B-Cells and T-Cells

These specialized cells are a critical part of the bodies humoral immune system. They recognize foreign antigens or invaders and mount a quick response. B-cells act quickly by developing antibodies to the antigen epitopes. T-cells react based on what serological class they are in. If it is a CD8 T-cell, its cytotoxic and can quickly fight and phagocytize the antigen, if it is a CD4 T-cell, it works in conjunction with B-cells and other T-cell subclasses to defend the host. This article will dive into B-cells, and every subclass of T-cells and how they work together to form the humoral branch of the immune system.

B-Cells

B-cells also known as B lymphocytes are a type of lymphocyte that functions as part of the humoral component of the adaptive immune system. It’s role is to secrete antibodies, but it also functions as an antigen-presenting cell (APC) that secretes cytokines. It possesses a B-cell receptor (BCR) on its surface that allows it to bind to a specific target antigen and initiate an immune response. B-cells develop from hematopoietic stem cells (HSCs) that originate within the bone marrow. They then develop into multipotent progenitor cells (MPP), which further differentiates into the common lymphoid progenitor (CLP). Development further progresses through several stages through various gene expression patterns and arrangements. Before maturation occurs, positive selection takes place to make sure that the pre-BCR and BCR can recognize and bind to specific ligands through antigen-independent signaling. If the cells are unable to bind, these B-cells cease to develop. Negative selection occurs through binding of self-antigen with the BCR. If the BCR is able to bind self-antigen it undergoes four fates; clonal deletion, receptor editing, anergy, or ignorance. Clonal deletion is the destruction of the B-cell through programmed cell death, in other words known as apoptosis. This is only for those B-cells that have expressed receptors for self-antigens. Receptor editing is exactly what the name suggests; editing of the BCR during the maturation process in an attempt to change the specificity the receptor to not recognize self-antigens. Anergy is used to describe lack of reaction by the bodies immune system. Its a way of saying that the B-cells that express BCRs for self-antigen will simply not be used. The last fate; ignorance means that the B-cell ignores the signal and continues through natural development. When negative selection is complete, the B-cells are now in a state of central tolerance. These mature B-cells do not bind with self antigens. From the bone marrow, B-cells migrate to the spleen as transitional B-cells. Within the spleen they become Follicular B-cells or Marginal zone B-cells depending on the signal received through the BCR. Once completely differentiated, they are now called naive B-cells.

B cell

B-Cell Activation

Activation usually occurs within the secondary lymphoid organs, such as the spleen and the lymph nodes. This is where naive B-cells are positioned once mature. When these naive immunocompetent B-cells encounter an antigen through its BCR, the antigen is internalized by receptor-mediated endocytosis, digested, and positioned on MHC II molecules on the B-cell surface. This allows the B-cell to act as an antigen-presenting cell to T-cells. T-cell dependent activation requires a T-cell helper, most commonly a follicular T-helper cell, to bind to the antigen-complexed MHC II molecule on the B-cell surface through its T-cell receptor (TCR) which drives T-cell activation. These T-cells express the surface protein CD40L and secrete cytokines IL-4, and IL-21 which bind to CD40 on the B-cell surface and act as co-stimulatory factors for B-cell activation. The co-stimulatory factors promote proliferation, immunoglobulin class switching, and somatic hypermutation. Activated T-cells then provide a secondary wave of activation that cause the B-cells to proliferate and form germinal centers. During the production of these germinal centers, activated B-cells may differentiate into plasma blasts, which can produce weak IgM antibodies. Within the germinal centers, B-cells differentiate into high affinity memory B-cells or long-lived plasma cells. The primary function of plasma cells is the secretion of clone-specific antibodies. There are very few antigens that can directly provide T-cell independent B-cell activation. Some components of bacterial cell walls (lipopolysaccharide), and bacterial flagellin are some to name a few. One other mechanism through which B-cell activation is enhanced is through the activity of CD21, CD19, and CD81; all three are surface proteins that form a complex. When the BCR binds to an antigen that is tagged with the complement protein C3, CD21 binds to C3, and downstream signaling lowers the activation threshold of the cell.

