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Lymphocytes are one of the five kinds of white blood cells (leukocytes) that circulate in the blood. Although mature lymphocytes all look pretty much alike, they are extraordinarily diverse in their functions. The most abundant lymphocytes are B lymphocytes (B cells) and T lymphocytes (T cells). B cells are produced in the bone marrow. The precursors of T cells are also produced in the bone marrow but leave the bone marrow and mature in the thymus (which accounts for their designation). Each B cell and T cell is specific for a particular antigen. What this means is that each is able to bind to a particular molecular structure. The specificity of binding resides in a receptor for antigen: the B cell receptor (BCR) for antigen and the T cell receptor (TCR) respectively.
Both BCRs and TCRs share these properties:
- They are integral membrane proteins.
- They are present in thousands of identical copies exposed at the cell surface.
- They are made before the cell ever encounters an antigen.
- They are encoded by genes assembled by the recombination of segments of DNA.
- They have a unique binding site.
- This site binds to a portion of the antigen called an antigenic determinant or epitope.
- The binding, like that between an enzyme and its substrate depends on complementarity of the surface of the receptor and the surface of the epitope.
- The binding occurs by non-covalent forces (again, like an enzyme binding to its substrate).
- Successful binding of the antigen receptor to the epitope, if accompanied by additional signals, results in:
- stimulation of the cell to leave G0 and enter the cell cycle.
- Repeated mitosis leads to the development of a clone of cells bearing the same antigen receptor; that is, a clone of cells of the identical specificity.
BCRs and TCRs differ in their structure, the genes that encode them and the type of epitope to which they bind.
Fig.184.108.40.206 B cell and lymphokines
- BCRs bind intact antigens (like diphtheria toxoid, the protein introduced into your body in the DTP vaccine). These may be
- soluble molecules present in the extracellular fluid
- intact molecules that the B cell plucks from the surface of antigen-presenting cells like macrophages and dendritic cells
- The bound antigen molecules are engulfed into the B cell by receptor-mediated endocytosis.
- The antigen is digested into fragments which are then displayed at the cell surface nestled inside a class II histocompatibility molecule.
- Helper T cells specific for this structure (i.e., with complementary TCRs) bind the B cell and secrete lymphokines that:
- stimulate the B cell to enter the cell cycle and develop, by repeated mitosis, into a clone of cells with identical BCRs
- switch from synthesizing their BCRs as integral membrane proteins to a soluble version
- differentiate into plasma cells that secrete these soluble BCRs, which we now call antibodies
The surface of each T cell also displays thousands of identical T cell receptors (TCRs). There are two types of T cells that differ in their TCR:
- alpha/beta (αβ) T cells. Their TCR is a heterodimer of an alpha chain with a beta chain. Each chain has a variable (V) region and a constant (C) region. The V regions each contain 3 hypervariable regions that make up the antigen-binding site.
- gamma/delta (γδ) T cells. Their TCR is also a heterodimer of a gamma chain paired with a delta chain.
The discussion that follows now concerns alpha/beta T cells. Gamma/delta T cells, which are less well understood, are discussed at the end.
The TCR (of alpha/beta T cells) binds a bimolecular complex displayed at the surface of some other cell called an antigen-presenting cell (APC). This complex consists of a fragment of an antigen lying within the groove of a histocompatibility molecule. The complex has been compared to a "hot dog in a bun".
Most of the T cells in the body belong to one of two subsets. These are distinguished by the presence on their surface of one or the other of two glycoproteins designated:
Which of these molecules is present determines what types of cells the T cell can bind to.
- CD8+ T cells bind epitopes that are part of class I histocompatibility molecules. Almost all the cells of the body express class I molecules.
- CD4+ T cells bind epitopes that are part of class II histocompatibility molecules. Only specialized antigen-presenting cells express class II molecules. These include dendritic cells, phagocytic cells like macrophages and B cells.
CD8+ T cells
The best understood CD8+ T cells are cytotoxic T lymphocytes (CTLs). They secrete molecules that destroy the cell to which they have bound.
This is a very useful function if the target cell is infected with a virus because the cell is usually destroyed before it can release a fresh crop of viruses able to infect other cells. An example will show the beauty and biological efficiency of this mechanism.
Every time you get a virus infection, say influenza (flu), the virus invades certain cells of your body (in this case cells of the respiratory passages). Once inside, the virus subverts the metabolism of the cell to make more virus. This involves synthesizing a number of different macromolecules encoded by the viral genome. In due course, these are assembled into a fresh crop of virus particles that leave the cell (often killing it in the process) and spread to new target cells. Except while in transit from their old homes to their new, the viruses work inside of your cells safe from any antibodies that might be present in blood, lymph, and secretions. But early in the process, infected cells display fragments of the viral proteins in their surface class I molecules. CTLs specific for that antigen will be able to bind to the infected cell and often will be able to destroy it before it can release a fresh crop of viruses.
In general, the role of the CD8+ T cells is to monitor all the cells of the body, ready to destroy any that express foreign antigen fragments in their class I molecules.
CD4+ T cells
CD4+ T cells bind an epitope consisting of an antigen fragment lying in the groove of a class II histocompatibility molecule. CD4+ T cells are essential for both the cell-mediated and antibody-mediated branches of the immune system:
- cell-mediated immunity: These CD4+ cells bind to antigen presented by antigen-presenting cells (APCs) like phagocytic macrophages and dendritic cells. The T cells then release lymphokines that attract other cells to the area. The result is inflammation: the accumulation of cells and molecules that attempt to wall off and destroy the antigenic material (an abscess is one example, the rash following exposure to poison ivy is another).
- antibody-mediated immunity: These CD4+ cells, called helper T cells, bind to antigen presented by B cells. The result is the development of clones of plasma cells secreting antibodies against the antigenic material.
AIDS provides a vivid illustration of the importance of CD4+ T cells in immunity. The human immunodeficiency virus (HIV) binds to CD4 molecules and thus is able to invade and infect CD4+ T cells. As the disease progresses, the number of CD4+ T cells declines below its normal level of about 1000 per microliter (µl). A partial explanation for this may be the unceasing efforts of the patient's CD8+ T cells to destroy the infected CD4+ cells. However, it turns out that only a small fraction of the patients CD4+ T cells are infected at any given time. How uninfected CD4+ cells may be induced to commit suicide is discussed in the page on apoptosis.
