Cellular Basis of Immune Response

Introduction:

During embryonic development the blood cell precursors originate mainly in yolk sac and fetal liver. These cells differentiate into myeloid and lymphoid series. Lymphoid series leads to the development of B-lymphoctes, T-lymphocytes and NK cells and myeloid series leads to formation of monocytes and macrophages, erytherocytes,  neutrophils, basophils , eosinophils, megakaryocytes \platelets and dendritic cells. The normal ratio of Ratio of T-lymphocytes: B- lymphocytes is T:B = 3:1. Lymphocytes, the cells competent to initiate immune responses, can be divided into two major groups: thymus-derived or T cells responsible for “cellular immunity” (e. g. delayed hypersensitivity reactions) and bursa (or bursa-equivalent) derived or B cells which produce immunoglobulin (antibody) molecules and are involved in “humoral immunity”. “Accessory” cells, such as monocytes (or macrophages), polymorphonuclear leucocytes and mast cells act in an auxiliary manner by facilitating antigen processing or presentation, or by liberating factors which modify the various manifestations of the immune response.

Diagrammatic representation of stem cell differentiation

Stem cell differentiation

T Lymphocytes:

T lymphocytes arise from a pluripotent stem cell in bone marrow. Unlike B-lymphocytes, which undergo continued maturation in bone marrow, T lymphocytes differentiate in the thymus. B-lymphocytes have immunoglobulins on their cell surface through which they recognize antigens but T lymphocytes are surface-negative for immunoglobulin and do not exhibit DNA rearrangements in immunoglobulin genes. Rather, T lymphocytes express a distinct membrane receptor for antigen that recognizes antigen in conjunction with membrane glycoproteins encoded in the major histocompatibility complex. T lymphocytes play a major role in the initiation and regulation of immune responses, and are key elements of cell-mediated immune responses against viruses, intracellular bacteria, and tumors. T lymphocytes express different surface membrane antigens at various stages of development and/or cell activation. These surface markers have been useful in the identification of phenotypic and functional diversity among T lymphocytes. These markers previously were identified by means of monoclonal antibodies, which recognize specific antigenic determinants (termed epitopes) within a given surface membrane protein. So, a leukocyte surface marker, which is reactive toward a group (or cluster) of monoclonal antibodies, is identified according to a cluster of differentiation (CD) number (e.g. CD2, CD8). Certain CD markers are expressed by virtually all peripheral blood T lymphocytes. This is the case with respect to CD2, a 50-kilodalton glycoprotein through which T lymphocytes form rosettes with sheep red blood cells. Other CD markers are useful in segregating T lymphocytes into a number of distinct subpopulations.

Thus, approximately 60 percent of peripheral blood T lymphocytes express CD4, a glycoprotein expressed on T cells whose activation is dependent on recognition of antigen in conjunction with class II major histocompatibility complex (MHC) molecules. Approximately 30 percent of peripheral blood T lymphocytes express CD8; a membrane protein expressed by T cells whose activation is dependent on recognition of antigen in conjunction with class I MHC molecules. The majority of circulating T lymphocytes express either CD4+ or CD8+, but not both. These two surface markers define subsets of T lymphocytes with significantly different effector functions. Notably, CD4+ T lymphocytes typically function as helper (designated TH) cells, providing “help” to other T lymphocytes as well as to immunoglobulin-producing B lymphocytes. On the other hand, CD8+ T cells exhibit cytotoxic /suppressor (Tc or Ts) activity. CD8 T lymphocytes also display cytotoxic activity toward numerous virally infected or tumor cells. It is not clear whether the suppressor and cytotoxic activities are mediated by the same or distinct subpopulations of CD8 T cells.

T-Lymphocyte ontogeny:

T cell development occurs in the thymus. T cell precursors however arrive to the thymus from the Bone Marrow. The stages of T cell development are identified by the expression of specific cell surface markers, such as TCR (T Cell Receptor), CD3 (which serves as the signal transduction component of TCR), and CD4/CD8. In the cortex of thymus within the cortical epithelium T-cells progenitors differentiate under the influence of thymic hormones (Thymosin and Thymopoietin) into T-cell sub population. All T-cells have CD-3 receptor on their surface in association with Antigen receptors. CD-3 is a complex of 5 trans-membranous proteins which takes information from outside to inside of the cell. Signal transduction is by Zeta-chain (transmembranous protein) which has tyrosin kinase activity.CD-4 has single trans-membranous polypeptide whereas CD-8 has 2 trans-membrane polypeptides. These may signal by tyrosine kinase.

