hematology2

Question 1

Transfusion related lung injury (TRALI)

Answer 1

is acute lung injury (development of diffuse lung infiltrates, problems breathing and difficulty maintaining peripheral oxygen saturation on room air) within 6 hours of a transfusion. Vigorous ventilatory support may be required but the syndrome resolves quickly in 90% of those affected. The risk is 1/5,000-1/3,000 per transfusion depending on the product (all products can cause this reaction).

Question 2

Transfusion associated circulatory overload (TACO)

Answer 2

is volume (fluid overload) related to excessive amounts of products and/or cardiac dysfunction. Diuretics will help resolve the problem.

Question 3

Monocytes

Answer 3

derived from myeloid/ monocyte precursor under stimulation of GM-CSF and M-CSF, these cells have a shorter development time in the bone marrow (7 days), move to the peripheral blood for 3-5 days. Some then emigrate to tissues where they develop into tissue based macrophages lasting for days-months. (liver-kuffer cell) Major functions of these cells is to a) migrate from blood to sites of infection and provide effector phagocytic cells to remove microbes, dead and dying inflammatory cells, and debris; b) filter out microbes from blood stream (spleen-macrophage); c) process and present antigens to the adaptive immune system; and, d) remove apoptotic cells.

Question 4

Neutrophil

Answer 4

The polymorphonuclear leukocyte (PMN), neutrophil or granulocyte is produced in the marrow (GM-CSF, G-CSF), remains there for a few days (10-14 days, under stress 5 days) in a storage pool held in reserve to fight infections, is subsequently released into peripheral blood (6hrs) where it may marginate between the post-capillary venules and the laminar blood flow. After 6 hrs, the neutrophil moves into the tissues where it turns over in 1-2 days. The neutrophil is a major component of the innate immune system migrating quickly to the site of infection where it ingests and kills microbes. It is the prototypic first responder but is also important in stimulating wound healing and tissue repair. Prolifer pool (mitotic compartment) is smaller in number of cells than the storage compartment where maturation occurs. Absolute neutrophil count=bands and segs.

Question 5

Eosinophil

Answer 5

Produced in the bone marrow under the influence of IL-5, its maturation and kinetics are like neutrophils. The eosinophil, however, is slightly larger, has prominent eosinophilic (red/orange) granules and bilobed nucleus. Mature cell 12-14 micron dia. After transversing the peripheral blood, eosinophils move to mucosal surfaces (GI tract, tracheobronchial tree, etc.) where they survive for weeks. These cells play a role in allergic reactions, parasitic infections and response to tumors. They can play the role of a phagocyte and be immunostimulatory or immunoinhibitory. Will release ROS onto large parasite without actualy engulfing them. Roles allergies, parasite infection, response to tumors: may be immuno-enhancing or immuno-suppressive.

Question 6

Basophil

Answer 6

After production in the marrow, basophils spend most of their life in tissues. Similar in size to eosinophils, they have prominent basophilic (blue-purple) primary granules and receptors for IgE. These cells appear to play a major role in hypersensitivity (allergic) reactions. Larger than neutrophil, smaller than monocyte.

Question 7

Neutropenia

Answer 7

is defined as a decrease in the absolute neutrophil count (bands and segs) below accepted norms. This may vary with age (<250/μl a very severe risk (sepsis, pneumonia). We work on the assumption that the decrease in blood levels represent a decrease in storage. This is not always true and you need bone marrow sample to check.

Question 8

Clinical and Labooratory Findings of neutropenia:

Answer 8

Once defined, the workup of neutropenia should include a thorough history investigating the duration or periodicity of the low counts and associated symptoms or signs; numbers, sites, and agents involved in infections; exposure to drugs and toxins; and family history of recurrent infections, immune disorders or hematologic abnormalities. A complete physical exam should be performed with special attention paid to infected sites; teeth and gums, lymph nodes, liver, spleen and other findings (nails, skin, dysmorphic features, etc.). Initial laboratory investigations include an initial CBC with a white blood cell differential and review of leukocyte morphology. For chronic neutropenia, counts twice weekly for six weeks will define the extent and persistence of the neutropenia. Blood chemistries (LDH, SGOT, SGPT, uric acid, alkaline phosphatase), neutrophil antibodies (due to related autoimmune disorder) and a bone marrow aspirate and biopsy may be essential to defining the specific cause of neutropenia. Other tests may be necessary to diagnose the disease or syndrome responsible for neutropenia.

Question 9

Classifications of neutropenia

Answer 9

Based on initial evaluation, neutropenias can be classified as those with a decreased bone marrow production (primary or secondary) or those with a normal or near normal reserve but increased turnover. Included in the former are Kostmann Syndrome, Shwachman-Diamond Syndrome, cyclic neutropenia and idiopathic neutropenia while the latter is usually associated with infection, drugs, antibody-associated neutropenia or hypersplenism. Infection or drug related neutropenia can be related to an increase in turnover or decreased production or both. A number of syndromes have normal production but increase in peripheral utilization or transit time through peripheral blood. Most do not have problems with severe, recurrent infections. Acute is less then three months, it is related to infections, antibiotics or other drugs. Chronic neutropenia are more complicated and congenital type last for your whole life.

Question 10

Infectious Complications of Neutropenia

Answer 10

Patients with severe neutropenia and production defects are predisposed to bacterial or fungal infections. Staphylococcus aureus and gram-negative bacteria are the most common pathogens, but other microbes can cause infection as well. The most common sites and types include septicemia, cellulitis, skin abscesses, pneumonia, and perirectal abscesses. Sinusitis, aphthous ulcers, gingivitis, and periodontal disease cause significant problems.

Question 11

Decreased production neutropenia

Answer 11

: Several acquired disorders which decrease the production and/or bone marrow reserve (storage pool) are included in this category. Chemotherapy drugs used for malignant conditions are a major cause of neutropenia related to direct effects on stem cells and myeloid precursors. Exposure to these drugs markedly reduces production and may leave the patient at high risk for severe infection. Other drugs (chloramphenicol, etc.) may also have a direct toxic effect on precursors and cause severe neutropenia. Immune mechanisms with antibiotics can cause neutropenia. Hypersensitivity to Dilantin or phenobarbital can cause neutropenia. One of the more common causes of acute neutropenia is viral infections (EBV, varicella, measles, CMV, hepatitis, HIV). The mechanism for infection include increased utilization, complement mediated margination, marrow suppression/failure (direct effect), cytokine/ chemokine induced margination, and antigbody production. These may directly suppress marrow production or cause an increase in turnover peripherally, either directly related to consumption in tissues or indirectly related to effects of antibodies. Finally, nutritional deficiencies, such as folate, B12, copper or protein/calorie malnutrition can cause ineffective myelopoiesis and neutropenia due to intramedullary death secondary to effects of deficiency on replication. As with some other disorders in this category, the neutropenia can be associated with other cytopenias (anemia, thrombocytopenia).

Question 12

Kostmann syndrome

Answer 12

with severe peripheral neutropenia and marked decrease in myeloid production beyond promyelocytes, leads to a high risk for infection and death before 2 years of age unless patients receive aggressive management. The mechanism is apoptosis of myeloid precursors associated with elastase (ELA-2) or HAX-1gene mutations, therefore in the marrow you see myeloid hypoplasia, severe maturation arrest promyelocyte/myelocyte stage. Rarely, defects in G-CSF receptor. Some patients survive to develop myeloid leukemia or myelodysplastic syndrome (MDS). Recent identification of elastase gene mutations and early apoptosis of precursors may provide a clue to the etiology of some cases. Inheritance may be autosomal recessive or dominant; or, in some cases, the disease has a sporadic presentation.

