blood and lymph2.2b

Question 1

T CELL-MEDIATED IMMUNITY & DELAYED HYPERSENSITIVITY.

Answer 1

Delayed-type hypersensitivity (DTH) is an old term for T cell-mediated events that are considered undesirable or injurious. When the very same mechanisms produce helpful immune responses, they are often referred to as (T) cell-mediated immunity. Since its hard to decide whether a reaction is good or bad, it is most reasonable to just say “T cell-mediated” or “Type IV” mechanisms. They are the only type of immunopathology (of Types I, II, III and IV) which do not require antibody or B cells. Since nothing in medicine is simple, real diseases often involve both Type IV and antibody-mediated phenomena; the underlying problem may be disordered immune regulation.

Question 2

Some examples where Type IV represents all or most of the mechanism

Answer 2

Rejection of allografts,
Graft-vs.-host disease (GvHD) - the reverse of allograft rejection, A positive tuberculin skin test,
Resistance to Mycobacterium tuberculosis,
Resistance to fungal infections,
Contact dermatitis, e.g., poison ivy,
Chronic beryllium disease, and Many autoimmune diseases, e.g. multiple sclerosis
Tumor immunity

Question 3

IMMUNIZATION AND EFFECTOR PHASES.

Answer 3

This is mostly a review of T cell mechanisms we’ve already considered, but now it’s very important that we have a clear understanding of the difference between the initiation of an immune response following first exposure to the antigen (immunization phase), and the elicitation of a reaction in a person who is already immunized (effector phase).

Question 4

Immunization phase

Answer 4

an immune response following first exposure to the antigen.

Question 5

Effector phase

Answer 5

elicitation of a reaction in a person who is already immunized.

Question 6

Tuberculin skin testing.

Answer 6

The Mantoux skin test is most commonly used in the USA. In it, 0.1 mL of PPD—purified protein derivative, a standardized preparation of M. tuberculosis antigens—is injected intradermally. The antigen is taken up by local macrophages and dendritic cells, and presented on MHC Class II. If the subject has an expanded number of anti-tuberculosis Th1 memory cells, they will come by and get stimulated, produce IFNγ, and attract macrophages. The test is read at 48 hours, and the diameter of the induration (firm raised part) is measured; 15 mm is always positive, and 10 or even 5 mm can be called positive under certain conditions, for example if a person is partly immunosuppressed. The induration is significant, since it represents a cellular infiltrate. One activated Th1 can attract 1000 macrophages, so these, not Th1, would be the predominant cell if you biopsied the site at 48 hours.

Question 7

Immunization to TB antigens

Answer 7

Immunization to TB antigens normally happens during a primary infection, which is usually unapparent to the patient, so a positive routine skin test usually comes as a surprise. Exposure to other species of Mycobacteria can occasionally produce a positive skin test. In many countries, Bacille Calmette-Guérin (BCG) vaccine—it is live attenuated bovine tuberculosis bacteria—is used, and most people so immunized have positive (cross-reactive) PPD skin tests.

Question 8

Initiation with poison ivy

Answer 8

Initiation with poison ivy, an example of contact dermatitis due to the oil of Toxicodendron (formerly Rhus) radicans. It contains the compound urushiol, which can penetrate intact skin and become associated with MHC on dendritic cells (either by binding directly to MHC, or by binding peptides which then get presented on MHC). The dendritic cell travels to the draining lymph nodes, where it presents its MHC plus antigen to the appropriate Th0 precursors, which develop into Th1 and Th17 cells. These begin to divide in the usual way, but by the time increased numbers of them are in the circulation, the antigen has been washed or worn off the skin, and there is no reaction. So at the time you became immunized (older word: “sensitized”) you probably didn’t know it happened.

