Cell-Mediated Immunity Definition

• CMI is an unique sort of acquired immune response that is not mediated by antibodies but rather by sensitised T cells.
• This type of immunity is passed from donor to recipient without the need of antisera, but rather with intact lymphocytes; therefore, it is referred to as cell-mediated immune reaction.
• Contrary to humoral immunity, cell-mediated immunity does not rely on antibodies to carry out adaptive immunological functions.
• Cell-mediated immunity is driven by mature T lymphocytes, macrophages, and the generation of cytokines in response to an antigen.
• T cells that participate in cell-mediated immunity rely on antigen-presenting cells with membrane-bound MHC class I proteins to detect intracellular target antigens.
• Dependent upon the binding specificity of MHC proteins to external antigens, the maturation and differentiation of naive T cells into helper or killer T cells.
• Infection of bodily cells by a virus, bacterium, or fungus activates cell-mediated immunity (intracellular invaders).
• With the use of MHC class I proteins, T lymphocytes may identify cancerous cells. There are three primary types of lymphocytes engaged in cell-mediated immunity: helper T cells, killer T cells, and macrophages.
• When a “helper” T cell encounters an antigen-presenting cell in the body, it secretes signalling substances known as cytokines.
• These cytokines attract “killer” T cells and macrophages to the antigen-presenting cell in an effort to destroy it.

Induction of Cell-Mediated Immunity

• Antigen processing and presentation involve the association of antigens with self-MHC molecules for presentation to T lymphocytes with the proper receptors.
• Proteins from foreign antigens, such as bacteria, are ingested by APCs, such as macrophages, via endocytic vesicles.
• They are then exposed to intracellular proteases in vesicles. Endosomal vesicles create peptides containing roughly 10 to 30 amino acid residues.
• Endosomal vesicles are thus capable of fusing with exocytic vesicles containing class II MHC molecules. The sequence of events involved in the induction of CMI is described below.

Presentation of foreign antigen by APCs to T lymphocytes

• CMI is initiated by the presentation of foreign antigen to T cells by APCs.
• T lymphocytes have T-cell receptors (TCRs), which are antigen recognition receptors that recognise foreign antigen and a self-MHC molecule on the surface of APCs.
• The sensitised T lymphocytes subsequently undergo blast transformation, clonal proliferation, and differentiation into memory cells and effector cells, including Th, Tc, Td, and Ts.
• Activated lymphocytes then release lymphokines, which are biologically active substances responsible for diverse CMI symptoms.

Recognition of antigen by T cells

• T cells only identify antigens when MHC molecules are present. CD8 cells identify the combination of foreign antigen and class I MHC molecule.
• After identification, these CD8 cells develop into Tc and Ts lymphocytes. On the other hand, CD4 cells develop into Th and Td cells upon recognising the combination of antigen and class II MHC antigen.
• As with other membrane glycoproteins, the class II MHC molecules are generated in the rough endoplasmic reticulum and subsequently exported via the Golgi apparatus.
• The invariant chain (Ii) is a third polypeptide that protects the binding site of the class II dimer until the decreased pH of the compartment generated following fusion with an endosomal vesicle causes its separation.
• The MHC class II peptide antigen complex is then delivered to the cell surface, where it is displayed and recognised by the TCR of a CD4 T cell.
• The lymphocyte attaches to the target cells after recognising antigen and class I MHC molecules.
• Class I MHC molecules prepare endogenous antigens, such as cytosolic viral proteins produced in an infected cell, for presentation.
• In summary, cytosolic proteins are degraded by the proteasome, a peptidase complex. Using peptide transporter systems, cytosolic peptides acquire access to developing MHC class I molecules in the rough endoplasmic reticulum (transporters associated with antigen processing; TAPs). Additionally, the TAP genes are encoded in the MHC.
• Because the binding groove of the class I molecule is more limited than that of the class II molecule, class I MHC molecules contain shorter peptides than class II MHC molecules.

Release of cytokines by Tc lymphocytes

• This causes Tc lymphocytes to release cytokines, leading in the destruction of target cells.
• The T cells then separate from the target cells and attach to new target cells, repeating the process. Interferon-gamma produced and secreted by Tc cells may also contribute in some way to macrophage activation.

Cytokines

• Cytokines are biologically active chemicals that are released by monocytes, lymphocytes, and other cells, and they play an active role in innate immunity, adoptive immunity, and inflammation.
• They actively participate in a vast array of biological processes, ranging from chemotaxis to cell activation.
• Cytokines were initially recognised as mediators and regulators of immunological processes produced by immune cells.
• It is now known that many cytokines are produced by cells other than immune cells, and that they can also affect nonimmune cells.
• Currently, cytokines are employed clinically as biological response modifiers to treat a variety of diseases.
• Typically, cytokines are not preserved as produced proteins. Their synthesis is rather initiated by gene transcription, and their mRNAs have a short half-life.
• As needed, they are created during immunological reactions. Many distinct cytokines are produced by numerous cell types and affect on numerous cell types (i.e., they are pleiotropic), and in many instances, they have comparable effects (i.e., they are redundant).
• Because of the nature of cytokine receptors, redundancy exists.