Memory B-cell Activation

Activation begins through detection and binding of the target antigen. When the antigen binds, it is taken up by the B-cell through receptor-mediated endocytosis, degraded, and presented onto the MHC II molecule within the B-cell surface. The memory B-cell then acts as an antigen-presenting cell that presents the antigen:MHC II complex to T-cells. Most commonly memory follicular T-helper cells that bind through their TCR. The memory B-cell is then activated and differentiates into either plasmablasts and plasma cells or generate germinal centers.

T-Cells

A T-cell is another lymphocyte, which is a subset of white blood cells. They are called T-cells because they mature in the thymus from thymocytes. There are several subsets of T-cells, each with a specific role in the immune system. These T-cells, just like B-cells originate from hematopoietic stem cells in the bone marrow. These lymphoid progenitor cells populate the thymus and expand by cell division to immature thymocytes. The earliest thymocytes do not express either CD4+ or CD8+ and are classified as double negative cells. Through progression they become double positive and then eventually differentiate into single positive cells, either becoming CD8+, or CD4+. Its interesting to note that there is a small population of double positive T-cells within the peripheral circulation, although their function is unknown. About 98% of thymocytes undergo apoptosis during the development process by failing either positive selection or negative selection. The 2% that survive leave the thymus and become mature immunocompetent T-cells. Lets review positive and negative selection again. Positive selection selects for T-cells that are capable of interacting with MHC molecules. During positive selection signals by double positive precursors express either MHC class I or II receptors. A thymocytes fate is determined during positive selection. Double positive CD4+/CD8+ cells that interact with MHC class II molecules eventually become CD4+ cells, and on the contrary thymocytes that interact well with MHC class I molecules mature into CD8+ cells. Negative selection removes thymocytes that are capable of strongly binding with self MHC peptides.

Difference-Between-T-cells-and-B-cells-

T-Helper Cells

T-helper cells do just what their name suggests, they help other cells in immunological processes. This is evident in the activation of B-cells talked about previously. These cells are also most well known as CD4+ T-cells because the highly express CD4 glycoprotein on their surfaces. These T-cells become activated when they are presented with peptide antigens or epitopes by MHC class II molecules, usually present on antigen-presenting cells. Once activated, these cells proliferate rapidly and secrete multiple cytokines. T-helper cells differentiate into several subtypes; TH1, TH2, TH3, TH17, TH9, and THF, each secreting different cytokines to facilitate different pathways of the immune response. This is an article for another time.

TCellSubsets3-01

Cytotoxic T-Cells

These killer T-cells destroy virus-infected cells and tumor cells. These cells are known as CD8+ T-cells since they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigen epitopes that are associated with MHC class I molecules. Cytotoxic T-cells are highly regulated by Regulatory T-cells through IL-10, adenosine, and other molecules. They can be inactivated to an anergic state, which prevents autoimmune diseases.

T-cell CD8

Memory T-Cells

These memory T-cells are long-lived and when presented with an antigen that is recognized they can quickly expand and differentiate into large numbers of effector T-cells. These memory T-cells can either be CD4+ or CD8+ T-cells. There are four subtypes of memory T-cells that will be discussed below.

Central memory T-cells express CD45RO, C-C chemokine receptor type 7 (CCR7) and L-selectin which are all surface protein markers. They have high expression of CD44, and is commonly found within the lymph nodes.

Effector memory T-cells express CD45RO, but lack expression of CCR7 and L-selectin. These T-cells also have high expression of CD44, but are not found in the lymph nodes. These T-cells are found in the peripheral circulation and tissues.

Tissue resident memory T-cells occupy tissues without recirculating. The one specific surface marker that is associated with these cells is integral aeB7.

Virtual memory T-cells differ from all other memory subsets in that they do not originate from a clonal expansion event. These cells reside at low frequencies.

Natural Killer T-cells (NK)

First off, it should be mentioned that these cells should not be confused with natural killer cells of the innate immune system. Unlike conventional T-cells that recognize antigen epitopes presented on MHC I/II molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. When these cells are activated, these cells perform functions from both T-helper cells and cytotoxic T-cells. These cells specialize in recognizing tumor cells and cells infected with herpes viruses.