Question 9

Elicitation of poison ivy

Answer 9

You again encounter poison ivy plants. The oil rubs off on your skin and urushiol again associates with MHC on antigen-presenting cells. This time though, memory T cells from the expanded clones are throughout the body, and get activated in the area where the oil has been deposited. They secrete interferon-γ which attracts and activates a large number of macrophages. The result is a firm red area of inflammation that, because of all the cellular events that need to take place, begins to be visible in 6 to 12 hours, and peaks at 24 to 48 hours, thus earning the label delayed- type hypersensitivity. Breakdown of the skin often leads to blistering.

Question 10

Memory T cells in elicitation

Answer 10

Memory T cells in elicitation are persisting cells in a clone that was expanded by contact with antigen. The key thing is that there are more of them than in a naïve person. They also have a lower activation threshold, so that it takes less antigen for elicitation of a reaction than it did to immunize in the first place.

Question 11

T CELL-MEDIATED IMMUNITY IN VITRO.

Answer 11

The lab can do a variety of tests. Whole blood or isolated white blood cells (both T cells and APCs like monocytes are needed) may be incubated with antigen in cell culture, and activation observed: one could count cell numbers for proliferation, look at cell size for activation (“blast transformation”), or at DNA synthesis using radiolabeled precursors. Cytokines released into the medium can be quantified, too. None of these is a routine test, however, except for the QuantiFERON-TB Gold test

Question 12

QuantiFERON-TB Gold test

Answer 12

QuantiFERON-TB Gold test is new, very nice, and is preferred to skin testing when the subject has had BCG immunization. Purified M. tuberculosis (human-specific) proteins are added to a sample of whole blood, and after incubation, interferon-γ is measured in the medium by an ELISA assay. Unlike the skin test, it remains negative in people vaccinated with BCG, (no cross-reactive antigen with BCG,) allowing you to distinguish infection from previous immunization.

Question 13

CYTOTOXIC T LYMPHOCYTES IN DTH.

Answer 13

There is no in vivo test for them. They probably take part in many manifestations of T cell-mediated immunity, and are quite important in many autoimmune diseases, tumor immunity, and transplant rejection. To demonstrate their presence, we need a suitable target cell (for example, an antigen-presenting cell exposed to the antigen, or any cell infected by it, if that is possible; sometime, normal cells can be soaked in an epitope-sized peptide which associates directly with MHC without having to be processed.) These are then mixed with the patient’s T cells (or purified CD8 cells) and after several hours, target cell death is measured, usually by the release of intracellular contents.

Question 14

CONTACT DERMATITIS.

Answer 14

Also called contact hypersensitivity or contact sensitivity or, incorrectly, contact allergy; ‘allergy’ should be reserved for IgE-mediated events. The classic example of this is poison ivy, but many other chemicals can cause it; the main requirements are that they pass through intact skin to reach antigen-presenting cells, and they associate with MHC Class II. Metals like nickel (used in plated goods, including jewelry, watch straps, garters); chemicals like paraphenylenediamine, the only permanent hair dye; latex in gloves; topical antibiotics like neomycin and bacitracin; plants, including poison oak and poison sumac; soaps, detergents and industrial chemicals. How do you treat these? Avoidance, and topical steroid creams or ointments.

Question 15

The TB skin test

Answer 15

The TB skin test implications about memory cells: The dose of PPD needed to elicit a positive reaction in an immune person is far lower that would be required to immunize him or her. Therefore, TB skin tests are not immunizing, and they can be repeated regularly without the subject becoming positive. Memory cells are long-lived, and after immunization with vaccine or by infection you may stay skin-test positive for years, though not necessarily forever. Exposure to many other environmental antigens can produce delayed-type hypersensitivity. So to determine if a patient has normal T cell function, we perform skin tests just like the Mantoux test, using a panel of common antigens, which may include tetanus toxoid, Candida (yeast) extract, mumps antigen, PPD, streptococcal proteins, and Trichophytin (from a common skin fungus). Studies have shown that over 95% of adults will have a positive DTH response to at least one of these, so a negative panel suggests “anergy” and requires follow-up investigation.