Question 13

Shwachman-Diamond syndrome

Answer 13

is characterized by neutropenia (90% at sometime during their clinical course), pancreatic insufficiency with fat malabsorption, bony abnormalities (metaphyseal chondrodysplasia and Erlenmeyer flask deformities of long bones), and growth delay. Half of patients develop aplastic anemia or MDS/leukemia due to stem cell failure. Autosomal recessive inheritance is described in most cases and the gene defect(s) have been recently described. Abnormalities include apoptosis in precursors and a possible defect in “nurse” cells in the marrow stroma providing support for the developing myeloid cells. Pancreatic function may improve over time. Defects in the Schwachman-Diamond gene have recently been reported. Many have defect in SBDS gene. 25% develop marrow aplasia, 25% develop MDS/AML. Management: Pancreatic enzyme replacement; G-CSF, Aggressive antibiotic therapy and supportive care for infection, BMT for severe complications.

Question 14

Cyclic neutropenia

Answer 14

is characterized by severe peripheral neutropenia for 5-7 days with specific periodicity (15-25 day cycles). Recurrent fevers and mouth ulcers may accompany infections during the time of neutropenia. At other times in the cycle, the ANC is normal and there is no greater risk for infection. This condition has also been linked to apoptosis in marrow precursors and mutations in the gene for elastase causing apoptosis in precursors and cyclic hematopoiesis. Autosomal recessive, dominant and sporadic patterns have been reported. Infections, mouth ulcers during neutropenia; improvement with age. Some have cycles in platelet and retic count. Management: Aggressive antibiotic and supportive care for infection, G-CSF daily or alternate days

Question 15

Chronic Idiopathic Neutropenia

Answer 15

Mechanism/Inheritance/Genetics: Myeloid hypoplasia and maturation arrest at myelocyte, metamyelocyte or band stage, No specific inheritance identified, sporadic. Clinical features: Moderate to severe neutropenia, Recurrent infections (skin, sino-pulmonary tract, etc.), No other associated findings, No neutrophil antibodies detected. Management: Usually responsive to G-CSF

Question 16

Chronic benign neutropenia of childhood

Answer 16

results from production of antibodies which cross-react with neutrophils. These children (median age 8-11 months, most before 14 months) have a very low ANC chronically but may increase their counts in association with infection. There is usually no increased risk for severe infections, and the neutropenia resolves after a median duration of 20 months (range 6-54 months). Supportive care and reassurance are the hallmarks of management.

Question 17

Autoimmune neutropenia

Answer 17

may be caused by antibodies to specific determinants on the neutrophil. Marrow production is normal to increased, storage pool is normal to slightly decreased. Neutropenia is due to increased turnover, vascular compartment decreased. It may be seen in association with Systemic Lupus Erythematosus (SLE), Evan’s Syndrome, or Felty’s Syndrome. May also find thrombocytopenia, autoimmune hemolytic anemia, or other hematologic antibodies. In these cases, antibodies to red cells, platelets or coagulation proteins (lupus anticoagulants) may also be seen. With passive transfer of antibody from mother’s circulation attacking baby’s cells causing neutropenia, alloimmune neutropenia shares a common pathophysiology with Rh hemolytic disease and alloimmune thrombocytopenia of the newborn. Transplacental passage of neonatal cells which contain antigens not expressed by maternal cells into the maternal circulation sensitizes the mother to produce antibodies against the infant’s antigens. Accumulation of IgG class antibodies by the fetus provides a pool of antibodies which bind the infant’s neutrophils and cause neutropenia. The neutropenia may last for 2-4 weeks and, occasionally, 3-4 months, and occasional infections are seen. Affected patients may be asymptomatic or may develop skin infections, and rarely pneumonia, sepsis or meningitis. Commonly confused with neutropenia caused by sepsis. Marrow shows myeloid hyperplasia with maturation arrest at mature precursors (don’t delete that storage pool). Some drugs cause antibody mediated neutropenia which resolves with discontinuation of the drug.

Question 18

Splenomegaly and hypersplenism

Answer 18

cause neutropenia related to excessive sequestration of neutrophils in the spleen and may be associated with sequestration of red cells and platelets as well. Severe infection, particularly with bacterial pathogens and activation of complement (specifically C5a) may result in excessive demargination of neutrophils and pseudoneutropenia.

Question 19

Management of Kostmanns’ Syndrome

Answer 19

Aggressive treatment of infections, G-CSF 3-100 mcg/kg/day to keep ANC >1,000/ml, Consider BMT for poor response to G-CSF; AML; MDS

Question 20

Infectious Complications

Answer 20

Patients with severe neutropenia and production defects are predisposed to bacterial or fungal infections. Staphylococcus aureus and gram-negative bacteria are the most common pathogens, but other microbes can cause infection as well. The most common sites and types include septicemia, cellulitis, skin abscesses, pneumonia, and perirectal abscesses. Sinusitis, aphthous ulcers, gingivitis, and periodontal disease cause significant problems.

Question 21

Management of secondary neutropenias

Answer 21

Withdraw unnecessary drugs and eliminate toxins, Treatment of underlying disorder, Replacement of specific deficiency, Aggressive management of infections, Supportive care including prophylactic antibiotics, G-CSF in some conditions (e.g., chemotherapy)

Question 22

Autoimmune neutropenia management

Answer 22

Treat primary autoimmune disorder and/or hematologic antibodies. G-CSF may be helpful if marrow storage pool depleted.

Question 23

Alloimmune neutropenia treatment

Answer 23

Antibiotics and supportive care for infections. IVIG infusion not always effective. Consider G-CSF in face of severe infection

Question 24

Treatment strategies for neutropenia

Answer 24

Define the type of neutropenia. For undefined cases or those with severe congenital syndromes associated with ANC 38.5°C, complete appropriate cultures (including blood) and treat with antibiotics. For severe infections, aggressive attempts at identifying infected site and involved organisms. Initiate broad spectrum antibiotics and change to specific antibiotics when organisms are identified. In some syndromes, prophylactic antibiotics are helpful. For severe cases, G-CSF given at a dose of 3-5 μg/kg subcutaneously every day will normalize production.

Question 25

Leukocytosis

Answer 25

an increase in total number of WBCs beyond normal values. Think of infection, inflammation, non-specific physiologic stress, or malignancy (leukemia). Left shift is a term referring to changes in the white cell differential with an increase in segs and bands and possibly some immature myeloid precursors usually only found in marrow (metamyelocytes or myelocytes). Specific implications for leukocytosis depends on the cell lines which are increased.

Question 26

Neutrophilia

Answer 26

is defined as an ANC >7,500 cells/μl outside the neonatal age range (7-13,000/μl is normal range for newborns). This may be caused by increased production (infection or inflammation, myeloproliferative disorders, drugs such as lithium, tumors, or stress-related leukemoid reactions). Other reasons include increased release from storage pool (e.g., steroids, acute infection, stress, endotoxin), decreased egress from the circulation (steroids, splenectomy, leukocyte adhesion deficiency) and reduced margination (epinephrine, exercise, stress).

Question 27

Basophilia

Answer 27

Increase in peripheral basophils is seen primarily in drug or food hypersensitivity or urticaria. It may also be seen in infection or inflammation (rheumatoid arthritis, ulcerative colitis, influenza, chickenpox, smallpox, tuberculosis) as well as myeloproliferative diseases (CML, myeloid metaplasia).

Question 28

Eosinophilia

Answer 28

An absolute count >350/ul is abnormal. The etiologies fall into three main categories: allergies/allergic disorders (asthma, atopic dermatitis, hay fever, hives, etc.), parasitic infections, and drug reactions (allergic). Rarer causes include pemphigus, tumors or malignancies, and other infections like chronic active hepatitis. Hypereosinophilic syndromes and eosinophilic leukemia are rare.