 

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Erythropoietin (EPO)

The role of red blood cells is to carry oxygen. Just like anything in the body, this is tightly regulated by a mechanism that monitors whether or not there is adequate oxygen getting to tissues and other cells. Hypoxia is detected by the peritubular fibroblasts of the kidneys which causes erythropoietin (EPO) to be released. The EPO gene has a hypoxia-sensing region in its 3’ regulatory component which causes hypoxia inducible factor-1 (HIF-1) transcription factor to be assembled and it interacts with the 3’ enhancer of the gene causing increased EPO mRNA and production of more EPO.  EPO is a true hormone, being produced in the kidneys, and acting upon another distant location being the bone marrow. When EPO binds to its ligand (receptor) on red blood cell progenitors it initiates a cascade which is mediated through the JAK2 signal transducers which ultimately effects the gene expression. EPO has three main physiological effects on the body; it allows early release of reticulocytes from the bone marrow, prevents apoptosis, and reduces the time needed for cells to mature in the bone marrow before release into the periphery. 

There are two mechanism for which EPO stimulates early release of red cell precurors into the bone marrow. It induces changes in the adventitial cell layer of the marrow sinuses that increases the width of the spaces that the red cells squeeze out of. It also down regulates red blood cell surface receptors for adhesive molecules that are located on the bone marrow stroma. As a result the red cells are able to pass through without the receptor so that they won’t bind to the stroma and delay release.

Apoptosis is programmed cell death. EPO inhibits apoptosis by removing the induction signal. Under normal physiology the bone marrow produces more CFU-Es than needed that are stored in the bone marrow which have a “head start” in the maturation process. About a 10 day head start in maturation. The CFU-Es (Colony-forming unit-erythroid) are red blood cell progenitor cells that develop from BFU-Es (Burst-forming unit-erythroid). Both BFU-E and CFU-E are red blood cell progenitor cells that develop into the pronormoblast, which is the first morphologically identifiable red blood cell precursor. If healthy, those cells live out there life span and undergo apoptosis. If there is a deficiency of red blood cell mass, those cells undergo maturation to be released, while simultaneously the apoptosis induction signal is inhibited. The normal death signal consists of a death receptor being FAS, on the membrane of the earliest red blood cell precursors (CFU-Es/BFU-Es), and FASL ligand on the maturing red blood cells precursors. When EPO levels are low, because there is adequate oxygen delivery the older FASL bearing cells cross-link with earlier FAS precursors which stimulates apoptosis. EPO is able to subdue apoptosis by stimulating the more mature precursors to be released from the marrow, especially in times of hypoxia. At which point there will no FASL bearing cells to cross-link the early FAS bearing precursors. Its a two fold effect, the more mature cells are released to help increase red cell mass in times of need, and the early precursor are allowed to mature and be released without undergoing apoptosis. When EPO binds to its ligand on the red blood cell activates the JAK2-STAT pathway, which ends in and up-regulation of transcription for BCL-2, which is an anti-apoptotic protein. This anti-apoptotic protein rests on the cell membrane and prevents the release of cytochrome c, which initiates apoptosis. 

JAKSTAT

EPO has an effect on the bone marrow transit time of a red blood cell precursor in two different ways; increased rate of cellular processes, and decreased cell cycle times. What this means is that EPO stimulates synthesis of red cell RNA, such as the production of hemoglobin. It also stimulates the production of egress-promoting surface molecules within the bone marrow which allow the red blood cells to flow through the marrow easier. EPO stimulates cells to enter cell cycle arrest earlier than normal, and as a result, spend less time maturing and are able to be released. These cells may appear larger in size and have a bluish tinge to their cytoplasm because of this.