Categories of cytokines

Cytokines can be categorised based on their functions or their source, however it is important to remember that any attempt to categorise them will be limited by the fact that they can be produced by several cell types and operate on numerous cell types. Cytokine classifications are as follows:

1. Mediators affecting lymphocytes.
2. Mediators affecting macrophages and monocytes.
3. Mediators affecting polymorphonuclear leukocytes.
4. Mediators affecting stem cells.
5. Mediators produced by macrophages that affect other cells.

Mediators affecting lymphocytes

Interleukin-1 (IL-1)

It is a protein produced by macrophages and monocytes that have been activated. Antigens, poisons, and inflammatory processes enhance its synthesis, whereas cyclosporine and corticosteroids suppress it. It is a significant interleukin that mediates a broad spectrum of metabolic, physiological, inflammatory, and haematological functions. It serves numerous crucial purposes, which are listed below:

• It induces T and B lymphocytes, neutrophils, epithelial cells, and fibroblasts to proliferate, differentiate, or produce particular products. For instance, it encourages helper T cells to produce IL-2 and B cells to proliferate and create antibodies.
• It causes fever associated with infections and other inflammatory responses by acting on the hypothalamus.

Interleukin-2 (IL-2)

• IL-2 is a protein predominantly generated by helper T cells. It is the predominant T-cell growth factor. It increases the growth of both helper and cytotoxic T lymphocytes.
• It can also stimulate natural killer (NK) cells and monocytes and boost the proliferation of B cells. IL-2 operates in an autocrine manner on T cells.
• T cell activation induces expression of IL-2R and production of IL-2. The binding of IL-2 to IL-R increases cell division.
• When T cells are no longer activated by antigen, the IL-2R will degrade and the proliferative phase will conclude.

Interleukin-4 (IL-4)

• It is a protein that is mostly generated by T-cell helper cells and macrophages.
• It boosts the growth of Th-2 cells, the fraction of T cells that produce IL-4 and IL-5 and enhance humoral immunity through antibody production.
• In antibody-producing cells, it is also essential for class (isotype) flipping from one antibody class to another.

Interleukin-5 (IL-5)

• It is a protein made by T-cell helper cells.
• It stimulates the expansion and maturation of B lymphocytes and eosinophils.
• It promotes the formation and activation of eosinophils as well as enhancing IgA synthesis.

Interleukin-6 (IL-6)

• It is a protein generated by T cells that provide assistance and macrophages.
• It promotes the liver’s production of acute phase proteins.
• Additionally, it operates on the hypothalamus to induce fever.

Other interleukins

• IL-10, IL-12, and IL-13 are the other lymphocyte-affecting interleukins. Activated macrophages and Th-2 cells generate IL-10. Primarily, it is an inhibiting cytokine.
• It inhibits the synthesis of interferon type I. It reduces interferon-gamma synthesis by Th-1 cells, hence shifting immunological responses to the Th-2 type.
• It also suppresses immunological responses by inhibiting cytokine production by activated macrophages and the expression of class II MHC and costimulatory molecules on macrophages. Activated macrophages and dendritic cells create IL-12.
• It promotes interferon-gamma production and encourages the differentiation of Th cells into Th-1 cells. Moreover, it improves the cytolytic capabilities of Tc and NK cells. Th-2 cells produce IL-13.
• It contributes to the pathophysiology of allergic airway disease (asthma). It contributes to the occurrence of asthmatic hyperresponsiveness.

Transforming growth factor-beta (TGF-)

• It is produced by T cells as well as numerous other cell types. Primarily, it is an inhibitory cytokine. It suppresses the growth of T lymphocytes and macrophage activation.
• Additionally, it inhibits the effects of proinflammatory cytokines on polymorphonuclear leukocytes and endothelial cells.
• In essence, it reduces the immune response after an infection when it is no longer necessary, so promoting the healing process.

Mediators affecting macrophages and monocytes 

Chemokines are a subclass of cytokines with a low molecular weight and a distinct structural arrangement. More than fifty chemokines containing 68 to 120 amino acids have been discovered. Activated mononuclear cells release alpha-chemokines such as IL-8, which attract neutrophils. Activated T cells release the beta-chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted) and MCAF (monocyte chemotactic and activating factor) that attract macrophages and monocytes. Endothelial cells, local macrophages, and other cells present at the site of an infection create chemokines.

• They attract either macrophages or neutrophils to the site of an infection, and are therefore implicated in chemotaxis, the chemical-induced migration of leukocytes. Monocytes and neutrophils include receptors for chemokines on their surfaces.
• In addition, they enhance white cell migration into the tissue to reach the diseased location. They accomplish this by activating integrins on the surface of neutrophils and macrophages, which then bind to intercellular adhesion molecule (ICAM) proteins on the surface of the endothelium.

Mediators affecting polymorphonuclear leukocytes 

Tumor necrosis factor (TNF-)

• It is created by activated macrophages in response to microorganisms, particularly Gram-negative bacteria’s lipopolysaccharide.
• It’s a key mediator of acute inflammation. By activating endothelial cells to create adhesion molecules, it facilitates the migration of neutrophils and macrophages to sites of infection.
• It also creates chemotactic cytokines known as chemokines. In addition to acting on the hypothalamus to induce fever, TNF- also stimulates the synthesis of acute phase proteins.