Question 29

Monocytosis

Answer 29

Usually thought of with an absolute monocyte count of >1,000/μl in newborns and >500/μl in children and adults. Monocytosis may be found in hematologic (pre) malignancies (AML, pre-leukemia states, lymphoma, Hodgkin’s disease), collagen vascular diseases (SLE, RA), granulomatous diseases (sarcoid, ulcerative colitis, Crohn’s disease), infections (subacute bacterial endocarditis, syphilis, tuberculosis, protozoal rickettsial, and Pertussis infections), and carcinoma.

Question 30

Neutrophil

Answer 30

Neutrophil function is critical to the first response of the host. Neutrophils move in the laminar flow of the blood but are initially pulled to areas of infection by interacting with endothelial cells in a rolling motion. This is followed by a more extensive process of firm adhesion mediated by a separate set of adhesion proteins. Passing through the junctions between endothelial cells (diapedesis), the cells move towards the offending organisms (chemotaxis), following the trail of chemoattractants (bacterial products, complement products such as C5a, cytokines and chemokines) up the concentration gradients to engage the microbial invader. At the site of infection, the microbe, properly opsonized with C3b or antibody, is enveloped by pseudopods which, like arms, embrace the organism. With fusion of the pseudopods, a phagosome is formed encasing the ingested particle in a small volume. Granules of each class fuse with the growing phagolysosome and the oxidase enzyme system is assembled in the membrane initiating the respiratory burst and generating reactive oxygen species (superoxide anion, O2-; hydrogen peroxide, H2O2; hypochlorous acid, HClO; and hydroxyl radical, .OH). Together, the reactive oxygen species (ROS) and oxygen independent mechanisms (defensins, lysozyme, cathepsins, proteases) are focused on the phagolysosome and lead to the death and dissolution of the microbe.

Question 31

Function of neutrophil

Answer 31

The function of neutrophils and other phagocytic cells can be arbitrarily divided into four phases: adherence, chemotaxis, ingestion and degranulation/microbicidal activity. Each of these is induced through engagement of specific receptors which press into action the function of subcellular organelles through physiologic and biochemical processes. There is overlap in mechanisms used for two or more functions. For example, chemotaxis with protrusion of the pseudopod in the direction of movement shares a number of processes and organelles with cell motility (e.g., C5a, actin cytoskeleton, actin assembly). The differences in signaling which drive chemotaxis as distinct from ingestion are not completely understood and lie in the multiplicity of signaling pathways and effector mechanisms (involve C3bi receptors, which overlaps with CD11b/CD18). These are, however, integrated into a continuum of specific events leading in the correct sequence to ingestion and destruction of the microbe.

Question 32

Chronic granulomatous disease (CGD)

Answer 32

a syndrome which represents abnormalities in oxidase components. Specific microbes are ingested normally but cannot be killed. Neutrophilia. Normal adherence, chemotaxis, ingestion and degranulation. Defect in oxidase enzyme system. No toxic oxygen metabolites produced. Absent or reduced ability to kill coagulase positive bacteria and fungi (e.g., staph, E-coli). Deficiency of gp91phox, p22phox, p47phox, or p67phox results in absence of respiratory burst and production of ROS. Recurrent infections, limit the lifespan of these patients, may be deeply in lung, liver, spleen, lymph nodes and bones. Infections may be local or disseminated, but production of granulomas in tissues is typical of the syndrome. MPO deficiency may be also categorized here because HClO production is decreased and efficient killing of Candida is limited.

Question 33

Leukocyte adhesion deficiency I

Answer 33

results in neutrophilia (cant adhere), decreased adherence to endothelial surface leading to a defect in movement of neutrophil to infected tissue site. The molecular defect is a complete or partial deficiency of CD18 resulting in lack of expression of CD11b/CD18. It is autosomal recessive. The clinical presentation includeds recurrent soft tissue infections (skin, mucous membranes), gingivitis, mucositis, peridontitis, cellulitis, abscesses, delayed separation of umbilical cord/omphalitis, and poor wound healing.

Question 34

Leukocyte adhesion deficiency II

Answer 34

Results in neutrophilia, decreased rolling on endothelial surface as a prelude to tight adherence. RBC are also affect from abnormal ABH antigens. The molecular defect is abnormal transferase resulting in abnormal fucosylation of adhesion molecules (causing other CNS problems) (Sialyl LeX) and poor interaction with selectins, autosomal recessive. The clinical presentation is recurrent infections, mental retardation, short stature, and craniofacial abnormalities.

Question 35

Actin dysfunction

Answer 35

results in decreased chemotaxis, ingestion, and spreading. Results in increase infections and poor wound healing. Defect in actin associated proteins can also cause similar problems.

Question 36

Specific granule deficiency

Answer 36

Results in decreased chemotaxis and microbicidal activity and milde neutropenia (abnormality in nuclear configuration). Have no specific granule. Results in recurrent skin and deep tissue infections. Molecular defect in failure to produce specific granules or their contents, defect in a transcription factor (CEBPε) results in decrease production of specific granule proteins, autosomal recessive.

Question 37

Myeloperoxidase deficiency

Answer 37

results in partial or complete deficiency of myeloperoxidase and mild defect in killing bacteria, significant defect in killing candida. The molecular defect is a post-translational modification defect in processing protein, autosomal recessive. Presentation is generally healthy, increase fungal infections when associated with diabetes.

Question 38

Chediak Higashi Syndrome

Answer 38

Results in neutropenia, giant granules (leaky) in all leukocytes, abnormal degranulation, and major defect in movement, also decreased degranulation and microbicidal activity. The molecular defects are alterations in membrane fusion with formation of giant, leaky granules and other metabolic abnormalities in microtubule assembly. It effects all cells with granules (neurons). CHS gene has been identified, autosomal recessive. The clinical presentations are oculocutaneous albanism, nystagmus photophobia, recurrent infections of skin, mucous membranes and respiratory tract by bacteria, lymphoproliferative phase associated with EBV infection, fever, hepatosplenomegaly and hemophagocytic disorder, and neurodegenerative syndrome.

Question 39

Respiratory Burst

Answer 39

plays an important role in the immune system. It is a crucial reaction that occurs in phagocytes to degrade internalized particles and bacteria. NADPH oxidase, an enzyme family in the vasculature (in particular, in vascular disease), produces superoxide, which spontaneously recombines with other molecules to produce reactive free radicals. The superoxide reacts with NO, resulting in the formation of peroxynitrite, reducing the bioactive NO needed to dilate terminal arterioles and feed arteries and resistance arteries. Superoxide anion, peroxynitrite, and other reactive oxygen species also lead to pathology via peroxidation of proteins and lipids, and via activation of redox-sensitive signaling cascades and protein nitrosylation. NADPH oxidase activation has been suggested to depend on prior PKC activation.[1] Myeloperoxidase uses the reactive oxygen species hydrogen peroxide to produce hypochlorous acid. Many vascular stimuli, including all those known to lead to insulin resistance, activate NADPH oxidase via both increased gene expression and complex activation mechanisms.

Question 40

Complement disorders

Answer 40

The complement system comprises a group of plasma proteins activated by lectins, bacterial proteins, or surface bound IgG through two different pathways (classical or alternative). Sequential proteolytic interactions of complement proteins in a cascade lead to activated fragments which attach to membranes opsonizing the target cells for phagocytosis or which serve as chemotactic stimulants. Activation of the terminal components (C5-9) forms an amphiphilic cylinder which is inserted into the plasma membrane of the target (bacterium, cell, etc.) permitting free flow of intracellular constituents and lysis. Deficiencies of factors 1q, 4, and 2 are associated with an increased risk of systemic lupus erythematosus and other autoimmune or inflammatory vascular diseases. Primary deficiency of C3 results in inefficient opsonization of bacteria and recurrent bacterial infections (pneumococcus, H. influenzae, etc.). Defects in components C5-C9 have an associated increase in risk for Neisseria bacteria (meningitis, arthritis, sepsis).