Iron Deficiency and Microcytic Anemias

Iron is an essential element for oxygen transport within hemoglobin. Oddly enough it is the element that is missed the most in regards to adequate intake and proper nutrition. Over 1.62 billion people in the world are effected by anemia, which is most commonly caused by iron deficiency. Iron deficiency can be caused by chronic blood loss, and is most common in women and teenagers from loss of blood due to menses. Iron loss leads to increased fatigue and depression, pallor, and dry and splitting hair. It can also lead to confusion cognitive effects. Hemoglobin is made of four polypeptide chains, two of which are alpha, and two are beta that come together to form a tetramer heme group with iron located in the middle. Ferrous iron within each heme molecule reversibly binds to one oxygen molecule. With iron deficiency, there becomes a hemoglobin deficiency. A decreased hemoglobin lowers oxygen-carrying capacity leading to anemia. Anemia by definition is a reduced oxygen-carrying ability. Tissue hypoxia can wreak havoc on almost every cell of the body, and can shift the oxygen dissociation curve in an unfavorable direction. The structure of hemoglobin and its function and key elements can be reviewed here.

To understand iron deficiency its important to recognize important aspects of iron metabolism and transportation in cells. Review the Iron Absorption and Metabolism article here for that information. There are also laboratory values that give a good picture of the iron status within the body that one should pay attention to. Transferrin; which is measured as the total iron binding capacity (TIBC) indicates how much or how little iron is being transported throughout the body. Serum iron is an important indicator of the tissue iron supply, and finally serum ferritin gives a picture of iron storage status within the bone marrow and cells.

Iron Deficiency Anemia

There are three stages within iron deficiency. Each comes with their own classic picture of laboratory results and worsen from stage to stage. In the first stage, there is storage iron depletion. This is mild and the patient may not even feel a difference physically. The patients hemoglobin is normal, normal serum iron, and TIBC. There is however decreased ferritin which indicates that there is decreased storage of iron. The second stage of iron deficiency is characterized by transport iron depletion. The hemoglobin may or may not be abnormal, but there is increased TIBC, and decreased serum iron. An increased TIBC, means that there are more substrate (iron) binding spots within the transferrin molecule. This implies that less iron is binding, which when coupled with a decreased serum iron makes sense. The patient may experience mild anemia which comes with increased fatigue and pallor. A peripheral blood smear will most often start to exhibit anisocytosis and poikilocytosis. These reference indicators represent abnormal sized red cells and abnormal shaped red blood cells respectively. A good indicator is an increased RDW, an increased RDW indicates some degree of anisocytosis. This is accurate because the red blood cell is realizing the loss of this oxygen-carrying capacity so its trying to release red blood cells as fast it can from the bone marrow to compensate for the loss, and as a result these red blood cells will appear smaller in diameter and hypochromic. Hypochromasia indicates that there is less hemoglobin within the cell and there is more of a central pallor. The thought is that even though there is less hemoglobin within each cell, if the bone marrow can produce more of these red blood cells than normal then that equals out. This leads to a microcytic anemia, micro meaning small. Stage three of iron deficiency is often referred to as functional iron deficiency. Within this stage there is an unmistakable decrease in hemoglobin, serum iron, and ferritin. There is also a large increase in TIBC.

The overall effect of iron deficiency anemia on the body and on the bone marrow is ineffective erythropoiesis. The red cell production within the bone marrow is compromised. As a result, the bone marrow becomes hypercellular with red cell precursors reducing the M:E (Myeloid:Erythroid) ratio.

Iron-deficiency_Anemia,_Peripheral_Blood_Smear_(4422704616)

This picture depicts how a peripheral blood smear would illustrate iron deficiency anemia. The red cells are smaller and there is more of a central pallor to them, indicating a loss of hemoglobin. This is also called hypochromia.

normalbloodsmear

This picture depicts a normal peripheral blood smear. The red blood cells are larger in size and they have more color to them.

Anemia of Chronic Disease

Anemia of chronic disease is another form of microcytic anemia similar to iron deficiency anemia. It usually arises from a chronic infection or from chronic inflammation, but its also associated with some malignancies. A buildup in inflammatory cytokines alters iron metabolism. IL-6, which is an inflammatory cytokine inhibits erythrocyte production. It also increases hepcidin production. Hepcidin blocks iron release from the macrophages and the hepatocytes by down-regulating ferroportin. Without ferroportin there is no transportation of iron throughout the body and no production of hemoglobin or red blood cells. Laboratory findings will usually demonstrate low serum iron, low TIBC, low transferrin, and an increased to normal ferritin. The reticulocyte count is also normal, and sometimes increased. Reticulocytes are released from the bone marrow in times of red cell shortages to compensate.