Chemokines and other chemotactic factors

• Chemokines and other chemotactic substances recruit neutrophils, basophils, and eosinophils specifically to the site of an infection.
• IL-8 and the C5a component of the complement, for instance, specifically attract neutrophils. 

Leukocyte-inhibiting factor

• It limits neutrophil migration, keeping the cells at the site of the infection.

Mediators affecting stem cells 

Stem cell mediators include (a) IL-3, (b) granulocyte macrophage colony-stimulating factor (GM-CSF), and (c) granulocyte colony-stimulating factor (GCSF) (G-CSF).

• Helper T cell-produced IL-3 inhibits the development and differentiation of bone marrow stem cells.
• The GM-CSF generated by macrophages and T lymphocytes increases granulocyte development.
• Macrophages, fibroblasts, and endothelial cells create G-CSF. It promotes the formation of neutrophils from stem cells and is therefore used to prevent infections in cancer treatment patients.

Mediators produced by macrophages that affect other cells 

Tumor necrosis factor (TNF-)

As its name suggests, TNF- induces tumour cell death and necrosis in experimental mice. It is also known as cachectin due to the fact that it inhibits lipoprotein lipase in adipose cells, decreasing the consumption of fatty acids and causing cachexia. TNF- has multiple functions:

• It stimulates respiratory burst in neutrophils, hence boosting the scavenging capabilities of phagocytes.
• It enhances neutrophil adherence to blood vessel endothelial cells.
• Additionally, it enhances the development of B cells and boosts the production of lymphokines by T cell helper cells.

Macrophage migration inhibition factor (MIF)

• It is generated by macrophages in response to endotoxin activity.
• It maintains macrophages at the infection site.
• It plays a crucial function in the development of septic shock.

Nitric oxide (NO)

• It is generated by macrophages in response to endotoxin activity.
• NO induces vasodilation, leading to hypotension in septic shock.

Tests for Detection of CMI 

The CMI can be detected by the following in vivo and in vitro tests:

CMI in vivo tests 

• Skin tests can detect delayed hypersensitivity reactions to common antigens that come into contact with the body.
• Antigens used for skin testing are a purified protein derivative in the tuberculin test, dinitrochlorobenzene, or dinitrofluorobenzene.
• The majority of healthy individuals exhibit delayed reactivity to these skin antigens. The absence of a reaction to these skin tests shows CMI impairment.

CMI in vitro tests

There are numerous in vitro assays for the detection of CMI. These include:

1. Migration inhibition factor (MIF) test

• This test is used to determine the CMI by doing a semiquantitative evaluation of leukocyte migration inhibition.
• This test is based on the premise that cultured T lymphocytes create macrophage migration inhibitory factor in response to antigen exposure.
• This test involves the incubation of human peripheral leukocytes in capillary tubes within a culture chamber containing culture fluid.
• In the absence of antigen, the leukocytes move to the open end of the tube and produce a fan-like pattern. In the presence of antigen, the migration of leukocytes is inhibited.

2. Lymphocyte blast transformation

• When exposed to specific mitogens, such as phytohemagglutinin and concavalin, a substantial proportion of T cells undergo blast transformation.
• On exposure to the specific antigen, sensitised T lymphocytes are converted into massive blast cells with an increase in DNA synthesis.
• Incorporation of tritiated thymidine is then used to quantify the increase in DNA synthesis.

3. Enumeration of T cells, B cells, and subpopulation

• A fluorescence-activated cell sorter is utilised to determine the quantity of T and B cells.
• In this technique, cells are labelled using monoclonal antibodies conjugated to fluorescent or rhodamine dyes.
• The number of fluorescent cells is determined by passing single cells through a laser light beam.
• Using antibodies labelled with fluorescein against all immunoglobulin classes, the total number of B cells can be determined.
• Counting T cells, CD4 helper cells, CD8 suppressor cells, and other cells is made possible using monoclonal antibodies directed to the T-cell marker.
• The ratio of CD4 to CD8 in healthy individuals is at least 1.5, however it is less than one in AIDS patients.

4. Rosette formation

• A lymphocyte on which three or more sheep erythrocytes are joined constitutes a rosette. When most T cells are combined with sheep erythrocytes, rosettes form.
• E-rosette refers to the T-cell rosette.
• E-rosettes can be counted to indicate the presence of T cells. Rosette development is useful for detecting T cells, and thus the host’s CMI.

Functions of Cell-Mediated Immunity

• It gives immunity against diseases caused by obligate intracellular bacteria (Mycobacterium tuberculosis, Mycobacterium leprae, Brucella, etc.), viruses (smallpox, measles, mumps, etc.), fungus (Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, etc.), and parasites (Toxoplasma gondii, Leishmania donovani, etc.).
• It contributes to immune surveillance and protection against cancer.
• It plays a crucial role in the pathogenesis of delayed hypersensitivity reactions and certain autoimmune disorders, such as autoimmune thyroiditis, encephalitis, and others.