Question 41

Phagocytes disorder clinical findings

Answer 41

High rate of bacterial and fungal infections. Infections with atypical or unusual microorganisms (e.g., Aspergillus, disseminated candida, lymphadenitis due to Serratia and other gram negative organisms, infections with Cepacia Burkholderi. Catalase positive organisms in patients with CGD. Infections of exceptional severity. Peridontal disease in childhood. Recurrent infections in areas of the body, which interface with the microbial world. Infections occur at interface areas: skin (cellulitis, abscesses), sinopulmonary infections (pneumonia, sinusitis, gingivitis, ulcers, periodontal disease), perirectal infections. Deep infections may also occur.

Question 42

Complement disorders clinical findings

Answer 42

Bacterial infections, which might be seen with antibody deficiency (e.g., pyogenic organisms, H. influenzae, S. pneumoniae). Terminal complement deficiencies (C5-C9) have problems with Neisseria organisms.

Question 43

Phagocytes disorder screening tests

Answer 43

CBC, differential; Review of morphology; Bactericidal activity (put patient blood plus bacteria then measure amount of bacteria left to see ability to kill bacteria); Chemotaxis assay (chamber separated with filter, in bottom you put chemattratant, on top look at cells. If there is a chemtatic defect they will only move a little bit into the filter due to random movement); Expression of CD11b/CD18; NBT dye reduction or DHR oxidation (lode cell with non flurescent compound and then expose to PAMP and cleaves compound making fluresence compound, shows how much oxidation occurs).

Question 44

Phagocytes disorder confirmatory/detailed tests

Answer 44

Adherence to inert surface or endothelial cells. Measurement of CD11b/CD18, L-selection, Sialyl LeX; Response to chemoattractants: shape change, change in direction, rate of movement. Actin assembly; Ingestion of labeled particles or bacteria. Degranulation of specific and azurophilic components; Bactericidal/candidicidal activity; Production of O2-, H2O2 other oxidants; Studies for specific molecular defects in oxidase or other cell constituents.

Question 45

Complement disorder screening tests

Answer 45

C3, CH50; Quantitative Ig’s, Lymphocyte numbers.

Question 46

Complement disorder confirmatory/detailed tests

Answer 46

Measurement of specific complement components: alternative and classical pathways; detailed evaluation of adaptive immune response.

Question 47

Discuss management strategies for patients with innate immune disorders.

Answer 47

Anticipation of infection and aggressive attempts to define the causative agent. Surgical procedures for infected sites may be both diagnostic and therapeutic. Prompt initiation of broad spectrum antibiotics covering a wide range of organisms, switching to specific coverage when microbial diagnosis is known. For severe quantitative disorders of neutrophils, G-CSF may be used at a dose of 3 μg/kg/day to resolve the neutropenia (review from previous lecture). Specific syndromes of neutrophil dysfunction may benefit from prophylactic antibiotics or cytokine therapy (e.g., INFγ for CGD). Transplantation with hematopoietic stem cells has the capability to reconstitute neutrophil numbers and/or function. Gene therapy: proof of concept studies has demonstrated reconstitution. Specific problems need to be resolved before a practical solution is achieved.

Question 48

Stem cells

Answer 48

are undifferentiated cells which, when they divide, give rise on average to another stem cell and a daughter committed to differentiation. They vary in their potential; the fertilized ovum is the pluripotential stem cell, eventually giving rise to all other differentiated cells.

Question 49

The hematopoietic stem cell (HSC)

Answer 49

is more restricted, as it gives rise to all the hematopoietic cells, including the brain’s microglia, but no other cells (it is multipotential). The two possible differentiated daughters of the HSC are the common lymphocyte progenitor (CLP) and the common myeloid progenitor (CMP).

Question 50

common lymphocyte progenitor (CLP)

Answer 50

The CLP gives rise to B and T cell progenitors.

Question 51

common myeloid progenitor (CMP)

Answer 51

The CMP’s descendants include the progenitors of erythrocytes, megakaryocytes (from which platelets bud off), eosinophils, mast cells/basophils, and the common granulocyte/monocyte progenitor from which neutrophils and macrophages develop.

Question 52

B cell development

Answer 52

B cells in mammals develop in the Bone marrow. B cell progenitors can be identified as such when they begin to synthesize immunoglobulin components. The first to be detectable is mu chain in the cytoplasm; then complete cytoplasmic IgM (cIgM). This indicates that B cells choose the mothers or fathers heavy chain and rearrange their heavy chain genes before their light chains (less to recombined). The pro-B cell divides a few times. Then one of the light chain genes rearranges, making IgM (mu in constant region makes IgM)

Question 53

Pre-B cell

Answer 53

A cell with cytoplasmic IgM but no surface IgM. Next to appear is surface IgM (sIgM), which is an IgM monomer with an extra membrane-embedded extension at the end of its Fc, it is now an immature B cell.

Question 54

Mature B cell

Answer 54

Finally, when the cell is fully mature, both IgM and IgD (of the same specificity, of course) are found on the cell surface. All of this results from alternative splicing of the VDJ-mu-delta primary RNA transcripts. A functionally and diagnostically important point: an immature B cell has sIgM only; a mature B cell has sIgM and sIgD; and is ready to release antibody.

Question 55

INTRODUCTION TO T CELL DEVELOPMENT

Answer 55

T cells are very interesting. They carry out their development in three different locations: the bone marrow, then the Thymus, and finally the peripheral lymphoid organs. In the bone marrow one finds pre-T cells, which do not yet have the characteristic surface markers that distinguish T cells from other cells (differentiation antigens), but are committed to expressing them in the right environment. These go to the thymus, where they rearrange their receptor genes (V(D)J, very much like B cells do in the bone marrow, but a completely separate set of genes) and then are selected for their responsiveness to “self plus antigen.” While B cells see free antigen in solution, T cells only see antigen on the surface of another cell, which could thus be called an antigen-presenting cell.

Question 56

Pre T cells

Answer 56

Newly-arrived pre-T cells divide energetically at the thymus periphery, while they are trying to make T cell receptors (TCR); at this stage they express neither of the markers, CD4 and CD8, but soon those that successfully made TCR become CD4+/CD8+ “double positives.” As these filter through the thymus from cortex to medulla, they undergo selection. The result is a few mature T cells that get exported as CD4+ (only) helper T cells, or CD8+ (only) cytotoxic T cells. About 99% of thymocytes don’t survive selection, and die within the thymus.

Question 57

Define the Bursa of Fabricius, and discuss where its functions take place in mammals

Answer 57

Where B cells develop in birds. In mammals it develops in the bone marrow

Question 58

Describe the sequence of appearance of cytoplasmic and surface immunoglobulins in developing B cells. Using these data, derive a model that could explain self-tolerance at the B cell level (“clonal abortion”).

Answer 58

If an immature B cell (sIgM but no sIgD) is similarly exposed to antigen, this signal causes the cell to try receptor editing; if that fails it activates a suicide program (apoptosis), and dies. It partially explains why we do not make antibody to self. In the bone marrow pre- B cells are differentiating into immature B cells; you can imagine that any cell whose receptors happen to be anti-self will be likely to encounter self in the environment of the bone marrow, and it will either make a new receptor, or be deleted. If it does not encounter antigen (because its receptors are not against self) then it will mature further so that it expresses both sIgM and sIgD. Then, when it meets antigen, it will be stimulated, not deleted. Please note, though, that many anti-self B cells (usually to scarce antigens, not seen in the marrow; or with low affinity to more common antigens) escape clonal deletion and other measures are necessary to keep them from becoming activated.