This is just a brief overview of iron deficiency anemia and other microcytic anemias. This is just the beginning, follow and look forward to more in-depth reviews of each microcytic anemia. Key differences to look for is the TIBC value. In iron deficiency anemia the TIBC is increased and in anemia of chronic disease the TIBC is decreased. Ferritin is increased in anemia of chronic disease because the stored iron can’t be released from cells and the bone marrow due to the increased hepcidin production. Also the degree of anemia is mild compared to the more severe iron deficiency anemia.

 

 

The Antibody

An antibody or immunoglobulin is a large Y-shaped protein produced primarily by plasma cells of the humoral immune system. They are used to recognize and neutralize any foreign antigens or pathogens. An antibody is identical to the B-cell receptor of the cell that secretes it except for a small portion of the C-terminus of the heavy-chain constant region. The difference is that a B-cell receptor C-terminus is a hydrophobic membrane-anchoring sequence and on an antibody, the C-terminus is a hydrophilic sequence that allows its secretion. The Y-portion of the consists of two arms that vary between the different antibody molecules, otherwise known as the V-region. The V-region is involved in antigen binding. The C-region is far less variable and is the part of the molecule that interacts with effector cells and other molecules. All antibodies are constructed in the same way paired from heavy and light polypeptide chains joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together.

There are two types of light chains, lambda and kappa. A given immunoglobulin has one or the either, never both. In humans the ratio of kappa to lambda; the two types of light chains in immunoglobulins is 2:1. The class, and the effector function of an antibody is defined by the structure of its heavy chain. There are five main heavy-chain isotypes. The five major immunoglobulin classes are IgM, IgD, IgG, IgA, and IgE. IgG is the most abundant immunoglobulin and has several subclasses (1, 2, 3, and 4 in humans). The distinctive functional properties are conferred by the carboxyl -terminal part of the heavy chain, where it is not bonded with the heavy chain.

Each chain of the immunoglobulin consists of a protein domain. Each protein domain consists of a series of similar, but not identical sequences about 110 amino acids long . The light chain is made up of two domains, and the heavy chain consists of four. The variable or V-domain of the heavy and light chains together consist of the V-region of the antibody allowing it to bind specific antigens. The constant domains of the heavy and light chains together make up the C-region. The V-region or the Y of the molecule, where the antigen binding activity takes place is called the Fab fragments. Fab stands for fragment antigen binding. The other part of the molecule, the constant region (C-region) contains no antigen-binding activity, and is called the Fc fragment. Fc stands for Fragment crystallizable. This is the part of the molecule that interacts with effector molecules and cells.

The immunoglobulin molecule is flexible. There is a hinge region that links the Fc and Fab regions of the molecule, allowing independent movement of the two Fab arms.

Recap

To recap. An antibody molecule is made up of four polypeptide chains, comprising of two identical light chains and two identical heavy chains, which can be thought of as forming a flexible Y-shaped structure. Each of the four chains has a variable (V) region at its amino terminus, which contributes to the antigen-binding site, and a constant (C) region, which determines the isotype of the immunoglobulin. The light chains are bound to the heavy chains are non-convalent disulfide bonds. The V-regions of the light and heavy chains pair together to form the Fab region on the arms of the Y-structure. The trunk of the Y-structure, consisting of the carboxyl-terminal domains of the heavy chains make up the Fc fragment. The Fc fragment determines the different isotype of the immunoglobulin and interacts with different effector molecules. There is a hinge region joining the Fab and Fc regions allowing the antibody independent movement to maximize its antigen binding capabilities.