When the number of CD4+ T cells drops below 400 per microliter, the ability of the patient to mount an immune response declines dangerously. Not only does the patient become hypersusceptible to pathogens that give all of us grief but also to microorganisms, especially viruses and fungi, that normally inhabit our tissues without harming us. These opportunistic infections can be fatal.
Building the T-cell Repertoire
T cells have receptors (TCRs) that bind to antigen fragments nestled in MHC molecules. But all cells express class I MHC molecules containing fragments derived from self proteins. Many cells express class II MHC molecules that also contain self peptides. This presents a risk to the animal of the T cells recognizing these self-peptide/self-MHC complexes and mounting an autoimmune attack against them. Fortunately, this is usually avoided by a process of selection that goes on in the thymus (where all T cells develop).
The process works like this:
- The precursors of T cells like all blood cells are formed in the bone marrow.
- These cells then migrate to the cortex of the thymus. At this time they have neither a complete TCR nor either CD4 or CD8 (thus are called "double-negative" or DN cells).
- In the cortex of the thymus they begin to form a TCR and synthesize both CD4 and CD8 (so now they are "double-positive" or DP cells). The cortical cells of the thymus express a wide variety of small molecules, usually a peptide of 6–8 amino acids derived from body proteins; that is, "self" proteins such as proteins within the cytosol and serum proteins - proteins circulating in the blood and lymph nestled in a histocompatibility molecule (encoded by the MHC).
- Most of the cells (~97%) will produce a TCR that does not bind to any of the peptide-MHC molecules present on the surface of the cortical cells. Unless they can try again with a new TCR, these cells die by "neglect" (by apoptosis, actually).
- Those remaining cells whose TCR has bound a peptide antigen presented in class II MHC molecule stop expressing CD8 and become CD4+ T cells. It is these cells that will go on to become
- Th1 cells in cell-mediated immune responses
- Th1 helper cells for cytotoxic T lymphocytes (CTLs)
- Th2 helper cells for B cells
- Those remaining cells whose TCR has bound a peptide antigen presented in class I MHC molecule stop expressing CD4 and become CD8+ T cells.
- Both sets of cells are said to have undergone positive selection.
- After positive selection, these cells migrate to the medulla of the thymus.
- There those cells whose TCR binds very strongly to complexes of self-peptide and self-MHC are destroyed (again by apoptosis).
- This process of negative selection is important as it eliminates cells that might otherwise mount an autoimmune attack. It is one of the ways in which tolerance to self antigens is achieved. [Link to discussion of T-cell tolerance.]
- The cells whose TCRs bind antigen at an affinity below the threshold that triggers apoptosis are free to leave the thymus and migrate throughout the immune system (lymph nodes, spleen, etc.)
- It is this population that we depend on to mount immune responses against foreign antigens. A TCR that binds self-peptide/self-MHC with low affinity may well bind a foreign-peptide in self MHC with high affinity.
Gamma/Delta (γδ) T Cells
Gamma/delta T cells differ from their alpha/beta cousins in several ways:
- Their TCR is encoded by different gene segments.
- Their TCR binds to
- antigens that can be intact proteins (just as antibodies do) as well as a variety of other types of organic molecules (often containing phosphorus atoms).
- antigens that are not "presented" within class I or class II histocompatibility molecules;
- antigens that are not presented by "professional" antigen-presenting cells (APCs) like dendritic cells.
- Most of these T cells have neither CD8 nor CD4 on their surface. This makes sense because they have no need to recognize class I and class II histocompatibility molecules.
- Gamma/Delta T cells, like alpha/beta T cells, develop in the thymus. However, they migrate from there into body tissues, especially epithelia (e.g., intestine, skin, lining of the vagina), and don't recirculate between blood and lymph nodes (they represent no more than 5% of the T cells in the blood and are even rarer in lymph nodes). They encounter antigens on the surface of the epithelial cells that surround them rather than relying on the APCs found in lymph nodes.
What is the Function of γδ T cells?
That is still something of a mystery. Situated as they are at the interfaces between the external and internal worlds, they may represent a first line of defense against invading pathogens. Their response does seem to be quicker than that of αβ T cells. Curiously, many of the antigens to which γδ T cells respond are found not only on certain types of invaders (e.g., Mycobacterium tuberculosis, the agent of tuberculosis) but also on host cells that are under attack by pathogens.
Knockout mice that cannot make γδ T cells are slower to heal injuries to their skin. They are also much more susceptible to skin cancers than normal mice. Perhaps immune surveillance is one of the functions of γδ T cells.
Early B cell development and commitment to the B cell lineage occurs in the foetal liver prenatally, before continuing in the bone marrow throughout life. B cells are at the centre of the adaptive humoral immune system and are responsible for mediating the production of antigen-specific immunoglobulin (Ig) directed against invasive pathogens (typically known as antibodies). The function of B cells was discovered in the 1960s by Max Cooper who demonstrated that antibody production was completely abrogated in irradiated chickens after surgical removal of the Bursa of Fabricius (the primary site of B-cell development in birds) from which the notation ‘B’ cell was derived. Several discrete B-cell subsets have been defined that possess distinct functions in both adaptive and innate humoral immune responses.B-cell development and B-cell subsets (Rebecca Newman)
Immunoglobulins consist of two identical heavy and light chains, which are joined by disulphide bonds. During B cell development, rearrangement of the Ig heavy chain occurs first, commencing with D-J recombination, which takes place in the common lymphoid progenitors (CLPs) and pre-pro B cells. This is followed by V-DJ recombination yielding a functional heavy chain protein (Ig) in large pre-B cells. The recombined heavy chain then associates with the surrogate light chains and the Ig/ dimer to form the pre-B cell receptor (pre-BCR) which is expressed on the cell surface. Signalling through the pre-BCR drives intense proliferation and differentiation into the small pre-B cell stage. Quiescent small pre-B cells then undergo V-J rearrangement of the Ig light chain, allowing the production of a complete functional BCR with a unique specificity that is expressed as IgM on the surface of immature B cells. In a bid to prevent autoreactivity, immature B cells which encounter Ag capable of cross-linking their newly expressed BCRs are eliminated by a variety of mechanisms. After production in the BM, immature surface IgM+ B cells migrate to the spleen where they differentiate through distinct transitional B cell stages termed T1 and T2, before differentiating into long-lived mature follicular (FO) or marginal zone (MZ) B cells. Thus, B cells experience both antigen-dependent and -independent phases of selection, which are tightly regulated through signalling events. T3 B cells do not give rise to mature B cells, but instead represent a subset of anergic B cells which have been selected away from the B cell developmental pathway.