Question 59

Describe the antibody response to a typical antigen in a primary and in a secondary response.

Answer 59

In response to secondary (booster) immunizations the IgM response is about the same as in a primary, but the IgG response, efficiently helped by T cells, is sooner, faster, higher and more prolonged. During primary (initial exposure) B cell responses to antigen, IgM is secreted first, then for most antigens, helper T cells get involved and there is a switch to IgG, or possibly to IgA or IgE. Therefore a cell that is producing IgG can no longer make IgM or IgD. The switch occurs at a DNA level therefore primary transcript is different. The helper T cells in the gut and lung preferentially drive the M to A switch. The ‘switch helper’ mechanism indicates that B cells in general do what helper T cells tell them to.

Question 60

Describe the relative IgG and IgM levels in a normal infant from conception to one year of age. Distinguish maternal from infant's antibodies.

Answer 60

The fetus makes IgM before birth, but only acquires the capacity to make IgG about 3-6 months postnatally. However, at birth the baby has as much IgG in its blood as does an adult; this IgG is maternal, because IgG crosses the placenta, by active transport, from mother to fetus (no other class of immunoglobulin does). The half- life of IgG is about 3 weeks, so in 7 half lives = 21 weeks after birth there is less than 1% of the starting amount of maternal IgG left; fortunately, the infant should be making reasonable amounts of its own IgG from about 12 weeks. IgA also starts about 2-3 months. Therefore from about 3-9 months they have the lowest number of antibodies. Only IgG is actively transported across the placenta.

Question 61

Discuss the decrease in diversity seen in the immune repertoire of older people.

Answer 61

Older people are more susceptible to most infectious diseases and usually get sicker and take longer to recover. The explanations are probably spread around just about all body systems; for example, the cilia in lungs beat less efficiently so bacteria aren’t as easily cleared. But T cells and B cells age, too, though the numbers in the blood do not decline. We know that the thymus gradually becomes replaced with fat, though there are islands of healthy-looking lymphoid tissue in it up to a great age. People can completely reconstitute their T cell numbers and diversity up to about 40 years of age, then diversity becomes increasingly limited, and more and more cells show a ‘memory’ phenotype while fewer are naïve; old people have fewer but larger clones than do the young. A similar change takes place in B cells, too, possibly a decade or two later. So older folks generally make good responses to antigens they saw in the past, but fail to respond well to completely new antigens. This may help explain why the recent SARS epidemic— featuring a brand new pathogen—was disproportionately fatal in the elderly, as is West Nile Fever, brand new in America since 1999. It may also suggest that flu shots in the elderly (unless they are cross-reactive with an earlier strain of virus) are not as useful as we would like to think. However, for the H1N1 virus formerly known as Swine, older folks had cross-reactive immunologic memory of a related virus, and generally did better than the young.

Question 62

Discuss the relative values of immunizing the young and the old in an epidemic of a novel respiratory virus.

Answer 62

It may not be worthwhile to vaccinate older people because they may not be able to become immune. If you vaccinate the young people then you don’t have to vaccinate the old.

Question 63

Lst the six main types of T cells

Answer 63

There are 5 main kinds of helper T cells, and one killer T cell. Let’s start with the helpers (so called because they ‘help’ other cells do things). They also all express the surface marker CD4.

Question 64

Th0

Answer 64

an undecided helper precursor cells. These cells are found in the paracortex of lymph nodes, and corresponding positions in other secondary lymphoid tissues. When their correct antigen is brought to them by dendritic cells (DC), they begin to divide and differentiate, becoming either Th1, Th17, Th2, Tfh, or Treg cells. The previous experience of the DC—the conditions in the periphery when it was stimulated, what TLR were engaged, what cytokines and chemokines predominated—is the main determinant of the Th0’s ultimate progeny. For most antigens you end up with some of each; the relative proportions decide the functional outcome of antigen exposure.

Question 65

Th1 cells

Answer 65

They were first called delayed hypersensitivity T cells, and that name is still sometime used. The modern practice is to simply call this cell Th1. After this cell has been activated and has proliferated in the lymph node, most of the daughters leave and circulate around the body. When they encounter antigen, say at the infection site, they secrete lymphokines. The most important lymphokine secreted by Th1 is interferon gamma (IFNg), which is pro-inflammatory, being chemotactic for blood monocytes, which become tissue macrophages. These cells move in large numbers into the area where the Th1 is recognizing antigen. They are also activated by IFNg, becoming classically-activated M1 or ‘angry’ macrophages which avidly ingest and kill bacteria or other foreign invaders. Th1 also secrete IL-2, which helps CTL (killer T cells) get fully activated after they recognize antigen.

Question 66

M1 macrophages

Answer 66

avidly ingest and kill bacteria or other foreign invaders. The macrophages release their own cytokines that intensify inflammation including tumor- necrosis factor alpha (TNFα) and IL-1. This division of labor is efficient¾the T cell recognizes, the macrophage removes¾but runs the risk of damage to local tissues by the enraged but imbecilic macrophages. It is useful to think of Th1 and Th17 as pro-inflammatory, leading to the accumulation of angry (classically activated) M1 macrophages at the site of infection; the tactic is a vigorous response to get dangerous pathogens under control quickly. This is highly desirable, but also can easily get out of control and if it becomes chronic can result in very significant tissue damage.

Question 67

Th17 CELLS

Answer 67

There is a newly-described, intensely-researched Th subset called Th17 because it makes the inflammatory lymphokine IL-17 among others. It resembles the Th1 in that its main job seems to be causing inflammation; not surprisingly, then, it has been implicated in several autoimmune diseases, as has the Th1. It must do something useful for a living, of course; and that is resistance to particularly difficult bacterial and yeast pathogens.

Question 68

Th2 CELLS

Answer 68

Activated Th2 cells derived from Th0 leave the lymph node as do Th1, and circulate through blood and lymph until they encounter their antigen again in the tissues. Here the IL-4 they make has other actions: it attracts and activates macrophages, but differently than IFNγ; such macrophages are called alternatively activated or M2, and are more involved in healing (debris removal, scar formation, walling off pathogens that angry macrophages have failed to kill). IL-4 is also chemotactic for eosinophils, cells specialized for killing parasites like protozoans and worms. So Th1 are the cells of active, urgent destruction of invaders, via the M1’s they stimulate; Th2 cells, which tend to appear a day or two later in sites of inflammation, are involved via M2’s in repair and healing. As the yin and yang of T cell immunity they are an awesome pair.

Question 69

FOLLICULAR HELPER T CELLS, Tfh

Answer 69

Soon after the arrival of antigen-presenting DC in the lymph node, some activated Th cells can be seen migrating into the follicles of the cortex, where B cells are abundant. These are referred to as follicular helpers, and their role is to help B cells that have recognized antigen become activated and differentiate into antibody-secreting plasma cells. Tfh secrete a variety of cytokines, and by direct contact they stimulate the B cells to switch from secreting IgM, to IgG, IgA, or IgE. They tend to be heterogeneous; the Tfh in the gut, for example, switch B cells preferentially to IgA; those in spleen switch B cells to IgG. It’s not yet clear whether Tfh are a separate lineage, or if some Th1 and some Th2 cells acquire the surface marker (the chemokine receptor CXCR5) that allows them to go into the follicle as Tfh, in this theory: Th1-like Tfh would orchestrate aggressive resistance through classically activated macrophages, killer T cells, and help for the complement-activating antibody classes. Th2-like Tfh would orchestrate healing, worm-killing, and walling-off via alternatively activated macrophages and eosinophils that they attract directly, or indirectly through activated mast cells triggered by Tfh-helped IgE production. The existence of these cells reminds us that the antibody you make (or don’t make) may be as much a read-out of T cell function as of B cells. If Tfh cells can’t communicate correctly with B cells, for example, you may have difficulty making any antibody class, especially those downstream from IgM.

