formed by the inner surface of the cell. The pocket contains antigen-presenting cells, such as dendritic cells, which engulf the antigens, then present them with MHC II molecules on the cell surface. The dendritic cells migrate to an underlying tissue called a Peyer’s patch. Antigen-presenting cells, T cells, and B cells aggregate within the Peyer’s patch, forming organized lymphoid follicles. There, some T cells and B cells are activated. Other antigen-loaded dendritic cells migrate through the lymphatic system where they activate B cells, T cells, and plasma cells in the lymph nodes. The activated cells then return to MALT tissue effector sites. IgA and other antibodies are secreted into the intestinal lumen.

MALT is a crucial component of a functional immune system because mucosal surfaces, such as the nasal passages, are the first tissues onto which inhaled or ingested pathogens are deposited. This allows the immune system to detect and deal with pathogens very quickly after they enter the body through various mucous membranes. The mucosal tissue includes the mouth, pharynx, and esophagus, along with the gastrointestinal, respiratory, and urogenital tracts.

Immune tolerance

The immune system has to be regulated to prevent wasteful, unnecessary responses to harmless substances and, more importantly, so that it does not attack “self.” The acquired ability to prevent an unnecessary or harmful immune response to a detected foreign substance known not to cause disease or to self-antigens is described as immune tolerance. The primary mechanism for developing immune tolerance to self-antigens occurs during the selection for weakly, self-binding cells during T and B lymphocyte maturation. Any T or B lymphocytes that recognize harmless foreign or “self” antigens are deleted before they can fully mature into immunocompetent cells.

There are populations of T cells that suppress the immune response to self-antigens. They also suppress the immune response after the infection has cleared to minimize host cell damage induced by inflammation and cell lysis. Immune tolerance is especially well developed in the mucosa of the upper digestive system because of the tremendous number of foreign substances (such as food proteins) that APCs of the oral cavity, pharynx, and gastrointestinal mucosa encounter. Immune tolerance is brought about by specialized APCs in the liver, lymph nodes, small intestine, and lung that present harmless antigens to a diverse population of regulatory T (Treg) cells: specialized lymphocytes that suppress local inflammation and inhibit the secretion of stimulatory immune factors. The combined result of Treg cells is to prevent immunologic activation and inflammation in undesired tissue compartments, allowing the immune system to focus on hazardous pathogens instead.

Nasal-associated lymphoid tissues (NALTs) support the recall but not priming of influenza virus-specific cytotoxic T cells

The lymphoid tissue that drains the upper respiratory tract represents an important induction site for cytotoxic T lymphocyte (CTL) immunity to airborne pathogens and intranasal vaccines. Here, we investigated the role of the nasal-associated lymphoid tissues (NALTs), which are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage, in the initial priming and recall expansion of CD8 + T cells following an upper respiratory tract infection with a pathogenic influenza virus and immunization with a live attenuated influenza virus vaccine. Whereas NALTs served as the induction site for the recall expansion of memory CD8 + T cells following influenza virus infection or vaccination, they failed to support activation of naïve CD8 + T cells. Strikingly, NALTs, unlike other lymphoid tissues, were not routinely surveyed during the steady state by circulating T cells. The selective recruitment of memory T cells into these lymphoid structures occurred in response to infection-induced elevation of the chemokine CXCL10, which attracted CXCR3 + memory CD8 + T cells. These results have significant implications for intranasal vaccines, which deliver antigen to mucosal-associated lymphoid tissue and aim to elicit protective CTL-mediated immunity.

Keywords: CD8 T-cell priming influenza virus nasal-associated lymphoid tissue respiratory tract.

Conflict of interest statement

The authors declare no conflict of interest.


Influenza antigen is present within…

Influenza antigen is present within the NALTs following intranasal influenza infection. ( A…

HEV in the NALTs stain positive for PNAd and Madcam-1. ( A and…

CTL priming occurs in the…

CTL priming occurs in the cervical lymph nodes but not NALTs following influenza…

NALTs serve as the recall…

NALTs serve as the recall site for memory CD8 + T-cell responses following…

NALTs serve as the recall…

NALTs serve as the recall site for memory CD8 T-cell responses following an…

NALTs support the recall expansion…

NALTs support the recall expansion of memory CD8 + T cells, but not…

Effector memory CD8 T cells…

Effector memory CD8 T cells preferentially migrate into the inflamed NALTs. ( A…

CXCR3 signaling promotes memory CD8…

CXCR3 signaling promotes memory CD8 + T-cell infiltration into the inflamed NALTs. (…

Elevation of Cxcl10 in the…

Elevation of Cxcl10 in the influenza virus-infected NALTs. Shown is the expression of…