In addition to FO and MZ B cells, a third population of mature B cells exists known as B1 cells. B1 cells are located in a number of tissues including the spleen, intestine, the peritoneal cavity and pleural cavities. B1 cells have distinct haematopoietic origins in the in the fetal liver, and the initial wave of lymphopoiesis in the embryo appears to be skewed towards B1 B cell development.
B cell responses to antigen
Mature FO B cells recirculate between secondary lymphoid organs in search of antigen. Following cognate antigen encounter, B cells receiving T cell help can enter a couple of different developmental possibilities. Firstly, the cells can undergo plasmacytic differentiation, form extrafollicular plasmablasts and form IgM secreting plasma cells. These cells do not have somatically mutated Ig genes and are short-lived but provide a rapid initial response to antigen. The second developmental possibility is the establishment of a germinal centre, a specialised structure within which B cells undergo rounds of proliferation accompanied by affinity maturation: an iterative process of Ig gene mutation and selection resulting in a B cell pool which can bind to antigen with the highest affinity. The cells also undergo class-switch recombination. Immunoglobulin class switching to IgG, IgA and IgE is a major mechanism to diversify B cell responses, and match antibody function to immune challenge. Memory B cells and plasma cells expressing somatically mutated and generally high affinity BCRs of switched isotypes exit the GC.
Normal bone marrow, blood, and lymph tissue
To understand leukemia, it helps to know about the blood and lymph systems.
Bone marrow is the soft inner part of certain bones. It is made up of blood-forming cells, fat cells, and supporting tissues. A small fraction of the blood-forming cells are blood stem cells.
Inside the bone marrow, blood stem cells go through a series of changes to make new blood cells. During this process, the cells develop into 1 of the 3 main types of blood cell components:
Red blood cells
Red blood cells (RBCs) carry oxygen from the lungs to all other tissues in the body, and take carbon dioxide back to the lungs to be removed.
Platelets are actually cell fragments made by a type of bone marrow cell called a megakaryocyte. Platelets are important in plugging up holes in blood vessels caused by cuts or bruises.
White blood cells
White blood cells (WBCs) help the body fight infections. The main types of WBCs include lymphocytes, granulocytes, and monocytes.
Lymphocytes are the main cells that make up lymph tissue, a major part of the immune system. Lymph tissue is found in lymph nodes, the thymus, the spleen, the tonsils and adenoids, and is scattered throughout the digestive and respiratory systems and the bone marrow.
Lymphocytes develop from cells called lymphoblasts to become mature, infection-fighting cells. There are 2 main types of lymphocytes:
- B lymphocytes (B cells): B cells help protect the body by making proteins called antibodies. The antibodies attach to germs (bacteria, viruses, and fungi) in the body, which helps the immune system destroy them.
- T lymphocytes (T cells): There are several types of T cells, each with a special job. Some T cells can destroy germs directly, while others play a role in either boosting or slowing the activity of other immune system cells.
ALL develops from early forms of lymphocytes. It can start in either early B cells or T cells at different stages of maturity. This is discussed in Acute Lymphocytic Leukemia (ALL) Subtypes and Prognostic Factors.
Granulocytes are WBCs that have granules in them, which are spots that can be seen under the microscope. These granules contain enzymes and other substances that can destroy germs, such as bacteria. The 3 types of granulocytes – neutrophils, basophils, and eosinophils – are distinguished by the size and color of their granules.
Monocytes also help protect the body against bacteria. After circulating in the bloodstream for about a day, monocytes enter body tissues to become macrophages, which can destroy some germs by surrounding and digesting them.
15.4C: B Cells and T Cells - Biology
Physical and Chemical Barriers (Innate Immunity)
- The skin has thick layer of dead cells in the epidermis which provides a physical barrier. Periodic shedding of the epidermis removes microbes.
- The mucous membranes produce mucus that trap microbes.
- Hair within the nose filters air containing microbes, dust, pollutants
- Cilia lines the upper respiratory tract traps and propels inhaled debris to throat
- Urine flushes microbes out of the urethra
- Defecation and vomiting -expel microorganisms.
- Lysozyme, an enzyme produced in tears, perspiration, and saliva can break down cell walls and thus acts as an antibiotic (kills bacteria)
- Gastric juice in the stomach destroys bacteria and most toxins because the gastric juice is highly acidic (pH 2-3)
- Saliva dilutes the number of microorganisms and washes the teeth and mouth
- Acidity on skin inhibit bacterial growth
- Sebum (unsaturated fatty acids) provides a protective film on the skin and inhibits growth
- Hyaluronic acid is a gelatinous substance that slows the spread of noxious agents
Nonspecific Resistance (Innate Immunity)
- Phagocytic cells ingest and destroy all microbes that pass into body tissues. For example macrophages are cells derived from monocytes (a type of white blood cell). Macrophages leave the bloodstream and enter body tissues to patrol for pathogens. When the macrophage encounters a microbe, this is what happens:
- The microbe attaches to the phagocyte.
- The phagocyte's plasma membrane extends and surrounds the microbe and takes the microbe into the cell in a vesicle.
- The vesicle merges with a lysosome, which contains digestive enzymes.
- The digestive enzymes begin to break down the microbe. The phagocyte uses any nutrients it can and leaves the rest as indigestible material and antigenic fragments within the vesicle.