Question 70

REGULATORY T CELLS, Treg

Answer 70

A small population of cells (about 5% of all Th cells) has been identified whose main job is to suppress the activation and function of all other Th cells; they are our 5th helper T cell type. Most regulatory T cells have the phenotype CD4+/CD25+, and make the transcription factor FoxP3. Surface CD4 puts them in the helper family. They produce TGFβ and IL-10. They are very potent; one can suppress 1000 Th cells. Mice that lack Treg, or part of their signaling pathways, get autoimmunity, and so do rare people with a similar genetic defect. But even without a (known) genetic problem inadequate Treg function is common and leads to overactive immune responses and self-reactivity. Although Treg respond specifically to their corresponding antigen, their suppression of other T cells is not antigen-specific; any nearby Th is suppressed.

Question 71

CYTOTOXIC (KILLER) T CELLS, CTL

Answer 71

In the few minutes of CTL-target contact, the killer gives the target the ‘kiss of death’ or lethal hit. It has signaled the target to commit suicide by activating a physiological cell death process (called apoptosis) that leads to rapid DNA fragmentation and nuclear collapse (this would be useful in preventing virus replication). There are two ways a CTL can signal a cell to undergo apoptosis. It can engage the ‘death receptor’ Fas (CD95) on the target (CTLs bear the Fas ligand, CD95L). Cross-linked Fas activates a latent apoptosis pathway. Or it can secrete the contents of certain ‘lytic granules’ which contain proteases called granzymes, and other proteins called perforins which seem to allow the penetration of the granzymes into the target cell. These proteases trigger apoptosis. CTL are activated in the lymph nodes after contact with an antigen-bearing DC. They also require, for activation, help from Th1 in the form of IL-2, and other factors for conversion into memory cells.

Question 72

SUBPOPULATION MARKERS

Answer 72

To distinguish the T cell subpopulations physically from each other and from B cells, we take advantage of unique surface molecules. We have antibodies against these, most of them monoclonal antibodies made in mice. These antibodies can be tagged with a fluorescent molecule to make it easy to see which cells they bind to.
B cells are readily distinguished using antibodies to immunoglobulins or their chains, or to the surface marker CD20. The most useful molecules on T cells are CD3, CD4, and CD8. CD stands for “cluster designation”. CD3 is on the surface of virtually all T cells; CD4 (they recognize MHC II) is on T helpers, CD8 (they recognize MHC I) is on CTL. These molecules play a role in T cell activation. There are no reliable surface antigens to distinguish Th1 from Th2; you have to look at the lymphokines they make.

Question 73

Cytokine

Answer 73

are important in cell signaling. They are released by cells and affect the behavior of other cells, and sometimes the releasing cell itself. Cytokines include chemokines, interferons, interleukins, lymphokines, tumour necrosis factor but generally not hormones or growth factors (despite some terminologic overlap). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell.

Question 74

Lymphokine

Answer 74

a subset of cytokines that are produced by a type of immune cell known as a lymphocyte. They are protein mediators typically produced by T cells to direct the immune system response by signalling between its cells. Lymphokines have many roles, including the attraction of other immune cells, including macrophages and other lymphocytes, to an infected site and their subsequent activation to prepare them to mount an immune response. Circulating lymphocytes can detect a very small concentration of lymphokine and then move up the concentration gradient towards where the immune response is required. Lymphokines aid B cells to produce antibodies.

Question 75

Chemokine

Answer 75

a family of small cytokines, or signaling proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines.

Question 76

interleukin 4 (IL-4)

Answer 76

stimulates class-switching in B cells and promotes their synthesis of IgE antibodies. acts as a positive-feedback device promoting more pre-Th cells to enter the Th2 pathway.

Question 77

lymphokines made by Th1

Answer 77

secretes interleukin 12 (IL-12), TNF-β, IFN-γ

Question 78

lymphokines made by Th2

Answer 78

IL-4, IL-13, IL-5

Question 79

mphokines made by Th17

Answer 79

release interleukin 17

Question 80

lymohokines made by Treg cells

Answer 80

IL-10 & TGF-β

Question 81

ANTIGEN PRESENTATION TO T CELLS

Answer 81

When an antigen enters the body¾ let’s use a virus as an example¾ it will infect locally, cause an innate response, and eventually it or its breakdown products will get ingested by a dendritic cell. Within the endosome viral proteins are broken down to peptides. The endosome fuses with other vesicles which have MHC molecules embedded in their membrane, facing in. Some of the peptides associate with the MHC molecules. The endosome recycles to the cell’s surface and fuses to the plasma membrane, thus exposing MHC molecules bearing antigenic peptides to the outside world. These are antigen-presenting cells, APC, and the mechanism the extrinsic pathway because it involves antigen from outside the APC. Dendritic cells are the best at this. It’s this MHC- antigen complex that is presented to the receptor of an appropriate helper T cell. The crystallized MHC shows two chains (α, β) folded so that a sort of cleft appeared in the end that would be facing towards a T cell. The cleft’s base is a beta sheet and its sides are two alpha helices.

Question 82

mitogen

Answer 82

Lectins are proteins made by many life forms including us, plants, and invertebrates. They have affinity for certain sugars usually ones that the organism they come from doesn’t itself have. They may function as primitive “immune” molecules, gumming up the surfaces of foreign invaders. Curiously, some of them bind to and stimulate T and B cells, and they are thus very useful in the research and clinical labs. For example, phytohemagglutinin, PHA, a bean lectin, stimulates all T cells to divide because it binds to CD3, fooling the cell into thinking it has bound antigen. Because it stimulates T cell mitosis it is called a mitogen. Pokeweed mitogen (PWM) stimulates both T and B cells (nonspecifically) to divide. The usefulness of these agents is considerable. For example, to see a person’s karyotype (metaphase chromosome picture) you need dividing cells. Take some blood leukocytes, add PHA, and in three days or so you have all the lovely metaphase mitotic figures you could want. Or if you want to see if someone’s T cells can divide normally, or make IL-4, but you don’t know what antigens she’s immune to, just add some PHA to her blood cells; it will stimulate all helpers and CTL without regard to their antigen specificity.

Question 83

Compare and contrast the antigen receptors of T and B cells.

Answer 83

Because T cells see antigen only when it is complexed with cell-surface MHC molecules, T cells focus their attention on cell surfaces, and do not interact with free antigen; that is a job for the B cell and its antibodies.

Question 84

T CELL RECEPTOR

Answer 84

The T cell receptor for antigen (TCR) is structurally reminiscent of antibody, and sequence data indicate common ancestral genes long ago. The two chains are called alpha and beta (don’t confuse these with the α and β of Class II MHC), and each has a constant and a variable portion. The T cell makes its receptor out of V, (D) and J regions recombined as in B cells, and like antibody, each chain has 3 CDRs; the process takes place in the thymus. Both alpha and beta chains have transmembrane domains, unlike surface Ig, in which only the heavy chains are transmembrane. Intimately associated with the TCR is a complex of molecules called CD3; it has at least 5 chains. It serves to transduce TCR signals for the T cell. This means that when a T cell binds the correct antigen + MHC with its TCR, the actual signal that turns the T cell on is transmitted by CD3. This complexity implies that careful control of the process is required. When a Th0 binds to a good APC like a dendritic cell, the DC gives the T cell an activating boost by secreting cytokines. The T cell thus gets two “hits”¾one via its TCR/CD3 complex, and one from dendritic cell cytokines. In addition, the T cell gets a
whole range of activating signals from interactions
between molecules on its surface and corresponding
molecules on the APC. If it doesn’t get all the right signals, it
may be turned off instead of on. Biochemical events
follow that are typical of cells being stimulated: a rise
in intracellular calcium, breakdown of membrane
phospholipids, and activation of protein kinases which
in turn activate transcription factors. IL-2 receptors are
upregulated. The cell goes into cycle (proliferation)
and begins to secrete lymphokines (differentiation).
What transcription factors, and what lymphokines, it
makes are largely determined by the information given the Th0 by the dendritic cell.