Question 16

GRAFT REJECTION.

Answer 16

Rejection is a complex phenomenon eventually involving most or all of the specific immune and nonspecific amplifying elements of the immune system. Allograft immunity shows specificity and memory.

Question 17

First set graft rejection

Answer 17

A skin graft from mouse strain A to strain M is rejected in 10-20 days. Remember that the recipient will have 5-10% of its T cells able to react with the foreign MHC, even before grafting, because some foreign MHCs look like self MHC + a peptide. It is these cells that cause graft rejection in 10-20 days. And as this process proceeds, the recipient’s response to A histocompatibility antigens is boosted, and it develops more anti-A Th1 and CTL.

Question 18

second set graft rejection

Answer 18

Another A skin graft is placed on same M recipient. It is rejected in 5-10 days. This is a secondary response and results from T cell memory developed during the first exposure, which is specific because a first graft from unrelated strain C will be rejected in 10-20 days.

Question 19

Hyperacute or “white graft” reactions.

Answer 19

If you keep putting A grafts onto B, eventually they will be rejected even before they heal in, that is, they stay white and bloodless. This is due to the development of antibodies to histocompatibility antigens. Hyperacute rejection is common when xenografts (from another species) are attempted. It’s usually because of pre-existing antibody to ubiquitous carbohydrate epitopes which are present in the foreign species but not in the human. People are going so far as to try to breed transgenic pigs that lack these carbohydrates, as potential organ donors for human patients.

Question 20

AUTOIMMUNE DISEASES.

Answer 20

Many conditions are clearly autoimmune, and T cells are involved in the pathogenesis. Some of these conditions also involve autoantibodies, and thus there is both Type II and Type IV immunopathology. Which comes first, or is most important? That is still controversial. For example, MULTIPLE SCLEROSIS, the demyelinating disease in which T cell reactivity to an autoantigen (myelin basic protein) was first shown, responds to therapies directed at T cells, such as the humanized monoclonal antibody natalizumab. But it also responds to the B cell-depleting monoclonal rituximab.

Question 21

is the brain an antigen?

Answer 21

The brain is, in fact, antigenic in its owner, but not immunogenic. So as long as you keep it in your head, you should not have a problem. Even if T cells find their way into the normal brain, they will not be stimulated, because that would require (at least) professional antigen-presenting cells, an innate response, and cell damage; common in skin but not in the well-defended brain. if you make brain into an immunogen by presenting its antigens to your immune response in the ‘proper’ way (that is, so they can be picked up by dendritic cells and carried to lymph nodes) then you will make activated T cells and they will have no trouble entering and attacking the corresponding organ, even if we have always thought that cells there were “sequestered,” for example behind the blood-brain barrier.

Question 22

MOLECULAR MIMICRY.

Answer 22

Several groups have studied the way myelin basic protein peptides sit in MHC Class II, and analyzed the distribution of positive and negative charges over the surface that would contact a T cell receptor. Using the information to scan a database of microbial proteins they have found several cases where a viral peptide, whose sequence is not necessarily the same as the MBP sequence, but which has close structural similarity, that is, distribution of charges and hydrophobicity, acts as a strong stimulator of clones of T cells derived from MS patients. A prior infection from a virus could activate T cells against myelin.

Question 23

HASHIMOTO THYROIDITIS

Answer 23

is characterized by a destructive attack by T cells on thyroid antigens. Almost 1.5 million people in the US have the disease, the most common cause of hypothyroidism. Like many autoimmune diseases, it has a familial tendency; and families with it also have increased incidence of other autoimmune diseases, like type 1 diabetes, vitiligo, and gluten-sensitive enteropathy (celiac disease). It is about 5 times more prevalent in women than men. Although most investigators think the T cells are pathogenic, Hashimoto’s also involves anti-thyroid antibodies, whose presence is commonly used to confirm the diagnosis. A variety of environmental agents have been proposed, to explain the huge increase in incidence in certain regions (notably, Sicily).