- The phagocyte makes protein markers, and they enter the vesicle.
- The indigestible material is removed by exocytosis.
- The antigenic fragments bind to the protein marker and are displayed on the plasma membrane surface. The macrophage then secretes interleukin-1 which activates the T cells to secrete interleukin 2, as described below under specific resistance .
- Inflammation is a localized tissue response that occurs when your tissues are damaged and in response to other stimuli. Inflammation brings more white blood cells to the site where the microbes have invaded. The inflammatory response produces swelling, redness, heat, pain
- Fever inhibits bacterial growth and increases the rate of tissue repair during an infection.
Specific Resistance (Acquired Immunity)
- When an antigen is detected by a macrophage (as describe above under phagocytosis), this causes the T-cells to become activated.
The activation of T-cells by a specific antigen is called cell-mediated immunity. The body contains millions of different T-cells, each able to respond to one specific antigen.
- The T-cells secrete interleukin 2. Interleukin 2 causes the proliferation of certain cytotoxic T cells and B cells.
- From here, the immune response follows 2 paths: one path uses cytotoxic T cells and the other uses B cells.
- The cytotoxic T cells are capable of recognizing antigens on the surface of infected body cells.
- The cytotoxic T cells bind to the infected cells and secrete cytotoxins that induce apoptosis (cell suicide) in the infected cell and perforins that cause perforations in the infected cells.
- Both of these mechanisms destroys the pathogen in the infected body cell.
Click here for an animation on cytotoxic T cells.
The animation is followed by practice questions. Click here for even more practice questions.
Activation of a helper T cell and its roles in immunity:
T Cell Pathway
- T-cells can either directly destroy the microbes or use chemical secretions to destroy them.
- At the same time, T cells stimulate B cells to divide, forming plasma cells that are able to produce antibodies and memory B cells.
- If the same antigen enters the body later, the memory B cells divide to make more plasma cells and memory cells that can protect against future attacks by the same antigen.
- When the T cells activate (stimulate) the B cells to divide into plasma cells, this is called antibody-mediated immunity.
Click here for an animation on the immune response.
The animation is followed by practice questions.
There are 3 major types of T cells:
These cells secrete interleukin 2 (I-2) which stimulates cell division of T cells and B cells. In other words, these cells recruit even more cells to help fight the pathogen.
These cells remain dormant after the initial exposure to an antigen. If the same antigen presents itself again, even if it is years later, the memory cells are stimulated to convert themselves into cytotoxic T cells and help fight the pathogen.
This material is based upon work supported by the Nursing, Allied Health and Other Health-related Educational Grant Program, a grant program funded with proceeds of the State&rsquos Tobacco Lawsuit Settlement and administered by the Texas Higher Education Coordinating Board.
What are monoclonal antibodies?
Antibodies have become the basis for some of the most useful medicines, as well as some of the most powerful lab techniques in biology, Goodman said. One of these clinical and therapeutic superstars is what's known as a monoclonal antibody.
To create a monoclonal antibody, researchers vaccinate an animal (or possibly a human) to stimulate the production of antibodies against a particular substance. The body will gradually make antibodies that are more and more effective against that antigen. These antibody-producing cells are then filtered out of white blood cells and put into a dish to see which cells bind the antigen best, Goodman said. The cell that binds the best is then isolated — it is an antibody-producing factory, specifically honed to churn out one super-selective antibody.
From there, that cell is fused to a blood cancer cell, producing something called a hybridoma. This hybridoma, or monoclone, is an inexhaustible generator of exactly the same antibody, over and over and over. (Researchers bind the monoclonal cell to a cancer cell because cancer just continues to reproduce.)
"It just produces and produces and produces, and it will never stop, and it's a cancer, so it's essentially immortal," Goodman said. What it produces is a monoclonal antibody.
Such cell lines have an incredibly diverse range of uses. There are millions of commercial monoclonal antibodies, which are used in labs to tag the tiniest, most specific cellular targets for study, Goodman said.
"They're incredible, they're amazingly precise tools," Goodman said.
Monoclonal antibodies also form the basis for many blockbuster drugs. For instance, the drug adalimumab (brand name Humira), is a monoclonal antibody that treats rheumatoid arthritis by inhibiting an inflammatory protein known as a cytokine. Another, called bevacizumab (Avastin), targets a molecule that fuels blood vessel growth by blocking this molecule, bevacizumab can slow the growth of lung, colon, kidney and some brain cancers.
And in the SARS-CoV-2 pandemic, doctors around the world are racing to create monoclonal antibodies that will hopefully neutralize the new coronavirus, Greene said. These antibodies are filtered from the plasma of people who have recovered from COVID-19 (also called convalescent serum). The hope is that by isolating the most effective antibodies, and then producing them en-masse, doctors can create a treatment that provides a temporary, "passive" immunity until the body can catch up and mount an effective, more long-lasting response on its own, Greene said.
By contrast, polyclonal antibodies are derived from multiple B-cells. Polyclonal antibodies are a library of antibodies that all bind to slightly different parts of the antigen, or target. Polyclonal antibodies are typically produced by injecting an animal with the antigen, stimulating an immune response, and then extracting the animals' plasma to produce antibodies en masse, according to a 2005 study in the Institute for Laboratory Animal Research (ILAR) journal.
Unlike monoclonal antibodies, which can take up to 6 months to produce, polyclonal antibodies can be made in 4 to 8 weeks, and require less technical expertise. In addition, for certain types of tests where you are trying to detect the antigen, polyclonal antibodies might have a better chance of binding to the target antigen, making them potentially more sensitive. The downside of polyclonal antibodies is that, because each individual animal might produce a different array of antibodies, making polyclonal antibodies that are consistent from batch to batch can be more challenging, and it isn't as easy to have a large supply, according to a 2005 study in the journal Biotechniques.
Cells of the Immune System
This tutorial provides an overview of the immune system, concentrating on the roles played by B and T lymphocytes, and on the antigen-presentation system.