Question 85

cross-presentation

Answer 85

The dendritic cell, which gets everything going, is special in that it allows peptides from antigens it’s eaten to leak over into its “intrinsic” pathway, so that it can present them on Class I as well as Class II MHC at the same time. Thus it can bring samples in from the periphery and arrange not just for a Th response, but also for CTL.

Question 86

CD4

Answer 86

is on Th1, Th17, Treg, Tfh, and Th2. It binds to MHC Class II on the APC the T cell is interacting with; not to the peptide- binding cleft, but to the unvarying “base” which is the same in everybody. Thus when a Th is seeing antigen + Class II (as it should), the CD4 will help by increasing the strength of the bond.

Question 87

CD8

Answer 87

binds to the base of Class I, increasing the binding affinity of CTL to antigen + Class I. Without prior TCR binding, the CD molecules binding to MHC can’t activate the cells¾that would lead to chaos. They just increase the affinity of cell binding that got started by specific recognition. CD4 and CD8 also transduce some activating signals.

Question 88

T cells helping B cells

Answer 88

The B cell binds the epitope that its receptor is specific for, on a foreign antigen. It then endocytoses the bound molecule, which is broken down in the endocytic vesicle. Peptide fragments bind to MHC Class II molecules brought in by other vesicles that fuse with the endosome, and the MHC-peptide complex moves to the surface; the B cell now displays antigen + Class II. Eventually, along comes the correct Tfh and sees its epitope + Class II on the B cell’s surface. It binds and focuses surface interactions and helper lymphokines on the B cell. Note that the epitope that the T cell sees does not have to be the same as the one the B cell saw, and it almost never is.

Question 89

MHC-restriction

Answer 89

T cells are restricted in their recognition of antigen, to antigen on the surface of cells (here, the target cells) genetically identical to themselves. That is, they do not “see” antigen alone, but only antigen presented to them on the surface of a genetically-identical cell. The T cell and the antigen-presenting cell must come from individuals who share alleles at a group of genetic loci collectively called MHC (for Major Histocompatibility Complex), which code for surface glycoprotein molecules. Another way of saying this is that the T cell is MHC-restricted. MHC antigens are very variable, that is, there are thousands of alleles in any population. The chances of yours being exactly the same as mine (or of A’s being the same as B’s) are small. The major histocompatibility complex is a large group of genes whose products have related functions. There are two kinds of MHC molecules: Class I and Class II. Class I products are on all nucleated cells. Class II products are expressed on the surfaces of dendritic cells, macrophage-type cells, and B cells, all of which are involved in some way in presenting antigenic peptides to Th cells. When antigen is endocytosed and presented by a dendritic cell (DC) it associates with Class II MHC molecules in the endocytic vesicle (the extrinsic pathway), and these complexes are what the DC presents to the T cell. Th1, Th17, Tfh, Treg, and Th2 are selected to recognize peptides on Class II molecules. Class I MHC molecules, on the other hand, associate best with peptides that are sampled from proteins synthesized within the cell itself, not taken up by endocytosis; this is the intrinsic pathway. Most peptides would be from normal ‘self’ proteins, but antigens would include abnormal (mutated) molecules and especially proteins produced by internal pathogens such as viruses. CTL are programmed to see antigen in association with MHC Class I molecules.

Question 90

Describe the role of T cells in ridding the body of a viral infection

Answer 90

Cytotoxic T cells find and destroy infected cells that have been turned into virus- making factories.

Question 91

Describe the characteristics of T-independent antigens

Answer 91

Some antigens are just as good with or without T cell help; they are thus T-independent. They tend to be molecules with the same epitope repeated over and over; rare in proteins but common in large carbohydrates like, for example, the capsular polysaccharides of Streptococcus pneumoniae. The response to T-independent antigens is almost all IgM; T cell help is needed to switch over to IgG, IgA, or IgE. This is important because it means that even if people are extremely deficient in T cells they will be able to make some antibody to carbohydrates. With protein antigens, a little IgM and no IgG is made without T cell help.

Question 92

Histocompatibility

Answer 92

describes when living tissue between two individuals are compatible. Compatibility is influenced by a region of genetic loci. If a tissue is rejected, these region codes for histocompatibility antigens, some of which are expressed on the surfaces of all nucleated cells. Studies in several species have shown that they all have their strongest histocompatibility antigens coded for by a family of genes on a single chromosome; the group was therefore called the Major Histocompatibility Complex, or MHC. In mice the MHC is H-2; in humans it is HLA (Human Leukocyte Antigen, because it was so easy to get leukocytes for typing). The most important loci within HLA are HLA-A, HLA-B, and the HLA-D group, of which DR is the one most of greatest concern to transplanters. An extraordinary feature of the human histocompatibility antigens is their genetic polymorphism (very large number of alleles at each locus within the species); the odds against any two unrelated humans having exactly the same combination of antigens might be about 1,000,000:1. No other genetic system is so diverse.

Question 93

MHC ANTIGEN STRUCTURE

Answer 93

Both class I and class II antigens are glycoproteins composed of two polypeptide chains. Class I antigens consist of an allelically variable chain associated with an invariant chain called beta2-microglobulin. Both of
the class II chains (α and β) are variable. There is enough sequence homology between classes I and II, and immunoglobulins and T cell receptors, to indicate that they all arose from a common ancestral gene, the famous immunoglobulin domain. It’s fascinating to consider that the molecule that does the recognizing, and the molecule that’s recognized, originated in the same early structure. HLA-A and -B (and -C) produce molecules which are very similar in structure and function, and one arose from the other by gene duplication. Humans have triplicated (at least) Class II loci as well, so that we have DR, DP, and DQ. We often just say ‘HLA-D’ when we’re thinking Class II; in most cases we really mean HLA-DR, the most significant in solid organ transplantation.

Question 94

Alloantigen

Answer 94

an antigen present only in some individuals (as of a particular blood group) of a species and capable of inducing the production of an alloantibody by individuals which lack it.

Question 95

haplotype

Answer 95

a contraction for haploid genotype. A haplotype is a collection of specific alleles (particular DNA sequences) in a cluster of tightly-linked genes on a chromosome that are likely to be inherited together. Put in simple words, haplotype is the group of genes that a progeny inherits from one parent.

Question 96

Distinguish Class I and Class II histocompatibility antigens

Answer 96

Several loci encode components of the complement system and certain cytokines; some people call these MHC Class III. HLA-C is a relatively minor Class I locus, A and B are more important Class I loci. D is class II. Like most genes, each of these loci is expressed codominantly, so that at the HLA-A locus you have both paternal and maternal alleles expressed, etc. A typical phenotype then might be HLA- A1, A3/ HLA-B6, B7/ HLA-DR3, DR4, as it is for the father in this example. The MHC gene set that you inherited from one parent is called a haplotype. The cells show their phenotypes, the actual proteins expressed on the surface of their cells. Every cell expresses both alleles. Note that since MHC Class II antigens (called here ‘HLA-D’ for simplicity) are displayed, the cells shown are antigen-presenting cells: dendritic cells, macrophages, or B cells.