Question 24

SJÖGREN SYNDROME

Answer 24

is said to be the second most common autoimmune disease, but that’s only an estimate as it is difficult to diagnose; symptoms are highly variable until the characteristic dry eyes and mouth develop, which can take years. It is an autoimmune reaction against exocrine glands, especially those that secrete tears and saliva; little is known about its etiology, and pathogenesis seems to involve CTL. Like all these conditions, Sjogren has genetic and environmental predispositions.

Question 25

JUVENILE DIABETES.

Answer 25

Autoimmunity is strongly implicated in Type 1, or juvenile, or insulin-dependent diabetes mellitus (T1D). Depending on the technique used, antibody to β-cells (the islet cells that produce insulin) can be detected in the serum of over 90% of patients at the time of diagnosis (normals, fewer than 2%). Tissue obtained at autopsy from an occasional patient early in the disease show antibody and typical inflammatory responses. Although antibody is a useful marker, and B cells may play a role as APCs to the harmful T cells, T cells are implicated in the pathogenesis of T1D. In children at high genetic risk for T1D antibodies appear in the blood well (sometime many years) before the beginning of diabetic symptoms. It brings up many interesting questions; for example, should newly-diagnosed antibody-positive but non-diabetic children be treated with immunosuppressive drugs? There is a strong HLA association between T1D and HLA-DR3 or HLA-DR4. These are in linkage disequilibrium with HLA-DQ2 and HLA-DQ8, respectively. The DQ genes are thought to be the problem; they have unusual amino acids placements in the antigen-binding groove that allows ready presentation of islet cell-associated peptides. The best animal model, the NOD mouse, has an unusual Class II MHC molecule (H2 I-Ag7) which is very similar to DQ8.

Question 26

GRAFT VERSUS HOST REACTIONS.

Answer 26

If a non-identical graft contains T cells (and, except for corneas, they usually do, because they contain blood or have tissue spaces where leukocytes can be hiding) there is a perfectly good possibility that these cells will recognize HLA antigens of the recipient (host) as foreign, and so the graft will try to reject the host. Normally, the host is a lot bigger than the graft, and will usually reject the grafted lymphocytes before they can begin to mount a serious reaction.

Question 27

Three conditions for graft-versus-host disease (GvHD) to occur

Answer 27

1. The graft must contain immunocompetent T cells (even bone marrow has mature T cells in it). 2. There must be at least one antigen in the host which the graft’s T cells can recognize (so, no worries with identical twins.) 3. The host must be relatively immunoincompetent or unable for genetic reasons to recognize the graft’s MHC antigens, otherwise the graft would be rejected too rapidly.

Question 28

Acute GvHD.

Answer 28

This develops in 2 to 10 weeks after bone marrow transplantation in humans. The symptoms include a nasty maculopapular skin rash; diarrhea; hepatic inflammation with jaundice; and infections (probably due to immunosuppression, as Tregs try to control the raging immune activation.) The treatment is with anti-inflammatory drugs like corticosteroids and, paradoxically, with immunosuppressives.

Question 29

Chronic GvHD.

Answer 29

This develops in months to years, even in patients with a perfect HLA match; therefore it is probably against minor histocompatibility antigens. With a lot of chronically activated T cells pouring out cytokines, regulation is compromised and autoimmunity can become an issue. In bone marrow transplantation, removing the T cells from the bone marrow may prevent acute GvH disease. Oddly, this usually results in a poorer engraftment of the bone marrow’s stem cells. It’s possible that a few activated T cells make hematopoietic-stimulating growth factors that improve graft success.

Question 30

‘Graft-versus-leukemia reaction’.