All animals possess a nonspecific defense system called the innate immune system, which includes macrophages in mammals. Vertebrates have an additional powerful immune response called adaptive immunity. This Click & Learn describes key elements of the adaptive immune system, including B cells and antibody molecules, helper T cells and cytotoxic T cells, and antigen presentation.
The accompanying worksheet guides students’ exploration.
Student Learning Targets
Describe the roles different immune cells play in defending the human body from infection.
Types - Sickle Cell Disease
People who have sickle cell disease have abnormal hemoglobin, called hemoglobin S or sickle hemoglobin, in their red blood cells . Hemoglobin is a protein in red blood cells that carries oxygen throughout the body. People who have sickle cell disease inherit two abnormal hemoglobin genes , one from each parent.
The types of sickle cell disease include the following:
- Hemoglobin Sβ0 thalassemia
- Hemoglobin Sβ+ thalassemia
- Hemoglobin SC
- Hemoglobin SD
- Hemoglobin SE
- Hemoglobin SS
In all types of sickle cell disease, at least one of the two abnormal genes causes a person’s body to make hemoglobin S. When a person has two hemoglobin S genes (hemoglobin SS), the disease is called sickle cell anemia. This is the most common and often most severe type of sickle cell disease. Hemoglobin SC disease and hemoglobin Sβ thalassemia are two other common types of sickle cell disease. Hemoglobin SD and hemoglobin SE are much less common.
15.4C: B Cells and T Cells - Biology
Large quantities of water molecules constantly move across cell membranes by simple diffusion, often facilitated by movement through membrane proteins, including aquaporins. In general, net movement of water into or out of cells is negligible. For example, it has been estimated that an amount of water equivalent to roughly 100 times the volume of the cell diffuses across the red blood cell membrane every second the cell doesn't lose or gain water because equal amounts go in and out.
There are, however, many cases in which net flow of water occurs across cell membranes and sheets of cells. An example of great importance to you is the secretion of and absorption of water in your small intestine. In such situations, water still moves across membranes by simple diffusion, but the process is important enough to warrant a distinct name - osmosis.
Osmosis and Net Movement of Water
Osmosis is the net movement of water across a selectively permeable membrane driven by a difference in solute concentrations on the two sides of the membrane. A selectively permiable membrane is one that allows unrestricted passage of water, but not solute molecules or ions.
Different concentrations of solute molecules leads to different concentrations of free water molecules on either side of the membrane. On the side of the membrane with higher free water concentration (i.e. a lower concentration of solute), more water molecules will strike the pores in the membrane in a give interval of time. More strikes equates to more molecules passing through the pores, which in turn results in net diffusion of water from the compartment with high concentration of free water to that with low concentration of free water.
The key to remember about osmosis is that water flows from the solution with the lower solute concentration into the solution with higher solute concentration. This means that water flows in response to differences in molarity across a membrane. The size of the solute particles does not influence osmosis . Equilibrium is reached once sufficient water has moved to equalize the solute concentration on both sides of the membrane, and at that point, net flow of water ceases. Here is a simple example to illustrate these principles:
|Two containers of equal volume are separated by a membrane that allows free passage of water, but totally restricts passage of solute molecules. Solution A has 3 molecules of the protein albumin (molecular weight 66,000) and Solution B contains 15 molecules of glucose (molecular weight 180). Into which compartment will water flow, or will there be no net movement of water? [ answer ]|
Additional examples are provided on how to determine which direction water will flow in different circumstances.
When thinking about osmosis, we are always comparing solute concentrations between two solutions, and some standard terminology is commonly used to describe these differences:
- Isotonic: The solutions being compared have equal concentration of solutes.
- Hypertonic: The solution with the higher concentration of solutes.
- Hypotonic: The solution with the lower concentration of solutes.
In the examples above, Solutions A and B are isotonic (with each other), Solutions A and B are both hypertonic compared to Solution C, and Solution C is hypotonic relative to Solutions A and B.
Diffusion of water across a membrane generates a pressure called osmotic pressure. If the pressure in the compartment into which water is flowing is raised to the equivalent of the osmotic pressure, movement of water will stop. This pressure is often called hydrostatic ('water-stopping') pressure . The term osmolarity is used to describe the number of solute particles in a volume of fluid. Osmoles are used to describe the concentration in terms of number of particles - a 1 osmolar solution contains 1 mole of osmotically-active particles (molecules and ions) per liter.
The classic demonstration of osmosis and osmotic pressure is to immerse red blood cells in solutions of varying osmolarity and watch what happens. Blood serum is isotonic with respect to the cytoplasm, and red cells in that solution assume the shape of a biconcave disk. To prepare the images shown below, red cells from your intrepid author were suspended in three types of solutions:
- Isotonic - the cells were diluted in serum: Note the beautiful biconcave shape of the cells as they circulate in blood.
- Hypotonic - the cells in serum were diluted in water: At 200 milliosmols (mOs), the cells are visibly swollen and have lost their biconcave shape, and at 100 mOs, most have swollen so much that they have ruptured, leaving what are called red blood cell ghosts. In a hypotonic solution, water rushes into cells.
- Hypertonic - A concentrated solution of NaCl was mixed with the cells and serum to increase osmolarity: At 400 mOs and especially at 500 mOs, water has flowed out of the cells, causing them to collapse and assume the spiky appearance you see.
Predict what would happen if you mixed sufficient water with the 500 mOs sample shown above to reduce its osmolarity to about 300 mOs.
Calculating Osmotic and Hydrostatic Pressure
The flow of water across a membrane in response to differing concentrations of solutes on either side - osmosis - generates a pressure across the membrane called osmotic pressure. Osmotic pressure is defined as the hydrostatic pressure required to stop the flow of water, and thus, osmotic and hydrostatic pressures are, for all intents and purposes, equivalent. The membrane being referred to here can be an artifical lipid bilayer, a plasma membrane or a layer of cells.
The osmotic pressure P of a dilute solution is approximated by the following:
where R is the gas constant (0.082 liter-atmosphere/degree-mole), T is the absolute temperature, and C1 . Cn are the molar concentrations of all solutes (ions and molecules).