T lymphocytes undergo differentiation in the thymus, irrespective of whether they express the CD4 or CD8 phenotype. The process of T lym­phocyte maturation begins with the migration of T-cell recursors from the bone marrow to the cor­tical regions of the thymus. It is thought that these cells are attracted to the thymus by chemical signals provided by thymic epithelial cells. The earliest recognizable thymocyte committed to the T cell lineage is the pro-T cell. These cells bear CD2 (the sheep erythrocyte receptor) but do not express the T cell antigen receptor. Pro-T cells are also surface-negative for CD4 and CD8 (Double Negative or DN cells).

Diagrammatic representation of T-cell development

T-cell-development

During the next phase of development, maturing cortical thymocytes proceed along one of two alternative pathways. In one instance, the thymocytes begin to rearrange DNA segments encoding for the variable and constant regions of an alternative form of the T-cell antigen receptor (designated λ / δ TCR), and express this receptor in conjunction with a tightly associated complex of five membrane glycoproteins that form the CD3 complex. Most cells expressing the λ / δ TCR fail to express either CD4 or CD8 during further development and  release into the peripheral circulation as “double-negative”  T lymphocytes. The precise function of these λ / δ TCR–expressing cells has not been defined. The majority of pro-T cells follow a different pathway of differentiation. These pre-T cells co-express both CD4 and CD8 ( Double positive or DP cells), but do not yet express an antigen receptor. Subsequently, these cells undergo DNA rearrangements of genes encoding the constant and variable regions of the α / β TCR, which is expressed by the majority of mature T lymphocytes in the periphery. At this stage, the thymocytes are CD4+ , CD8 +, and express the α / β TCR in conjunction with the CD3 complex. During the first stages of development, which occur in the thymic medulla, two important events take place called as thymic education.

Thymic education:

It is an important step in maturation of T-cells. First, the cells progressively lose either CD4 or CD8. Secondly, these cells are “educated” by thymic epithelial cells to learn to differentiate between “self’ and “nonself’-MHC gene products. Those cells, which are auto-reactive toward self-MHC mole‑cules, are eliminated by a process of clonal deletion, an important mechanism of self-tolerance. As a consequence of this selection process, only about 10 percent of the immature T cells which enter the thymus eventually reach the peripheral circulation.

Positive selection:

Since TCR’s recognize antigen only in the context of MHC’s, T cells must be tuned to recognize host MHC first. During positive selection Double-Positive T cells that can recognize self MHC’s are selected for proliferation, and those T cells that do not recognize self MHC die via apoptosis. Positive selection also assures that the right TCR selection will go with the appropriate CD4 or CD8. For example, TCR’s specific for MHC II need to retain CD4, and lose CD8. If the reverse occurs, they will die via apoptosis. The same is true for the T cells that are specific for MHC I, which need to retain CD8, and lose CD4.

T-cell differentiation and maturation

T-cell differentiation and maturation

Negative selection:

At this point, those T cells that are strongly activated by self  MHC plus self peptides need to be eliminated in the thymus. If they escape this elimination, they may subsequently react against self antigens, and cause autoimmune disease. 

In summary, Positive selection selects for those T cells that react with MHC: self antigen. Negative selection eliminates those that react strongly with MHC: self antigen. Thus, successful T cell differentiation selects for MHC restricted TCR’s with low affinity for self antigens. Cells that fall outside this range primarily die via apoptosis. The rationale here is that a T cell that binds weakly to self MHC/self Antigen will not be activated but will be activated by a stronger binding to self MHC/ foreign Antigen complex.

Mature T cells which survive this selection process leave the thymic medulla through the walls of postcapillary venules. After circulating for a time, these T lymphocytes distribute among various peripheral lymphoid tissues including thymus-dependent regions of the inner cortex of lymph nodes, the spleen, and mucosa-associated lymphoid tissue (e.g., Peyer’s patches in the colon). It is presently unknown what signal(s) drives proliferation and differentiation of immature thymocytes. These cells do express receptors for certain cytokines, which exhibit growth factor activity, including interleukin-2 (IL-2) and interleukin-7 produced by thymocytes and stromal cells, respectively. Alternatively, thymic epithelial cells may induce thymocyte activation via the CD2 molecule, which recognizes a cell adhesion molecule (LFA-3) expressed on the epithelial cells.