 

 

 

Polycythemia Vera

Polycythemia vera is an uncommon neoplasm or blood cancer where the bone marrow produces too many erythrocytes, megakaryocytes, and granulocytes, resulting in panmyelosis. The cancer is caused by a mutation in the JAK2 gene. Janus Kinase 2 (JAK2) is a non-receptor tyrosine kinase that plays a role in signaling in the type II cytokine receptor family. Members of that family include interferon receptors, GM-CSF receptor family, gp130 receptors, and the single chain receptors (EPO-R, etc). The function of those receptors are not important. The most important receptor for this article is the EPO-R receptor. The erythropoietin receptor (EPO-R) is a protein encoded by the EPOR gene that pre-exists in a dimerized state. When the ligand erythropoietin binds to the EPO-R receptor it induces a conformational change that results in the autophosphorylation of the JAK2 kinases. This establishes the function of EPO-R which is to promote proliferation and the rescue of erythroid progenitors from apoptosis. EPO-R induces JAK2-STAT5 signaling and with help from the transcription factor GATA-1 induces the transcription of the protein BCL-XL which is anti-apoptotic and promotes red cell survival.

In polycythemia vera (PV) there is a JAK2V617F mutation that causes independent continuous expression of the JAK2 kinase without erythropoietin (EPO) that acts on signaling pathways involving the EPO-R or hyperexpression in the presence of EPO. This causes increased gene expression for erythroid precursor cell proliferation and differentiation. It up regulates BCL-XL, which as mentioned above is an anti-apoptotic. This causes an abnormal accumulation of red cells in the peripheral blood. Its important to note that the accumulation of the red cells is due to lack of apoptosis, NOT because they are dividing quicker. Also there is a difference between primary PV and secondary PV. In primary PV there is a decreased expression of EPO, this is a compensation method for the body. As there is autophosphorylation of the EPO-Receptor, the body tries to reverse the process by down regulating the expression of erythropoietin (EPO). In secondary PV, there is normal to increased expression of EPO.

P-vera

Diagnosis

Diagnosis of PV according to the World Heath Organization (WHO) has to satisfy both major and minor criteria. The major criteria that has to be observed is a hemoglobin higher than 18.5 g/dL in men, and greater than 16.5 g/dL in women. There also has to be the presence of the JAK2 mutation. Minor criteria include presence of bone marrow hypercellularity demonstrating panmyelosis, serum EPO levels decreased, and a demonstration of endogenous erythroid colony growth in vitro. Meaning that there is presence of red cell growth in the laboratory using EPO from the patient, which assumes there is an issue with the downstream signaling of EPO, not EPO itself.

Laboratory results illustrate an increased hemoglobin, hematocrit, and MCV. There is an increased red cell count, platelet count, and white blood cell count. The leukocyte alkaline phosphatase is also increased. Its important to know that although the platelet count is increased, there is also an altered function of the platelets. The erythrocyte sedimentation rate will be decreased due to the decrease in the zeta potential. The zeta potential is the electrokinetic potential between the red cells that stops them from stacking or from sticking to one another. One classic characteristic of PV is erythromelalgia. This is a burning sensation in the pain and feet, with a reddish or bluish discoloration. This is caused by an increased platelet agglutination, from being dysfunctional that results in microvascular blood clots.

Treatment

If untreated, PV can be fatal. Although the disease can’t be cured, it can be controlled and the life expectancy has risen with modern advances in medicine. Phlebotomy is recommended to reduce the hemoglobin and hematocrit levels, but can induce iron deficiency anemia if not monitored. Low dose aspirin is prescribed to reduce the risk of thrombotic events. The accumulation of the red cells increases the risk for the patient to develop thrombotic events because the blood is “thick”. Chemotherapy can be used, but is not normally indicated, unless therapeutic phlebotomy is unable to maintain a normal hemoglobin or hematocrit or when there is significant thrombocytosis. It is dangerous because of the risk for transformation to acute myeloid leukemia (AML).

To recap; its important to know the mutation in the JAK2 kinase that induces polycythemia vera. Although this mutation is demonstrated in 90% of cases, its possible that its absent. Panmyelosis and elevation of RBC indices is a diagnostic finding. Its important to know the major and minor criteria for the diagnosis of PV. Treatment is therapeutic phlebotomy and chemotherapy in rare cases, only when prior treatment has failed.