Similarly, the osmotic pressure across of membrane separating two solutions is:
where &DeltaC is the difference in solute concentration between the two solutions. Thus, if the membrane is permeable to water and not solutes, osmotic pressure is proportional to the difference in solute concentration across the membrane (the proportionality factor is RT).
Transplant problems that may show up later
The type of problems that can happen after a transplant depend on many factors, such as the type of transplant done, the pre-transplant chemo or radiation treatment used, the patient’s overall health, the patient’s age when the transplant was done, the length and degree of immune system suppression, and whether chronic graft-versus-host-disease (GVHD) is present and how bad it is. The problems can be caused by the conditioning treatment (the pre-transplant chemotherapy and radiation therapy), especially total body irradiation, or by other drugs used during transplant (such as the drugs that may be needed to suppress the immune system after transplant). Possible long-term risks of transplant include:
- Organ damage
- Relapse (the cancer comes back)
- Secondary (new) cancers
- Abnormal growth of lymph tissues
- Infertility (the inability to produce children)
- Hormone changes, such as changes in the thyroid or pituitary gland
- Cataracts (clouding of the lens of the eye, which causes vision loss)
The medicines used in transplants can harm the body’s organs, such as the heart, lungs, kidneys, liver, bones/joints, and nervous system. You may need careful follow-up with close monitoring and treatment of the long-term organ problems that the transplant can cause. Some of these, like infertility, should be discussed before the transplant, so you can prepare for them.
It’s important to find and quickly treat any long-term problems. Tell your doctor right away if you notice any changes or problems. Physical exams by your doctor, blood work, imaging tests, lung/breathing studies, and other tests will help look for and keep tabs on organ problems.
As transplant methods have improved, more people are living longer and doctors are learning more about the long-term results of stem cell transplant. Researchers continue to look for better ways to care for these survivors to give them the best possible quality of life.
Cancer that comes back
The goal of a stem cell transplant in cancer is to prolong life and, in many cases, even cure the cancer. But in some cases, the cancer comes back (sometimes called relapse or recurrence depending on when it might occur after a transplant). Relapse or recurrence can happen a few months to a few years after transplant. It happens much more rarely 5 or more years after transplant.
If cancer comes back, treatment options are often quite limited. A lot depends on your overall health at that point, and whether the type of cancer you have responds well to drug treatment. Treatment for those who are otherwise healthy and strong may include chemotherapy or targeted therapy. Some patients who have had allogeneic transplants may be helped by getting white blood cells from the same donor (this is called donor lymphocyte infusion) to boost the graft-versus-cancer effect. Sometimes a second transplant is possible. But most of these treatments pose serious risks even to healthier patients, so those who are frail, older, or have chronic health problems are often unable to have them.
Other options may include palliative (comfort) care, or a clinical trial of an investigational treatment. It’s important to know what the expected outcome of any further treatment might be, so talk with your doctor about the purpose of the treatment. Be sure you understand the benefits and risks before you decide.
Second cancers (new cancers caused by treatment)
Along with the possibility of the original cancer coming back (relapse) after it was treated with a stem cell transplant, there is also a chance of having a second cancer after transplant. Studies have shown that people who have had allogeneic transplants have a higher risk of second cancer than people who got a different type of stem cell transplant.
A cancer called post-transplant lymphoproliferative disease (PTLD), if it occurs, usually develops within the first year after the transplant. Other conditions and cancers that can happen are solid tumor cancers in different organs, leukemia, and myelodysplastic syndromes. These other conditions, if they occur, tend to develop a few years or longer after the transplant.
Risk factors for developing a second cancer are being studied and may include:
- Radiation (such as total body irradiation) and high-dose chemo as part of the conditioning treatment
- Previous chemo or radiation treatment that was not part of the transplant process the younger a person is when radiation is given, the more that person is at risk for certain types of cancer.
- Immune system problems (such as graft-versus-host disease, HLA-mismatched allogeneic transplant, and immunosuppressant therapy)
- Infection with viruses such as Epstein-Barr (EBV), cytomegalovirus (CMV), hepatitis B (HBV), or hepatitis C (HCV)
- The type of cancer you received the transplant for: for people who had their transplant when younger than 30 years old, those who had certain leukemias had a higher risk of having another cancer than people who did not have these leukemias.
Successfully treating a first cancer gives a second cancer time (and the chance) to develop. No matter what type of cancer is treated, and even without the high doses used for transplant, treatments like radiation and chemo can lead to a second cancer in the future.
Post-transplant lymphoproliferative disorder
Post-transplant lymphoproliferative disorder (PTLD) is an out-of-control growth of lymph cells, actually a type of lymphoma, that can develop after an allogeneic stem cell transplant. It’s linked to T-cells (a type of white blood cell that is part of the immune system) and the presence of Epstein-Barr virus (EBV). T-cells normally help rid the body of cells that contain viruses. When the T-cells aren’t working well, EBV-infected B-lymphocytes (a type of white blood cell) can grow and multiply. Most people are infected with EBV at some time during their lives, but the infection is controlled by a healthy immune system. The pre-transplant treatment given weakens the immune system, allowing the EBV infection to get out of control, which can lead to a PTLD.
Still, PTLD after allogeneic stem cell transplant is fairly rare. It most often develops within 1 to 6 months after allogeneic stem cell transplant, when the immune system is still very weak.
PTLD is life-threatening. It may show up as lymph node swelling, fever, and chills. There’s no one standard treatment, but it’s often treated by cutting back on immunosuppressant drugs to let the patient’s immune system fight back. Other treatments include white blood cell (lymphocyte) transfusions to boost the immune response, using drugs like rituximab to kill the B cells, and giving anti-viral drugs to treat the EBV.
Even though PTLD doesn’t often happen after transplant, it’s more likely to occur with less well-matched donors and when strong suppression of the immune system is needed. Studies are being done to identify risk factors for PTLD and look for ways to prevent it in transplant patients who are at risk.
15.4C: B Cells and T Cells - Biology
I. TYPES OF CELLS GROWN IN CULTURE
Tissue culture is often a generic term that refers to both organ culture and cell culture and the terms are often used interchangeably. Cell cultures are derived from either primary tissue explants or cell suspensions. Primary cell cultures typically will have a finite life span in culture whereas continuous cell lines are, by definition, abnormal and are often transformed cell lines.