Question 97

Identify the chromosome on which the MHC is found in humans

Answer 97

Chromosome 6

Question 98

Class I

Answer 98

HLA-A, HLA-B

Question 99

Class II

Answer 99

HLA-DR, HLA-DP, HLA-DQ

Question 100

Discuss HLA-A and B typing in terms of how many antigens a person expresses at each locus; given two unrelated parents' haplotypes, predict their children's phenotypes.

Answer 100

Typing at the HLA-A and HLA-B loci used to be done by treating the patient’s leukocytes with a panel of antisera to specific HLA alleles, and complement. If the cells expressed the allele that the antibody recognized, the complement lysed them, which was easy to observe. Nowadays, it is actually easier (and much more informative) to sequence the HLA genes themselves for typing.

Question 101

Distinguish between “HLA-D” and HLA-DR, -DP, -DQ

Answer 101

HLA-A and -B (and -C), the ‘original’ transplantation antigens, were fairly simple to study because they are on all nucleated cells and antisera to them were quite easily obtained. They are the human Class I MHC loci. But in 1965 Fritz Bach noted that most grafts perfectly matched at HLA-A and -B were nevertheless rapidly rejected; there must be another major locus involved. By accident it was observed that if leukocytes from the donor were mixed in cell culture with leukocytes from the recipient, there was, after a day or two, a burst of cell division in the culture, the MIXED LEUKOCYTE REACTION, or MLR, was positive. T cells were recognizing, and being stimulated to proliferate by, an antigen on the other person’s white cells that Bach did not have antisera against. Further studies identified the antigens being recognized as Class II MHC. The MLR in this form doesn’t help us know how strongly the recipient’s T cells are responding against donor Class II, because the recognition is bidirectional. But for transplanting what we want to know is: How strongly do the recipient’s T cells recognize the Class II of this potential donor versus that one? So we create a ‘one-way’ MLR, in which the cells from the donor are treated (DNA synthesis inhibitors or radiation) to prevent their division (after all, you really want to know, can the recipient recognize the donor’s MHC?). What you then observe is recipient’s Th cells dividing in response to the donor’s HLA-D (mostly DR, on monocytes). A strong reaction may preclude doing the transplant.

Question 102

Graft rejection

Answer 102

Grafts are rejected by T cell mechanisms with which you are already familiar, the most important being Th1 cells (via their lymphokines and the monocyte/macrophage inflammatory response) and CTL. Macrophage-derived inflammatory cytokines makes the reaction even more intense. As we have developed better immunosuppression regimens and drugs, graft survival has increased and morbidity decreased; but it’s almost never been possible to taper and stop a patient’s drugs without rejection. Antibody and complement are not thought to be important rejection mechanisms, except in hyperacute rejection. In this case, a graft is given to a patient who has preexisting antibody, IgG or IgM, to it (either to its HLA, because of a prior graft or transfusions, or, in a mismatch, to ABO blood group antigens). Antibody immediately binds to the endothelial cells of the graft’s blood vessels. Complement is activated and vasospasm results, via anaphylatoxins and histamine; the organ may never even become perfused with blood (a ‘white graft’). This catastrophe can be avoided by making sure there are no cytotoxic (complement-activating) antibodies in the serum of the recipient when tested on the donor’s leukocytes.

Question 103

T CELL INTERACTION IN REJECTION

Answer 103

Th1 recognize foreign MHC antigens of the Class II, HLA-DR loci; killer T cells (CTL) recognize foreign MHC antigens of the Class I, HLA-A and HLA-B loci. In rejection, what first happens is that Th1 cells recognize foreign HLA-DR on graft cells. Remember, not all cells express HLA-DR; the cells which do so are primarily macrophages and dendritic cells, of which most grafts have plenty. The Th1 proliferate (the phenomenon which is measured in the mixed leukocyte reaction). They will also secrete lymphokines (like IFNg) that attract a macrophage inflammatory response. The macrophages will mostly be the graft recipient’s. Meanwhile, CTL nearby are recognizing foreign HLA-A and HLA-B, which are on all the graft cells; this recognition is usually insufficient to activate them, though; they also require Th1- derived IL-2 as a second signal. Once activated, the CTL become highly cytotoxic; they may proliferate although they don’t have to, and they start killing cells in the graft. This whole sequence of events is exactly parallel to the recognition of antigen in a normal immune response, for example to a virus. The difference is that in a normal response, it’s peptide plus self-MHC that’s recognized; in rejection, it’s foreign MHC. If the donor and recipient are identical at HLA-A and B and different at HLA-DR, you will activate Th1, but no CTL will be generated (because there’s no antigenic difference between donor and host Class I). You will still reject the graft, but since only Th1 and not CTL are involved, rejection may be slower. If the donor and recipient are different at HLA-A/B but identical at HLA-DR, you will get no MLR (that is, no Th1 will be activated), so no IL-2 will be generated, and fewer CTL will be activated. So a good DR match is the most desirable thing, if you can find it.

Question 104

LINKING ANTIGEN RECOGNITION AND ALLOREACTIVITY

Answer 104

Th cells normally see MHC Class II + peptide; they also are the ones that see foreign MHC Class II. CTL normally see peptide + Class I, and they also can see foreign Class I. The recognition of foreign MHC is a chance cross-reaction; the receptors are actually selected to recognize self-MHC + antigen (Figure 4, below). In fact, if you make a cloned T cell line specific for the donor’s own HLA plus antigen X, the same cells can quite often be shown to also react with some foreign HLA (probably loaded with a self peptide, antigen Y).

Question 105

HLA-ASSOCIATED DISEASES

Answer 105

Many diseases have been shown to be associated with a particular HLA allele. These can be Class I or Class II. Let us take ankylosing spondylitis as an example. This is an arthritic condition in which there is inflammation of the insertions of tendons into bones, and the fibrous joints of the spine and pelvis, followed eventually by calcification so that affected joints may become inflexible (ankylosed). About 92% of people with ankylosing spondylitis are HLA-B27 (8% of people in the USA without the disease are B27). Your risk of getting the disease is 90 times greater if you are B27 than if you are not. Rats made transgenic for the human HLA-B27 gene develop arthritis similar to ankylosing spondylitis. Alternatively, the antigen might cross-react with B27, so that a response to the foreign antigen might somehow lead to autoimmunity There is some intriguing evidence of cross-reaction between certain Klebsiella bacteria and B27 (the ‘arthritogenic peptide’ hypothesis). Finally, HLA-B27 protein is prone to misfolding; could this, like a prion, cause inflammation? The second most-relevant risk locus is the ERAP1 gene, coding for an endopeptidase that affects how HLA-B27 loads with endogenous peptides; this could be seen as supporting the misfolding idea.

Question 106

Type 1 diabetes

Answer 106

Another interesting association is between HLA-DR3 and -DR4 and Type 1 juvenile (insulin- dependent) diabetes. The relative risk factor is 5 if a child has one of these antigens. HLA-DR2 seems to protect against Type 1 diabetes. In all these cases, the real culprit seems to be certain HLA-DQ alleles which are in strong linkage disequilibrium with those HLA-DR alleles.

Question 107

neoantigens

Answer 107

A growing body of evidence suggests that modifications of self-proteins (citrullination and deamidation in rheumatoid arthritis (RA) and celiac disease, respectively) may create novel epitopes that associate strongly with certain MHC alleles; the cells that respond to these ‘neoantigens’ cross-react with the normal protein. These modifications are probably more environmental than genetic; RA is associated with airborne pollution and smoking, and celiac with consumption of the grain protein group called gluten. In genome-wide surveys of single-nucleotide polymorphisms in various immunological diseases, by far the most “hits” are in the MHC. Clearly many genes affect disease risk, but the immune system is always walking on eggshells, trying to attack foreign molecules effectively while avoiding reaction against self.