Naïve Th cell:

Naïve Th cell is a T-cell that has differentiated in bone marrow, and successfully undergone the positive and negative selection processes in the thymus. A naïve T-cell is considered to be mature and unlike activated T-cells or memory T-cells it has not encountered its cognate antigen within the periphery. Upon activation, naïve Th cells become Th0, since they have both Th1 and Th2 characteristics, with further stimulation Th0 cells deviate either towards Th1 or Th2. Th1 and Th2 cells are classified based on the pattern of cytokines that they secrete. If the Th cell secretes mainly IL2 and INF-γ, it is termed a Th1 cell. If the T cell secretes mainly IL4, IL10, and IL13 it is termed a Th2 cell. 

Natural killer (NK) cells:

Natural killer (NK) cells are granular lymphocytes which do not pass through thymus. They don’t have CD-4 or CD-8 proteins and an antigen receptor. They kill virus infected and tumor cells without requirement of antigen presentation or MHC proteins. NK cells are activated by IL -2.  Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in early defenses against both allogeneic (nonself) cells and autologous cells undergoing various forms of stress, such as infection with viruses, bacteria, or parasites or malignant transformation. Although NK cells do not express classical antigen receptors of the immunoglobulin gene family, such as the antibodies produced by B cells or the T cell receptor expressed by T cells, they are equipped with various receptors whose engagement allows them to discriminate between target and nontarget cells. Activating receptors bind ligands on the target cell surface and trigger NK cell activation and target cell lysis. However Inhibitory receptors recognize MHC class I molecules (HLA) and inhibit killing by NK cells by overruling the actions of the activating receptors. This inhibitory signal is lost when the target cells do not express MHC class I and perhaps also in cells infected with virus, which might inhibit MHC class I exprssion or alter its conformation. The mechanism of NK cell killing is the same as that used by the cytotoxic T cells generated in an adaptive immune response; cytotoxic granules are released onto the surface of the bound target cell, and the effector proteins they contain penetrate the cell membrane and induce programmed cell death.

Dendritic cells:

Dendritic cells (DCs) were originally identified by Steinman and his colleagues in 1972. They are potent antigen presenting cells (APCs) that possess the ability to stimulate naïve T cells. They comprise a system of leukocytes widely distributed in all tissues, especially in those that provide an environmental interface. DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens (Ags), and their subsequent presentation on the cell surface linked to major histocompatibility (MHC) molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues where they present Ag to T cells and induce an immune response.

Macrophages:

Macrophages are part of the innate immune response. Unlike T and B cells, they do not contain any specific receptors. Macrophages have important function of homeostasis as they continuously phagocytose self-proteins and cells in their vicinity, during normal tissue repair and aging (e.g. old red blood cells). All of these proteins are degraded and presented on MHC-II. As they are self-proteins so they do not activate T cells, because in the absence of infection, macrophages express low levels of MHC-II, and almost no co-stimulator (B7). Further, T cells with high affinity receptor for self-peptides have been deleted during T cell development in the thymus. 
When there is infection, macrophages posses certain types of receptors that recognize differential carbohydrate patterns on foreign cells. They also have receptors for specific bacterial products such as lipopolysaccharide (LPS) (endotoxin). When these molecules bind their bacterial ligands, macrophages become strong antigen presenting cells because of up regulation of MHC-II and B7. They also start to secrete cytokines that aid in their functions (IL-1, 6, 8, 12 and TNF-α). It is at this point that antigen presentation by MHC II will activate Th cells.

B-cells:

B lymphocytes are generated from the common lymphoid progenitors, which are originated from the differentiation of the haematopoietic stem cells in bone marrow. The yolk sac and  fetal liver are the sites in the body where these cells are generated and developed during earlydevelopment.