II. WORK AREA AND EQUIPMENT
A. Laminar flow hoods. There are two types of laminar flow hoods, vertical and horizontal. The vertical hood, also known as a biology safety cabinet, is best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment. Horizontal hoods are designed such that the air flows directly at the operator hence they are not useful for working with hazardous organisms but are the best protection for your cultures. Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air. In a vertical hood, the filtered air blows down from the top of the cabinet in a horizontal hood, the filtered air blows out at the operator in a horizontal fashion. NOTE: these are not fume hoods and should not be used for volatile or explosive chemicals. They should also never be used for bacterial or fungal work. The hoods are equipped with a short-wave UV light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will be accessible to the UV light. Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage. The hoods should be turned on about 10-20 minutes before being used. Wipe down all surfaces with ethanol before and after each use. Keep the hood as free of clutter as possible because this will interfere with the laminar flow air pattern.
B. CO2Incubators. The cells are grown in an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained. Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be left out of the incubator for as little time as possible and the incubator doors should not be opened for very long. The humidity must also be maintained for those cells growing in tissue culture dishes so a pan of water is kept filled at all times.
C. Microscopes. Inverted phase contrast microscopes are used for visualizing the cells. Microscopes should be kept covered and the lights turned down when not in use. Before using the microscope or whenever an objective is changed, check that the phase rings are aligned.
D. Preservation. Cells are stored in liquid nitrogen (see Section III- Preservation and storage).
E. Vessels. Anchorage dependent cells require a nontoxic, biologically inert, and optically transparent surface that will allow cells to attach and allow movement for growth. The most convenient vessels are specially-treated polystyrene plastic that are supplied sterile and are disposable. These include petri dishes, multi-well plates, microtiter plates, roller bottles, and screwcap flasks - T-25, T-75, T-150 (cm 2 of surface area). Suspension cells are either shaken, stirred, or grown in vessels identical to those used for anchorage-dependent cells.
III. PRESERVATION AND STORAGE. Liquid N2 is used to preserve tissue culture cells, either in the liquid phase (-196°C) or in the vapor phase (-156°C). Freezing can be lethal to cells due to the effects of damage by ice crystals, alterations in the concentration of electrolytes, dehydration, and changes in pH. To minimize the effects of freezing, several precautions are taken. First, a cryoprotective agent which lowers the freezing point, such as glycerol or DMSO, is added. A typical freezing medium is 90% serum, 10% DMSO. In addition, it is best to use healthy cells that are growing in log phase and to replace the medium 24 hours before freezing. Also, the cells are slowly cooled from room temperature to -80°C to allow the water to move out of the cells before it freezes. The optimal rate of cooling is 1°-3°C per minute. Some labs have fancy freezing chambers to regulate the freezing at the optimal rate by periodically pulsing in liquid nitrogen. We use a low tech device called a Mr. Frosty (C#1562 -Nalgene, available from Sigma). The Mr. Frosty is filled with 200 ml of isopropanol at room temperature and the freezing vials containing the cells are placed in the container and the container is placed in the -80°C freezer. The effect of the isopropanol is to allow the tubes to come to the temperature of the freezer slowly, at about 1°C per minute. Once the container has reached -80°C (about 4 hours or, more conveniently, overnight) the vials are removed from the Mr. Frosty and immediately placed in the liquid nitrogen storage tank. Cells are stored at liquid nitrogen temperatures because the growth of ice crystals is retarded below -130°C. To maximize recovery of the cells when thawing, the cells are warmed very quickly by placing the tube directly from the liquid nitrogen container into a 37°C water bath with moderate shaking. As soon as the last ice crystal is melted, the cells are immediately diluted into prewarmed medium.
Cultures should be examined daily, observing the morphology, the color of the medium and the density of the cells. A tissue culture log should be maintained that is separate from your regular laboratory notebook. The log should contain: the name of the cell line, the medium components and any alterations to the standard medium, the dates on which the cells were split and/or fed, a calculation of the doubling time of the culture (this should be done at least once during the semester), and any observations relative to the morphology, etc.
A. Growth pattern. Cells will initially go through a quiescent or lag phase that depends on the cell type, the seeding density, the media components, and previous handling. The cells will then go into exponential growth where they have the highest metabolic activity. The cells will then enter into stationary phase where the number of cells is constant, this is characteristic of a confluent population (where all growth surfaces are covered).
B. Harvesting. Cells are harvested when the cells have reached a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cells that are not passaged and are allowed to grow to a confluent state can sometime lag for a long period of time and some may never recover. It is also essential to keep your cells as happy as possible to maximize the efficiency of transformation. Most cells are passaged (or at least fed) three times a week.
1. Suspension culture. Suspension cultures are fed by dilution into fresh medium.
2. Adherent cultures. Adherent cultures that do not need to be divided can simply be fed by removing the old medium and replacing it with fresh medium.
When the cells become semi-confluent, several methods are used to remove the cells from the growing surface so that they can be diluted:
- Mechanical - A rubber spatula can be used to physically remove the cells from the growth surface. This method is quick and easy but is also disruptive to the cells and may result in significant cell death. This method is best when harvesting many different samples of cells for preparing extracts, i.e., when viability is not important.
- Proteolytic enzymes - Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum
- EDTA - EDTA alone can also be used to detach cells and seems to be gentler on the cells than trypsin. The standard procedure for detaching adherent cells is as follows:
C. Media and growth requirements
1. Physiological parameters
A. temperature - 37C for cells from homeother
B. pH - 7.2-7.5 and osmolality of medium must be maintained
D. gas phase - bicarbonate conc. and CO2 tension in equilibrium
E. visible light - can have an adverse effect on cells light induced production of toxic compounds can occur in some media cells should be cultured in the dark and exposed to room light as little as possible
2. Medium requirements: (often empirical)
A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2
B. Trace elements - iron, zinc, selenium
C. sugars - glucose is the most common
D. amino acids - 13 essential
E. vitamins - B, etc.
F. choline, inositol
G. serum - contains a large number of growth promoting activities such as buffering toxic nutrients by binding them, neutralizes trypsin and other proteases, has undefined effects on the interaction between cells and substrate, and contains peptide hormones or hormone-like growth factors that promote healthy growth.
H. antibiotics - although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.
4. Measurement of growth and viability. The viability of cells can be observed visually using an inverted phase contrast microscope. Live cells are phase bright suspension cells are typically rounded and somewhat symmetrical adherent cells will form projections when they attach to the growth surface. Viability can also be assessed using the vital dye, trypan blue, which is excluded by live cells but accumulates in dead cells. Cell numbers are determined using a hemocytometer.
V. SAFETY CONSIDERATIONS
R. Ian Freshney, Culture of Animal cells: A manual of basic techniques, Wiley-Liss, 1987.
VI. TISSUE CULTURE PROCEDURES
Each student should maintain his own cells throughout the course of the experiment. These cells should be monitored daily for morphology and growth characteristics, fed every 2 to 3 days, and subcultured when necessary. A minimum of two 25 cm 2 flasks should be carried for each cell line these cells should be expanded as necessary for the transfection experiments. Each time the cells are subcultured, a viable cell count should be done, the subculture dilutions should be noted, and, after several passages, a doubling time determined. As soon as you have enough cells, several vials should be frozen away and stored in liquid N2. One vial from each freeze down should be thawed 1-2 weeks after freezing to check for viability. These frozen stocks will prove to be vital if any of your cultures become contaminated.
Procedures:1. Media preparation. Each student will be responsible for maintaining his own stock of cell culture media the particular type of media, the sera type and concentration, and other supplements will depend on the cell line. Do not share media with you partner (or anyone else) because if a culture or a bottle of media gets contaminated, you have no back-up. Most of the media components will be purchased prepared and sterile. In general, all you need to do is sterily combine several sterile solutions. To test for sterility after adding all components, pipet several mls from each media bottle into a small sterile petri dish or culture tube and incubate at 37EC for several days. Use only media that has been sterility tested. For this reason, you must anticipate your culture needs in advance so you can prepare the reagents necessary. But, please try not to waste media. Anticipate your needs but don't make more than you need. Tissue culture reagents are very expensive for example, bovine fetal calf serum cost
$200/500 ml. Some cell culture additives will be provided in a powdered form. These should be reconstituted to the appropriate concentration with double-distilled water (or medium, as appropriate) and filtered (in a sterile hood) through a 0-22 μm filter.
All media preparation and other cell culture work must be performed in a laminar flow hood. Before beginning your work, turn on blower for several minutes, wipe down all surfaces with 70% ethanol, and ethanol wash your clean hands. Use only sterile pipets, disposable test tubes and autoclaved pipet tips for cell culture. All culture vessels, test tubes, pipet tip boxes, stocks of sterile eppendorfs, etc. should be opened only in the laminar flow hood. If something is opened elsewhere in the lab by accident, you can probably assume its contaminated. If something does become contaminated, immediately discard the contaminated materials into the biohazard container and notify the instructor.
2. Growth and morphology. Visually inspect cells frequently. Cell culture is sometimes more an art than a science. Get to know what makes your cells happy. Frequent feeding is important for maintaining the pH balance of the medium and for eliminating waste products. Cells do not typically like to be too confluent so they should be subcultured when they are in a semi-confluent state. In general, mammalian cells should be handled gently. They should not be vortexed, vigorously pipetted or centrifuged at greater than 1500 g.
3. Cell feeding. Use prewarmed media and have cells out of the incubator for as little time as possible. Use 10-15 ml for T-25's, 25-35 ml for T-75's and 50-60 ml for T-150's. a. Suspension cultures. Feeding and subculturing suspension cultures are done simultaneously. About every 2-3 days, dilute the cells into fresh media. The dilution you use will depend on the density of the cells and how quickly they divide, which only you can determine. Typically 1:4 to 1:20 dilutions are appropriate for most cell lines. b. Adherent cells. About every 2-3 days, pour off old media from culture flasks and replace with fresh media. Subculture cells as described below before confluency is reached.
4. Subculturing adherent cells. When adherent cells become semi-confluent, subculture using 2 mM EDTA or trypsin/EDTA.
7. Viable cell counts. USING A HEMOCYTOMETER TO DETERMINE TOTAL CELL COUNTS AND VIABLE CELL NUMBERS (Reference: Sigma catalogue)Trypan blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live cells do not take up certain dyes, whereas dead cells do.
1. Prepare a cell suspension, either directly from a cell culture or from a concentrated or diluted suspension (depending on the cell density) and combine 20 μl of cells with 20 μl of trypan blue suspension (0.4%). Mix thoroughly and allow to stand for 5-15 minutes.
2. With the cover slip in place, transfer a small amount of trypan blue-cell suspension to both chambers of the hemocytometer by carefully touching the edge of the cover slip with the pipette tip and allowing each chamber to fill by capillary action. Do not overfill or underfill the chambers.3. Starting with 1 chamber of the hemocytometer, count all the cells in the 1 mm center square and four 1 mm corner square. Keep a separate count of viable and non-viable cells.4. If there are too many or too few cells to count, repeat the procedure either concentrating or diluting the original suspension as appropriate.5. The circle indicates the approximate area covered at 100X microscope magnification (10X ocular and 10X objective). Include cells on top and left touching middle line. Do not count cells touching middle line at bottom and right. Count 4 corner squares and middle square in both chambers and calculate the average.6. Each large square of the hemocytometer, with cover-slip in place, represents a total volume of 0.1 mm 3 or 10 -4 cm 3 . Since 1 cm 3 is equivalent to approximately 1 ml, the total number of cells per ml will be determined using the following calculations:Cells/ml = average cell count per square x dilution factor x 10 4
Total cells = cells/ml x the original volume of fluid from which the cell sample was removed % Cell viability = total viable cells (unstained)/total cells x 100.
This Web page is maintained by Julie B. Wolf, UMBC
Last updated on 3/2/2010
is designed for students interested in careers in industrial and biomedical sciences.