B lymphocytes play crucial roles in host defense against infection via a series of highly coordinated processes that include cell homing, antigen recognition, antibody secretion, antigen presentation, and/or cytokine release. The steps of maturation of B-cells are as follows:

In bone marrow in early stages, the B cell precursors (pre-proB cells) must interact physically with the stromal cells in order for proliferation and maturation to occur. Later stages (late pro-B cells) merely need the soluble growth factors produced by stromal cells. Stromal cells produce several necessary growth factors and cell-cell adhesion molecules. One key growth factor for B lymphopoiesis is interleukin-7.

The earliest identifiable stage of B cell differentiation is the pre-pro B cell. In this stage, the process of Ig gene rearrangement begins. The first rearrangement entails the joining of the D segment to the J segment of the Ig heavy chain gene (IgH). Subsequent rearrangements bring the V region juxtaposed to the DJ portion. After this, surrogate light chains can combine with the μ heavy chain protein in pro- and pre-B cells, forming a structure referred to as the pre-B cell receptor.

Diagrammatic representation of B-cell maturation

B-Cell maturaion

A newly formed B cell displays IgM on the cell surface. At this stage, the B cell is still immature and responds to antigen differently from a mature B cell. Immature B cells can be functionally removed by interaction with self antigen, either by undergoing programmed cell death (apoptosis) or by anergy, in which the cell is rendered nonresonsive in the presence of the antigen. Thus, similar to T cells, immature B lymphocyte undergo a process of “negative selection” to delete cells that are reactive to “self” antigen.

Immature B cells that are not removed by the processes of negative selection leave the bone marrow and migrate to peripheral or secondary lymphoid tissues such as the spleen and lymph nodes. Here further maturation takes place and the newly formed B cells express IgD in addition to IgM, on the cell surface. The mature B cells are now fully responsive to antigens and interaction with T cells.

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Tumor Immunity:

In animal studies it has been shown that they develop resistance to the chemically or virally induced tumors. This is because during tumor formation new antigens called as tumor associated antigens (TAA) develop on the surface of cells which are recognized as foreign or ‘non self’. Chemically induced tumors have TAA’s which are specific for a particular tumor and differ from other tumor. Virally induced TAA’s are non specific for a tumor and cross react with two or more tumor induced by same virus. TAA’s induce on cell surface by different viral cells do not cross react.  Immune surveillance detects the newly arising clones of malignant cells. This immune response is weak and can be overcome by large dose of tumor cells. Tumor cells can escape surveillance by modulation i.e. Internalization of surface antigen’s so that they no longer present to immune attack. Immune response against tumor include (in vivo unkown)

  1. NK cells- which act without antibodies.
  2. Killer cell which mediate antibody dependent cytolysis
  3. Cytotoxic (CD8)T-cells
  4. Activated macrophages.

Two types of antibodies are produced against tumor antigens; cytotoxic & blocking antibodies. The blocking antibodies induce tumor growth as these hinder the reorganization of tumor antigen by antibodies. One interferon TNF-α is found effective against many solid tumors. In addition lymphocytes activated by IL-2(Lymphokines activated cells)i.e. LAK cells are useful in cancer immunotherapy.

TIL-Tumor infiltrating lymphocytes:

Another cancer immunotherapy involves use of TIL- Tumor infiltrating lymphocytes. Basis of this therapy is that some cancers are infiltrated by NK cells and cytotoxic T-cells that seem to destroy tumor cells. The lymphocytes are surgically removed from cancer and grown in cultures until large number of cells are produced and activated with IL-2. These are then injected in same patient.

Some tumors contain Antigen’s that normally occur in fetal but not in adult cells

  • Carcinoembryonic antigen: It is elevated in serum of patient’s with carcinoma colon, pancreas, breast & liver. It is found in gut, liver, pancreas or fetus in small quantities. This antigen is not helpful in diagnosis but it is helpful in management of tumor. If its level decline after surgery, means that tumor is not spreading but elevated suggest recurrence.
  • α-Fetoprotein: Elevated in patients of hepatoma. It is found in fetal liver and can rise in many other specific malignant or non malignant diseases.

 

References:

  1. Immunology: Understanding The Immune System. By Klaus D. Elgert
  2. Immunology: A Short Course. By Richard Coico, Geoffrey Sunshine.
  3. Immunology.  By David K. Male.
  4. Immunology: Essential And Fundamental. By Sulabha Pathak, Urmi Palan.

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