Answer 30

For leukemia that has stopped responding to conventional therapy, one treatment is to give patients large doses of drugs or radiation, which would in themselves probably be fatal. One then can take marrow from the best matched allogeneic donor one can find, and give it to the patient after the high-dose (“myeloablative,” because it destroys all the bone marrow) therapy. Many studies have shown that leukemia patients who receive stored, pre-treatment bone marrow from themselves, or T-depleted allogeneic marrow, have the fewest GvH symptoms; but they also have a higher rate of leukemia relapse compared to those who get allogeneic marrow that still has some T cells. So it is assumed that somehow, a “graft- versus-leukemia” reaction is an important part of the success of the bone marrow transplant. Now more centers are using less harsh pretreatment, and trying to optimize the GvL effect while minimizing GvH. This is a difficult tightrope to walk, especially since we still don’t really understand what’s going on.

Question 31

Th2 CELLS IN IMMUNOPATHOLOGY.

Answer 31

Activated Th2 cells are found in the periphery in certain inflammatory and infectious states, especially asthma and chronic worm infestation. They activate macrophages (alternatively activated or M2) which produce fibrosis under these chronic conditions; and also attract eosinophils which, in excess, make inflammation more intense. So although this raises the question of whether allergy and asthma might really be T cell diseases, for now most people refer to asthma as a Type I condition because of the role played by IgE. But the terminology could be in for a change.

Question 32

Mycobacterium leprae

Answer 32

the causative organism of leprosy, has strange effects on dendritic cells, with the result that some people make strong Th1 responses against M. leprae, and others make Th2 responses. Both get immunopathology as the organism is extremely difficult to clear from the body. Those with Th1 responses get tuberculoid leprosy with large skin and nerve lesions, but they contain the infection. If the response is dominated by Th2 the initially-uncontrolled infection is widely disseminated in many small granulomas (lepromatous leprosy).

Question 33

Abacavir hypersensitivity syndrome

Answer 33

Up to 8% of people who are given abacavir, a nucleoside reverse transcriptase inhibitor, for HIV, develop abacavir hypersensitivity syndrome which is quite awful and difficult to diagnose correctly. Nearly all people with the syndrome are HLA-B*5701. We now test for this allele before offering the drug. HLA-B*5701 is Class I, not the Class II which is recognized by Th1. This is predominantly a CLT problem. Abacavir changes the structure of HLA-B*5701 so that it binds certain self-peptides that are not, of course, normally presented; the syndrome is actually a drug-induced autoimmune reaction

Question 34

Carbamazepine in the Han Chinese population

Answer 34

The strongest association (OR > 1,000) between HLA alleles and drug-induced hypersensitivity has been detected for carbamazepine in the Han Chinese population. The association is also in Thai, Malay, and Indian populations, but not in Caucasians. The allele is HLA-B*1502 and the correlation is specifically with a nasty condition called Stephens-Johnson Syndrome, or similar though nastier Toxic Epidermal Necrolysis, both of which are probably CTL-dominated forms of Type IV immunopathology.

Question 35

Capsule

Answer 35

usually thin and fibrous. Can be thickened and fibrotic in reactive conditions (i.e. syphilitic lymphadenitis) or neoplastic processes (i.e. nodular sclerosis Hodgkin lymphoma)

Question 36

Cortex

Answer 36

lymphoid follicles (primary and secondary), and paracortex (interfollicular T-cell zone)


Question 37

Medulla

Answer 37

medullary cords (lymphocytes, plasma cells, macrophages, and dendritic cells) and medullary sinuses

Question 38

Sinuses

Answer 38

subcapsular, cortical and medullary

Question 39

Secondary lymphoid follicle

Answer 39

Mantle zone: small cells surrounding the germinal center. Germinal center: Dark zone (upper): mostly centroblasts and Light zone (lower): mixed centrocytes, T-cells, dendritic cells and histiocytes

Question 40

Paracortex

Answer 40

consists of mixed lymphocytes, histiocytes, macrophages, Langerhans cell, interdigitating dendritic cells and high endothelial venules (HEV)

Question 41

Lymph node sinuses

Answer 41

usually patent containing lymphocytes, plasma cells and histiocytes.

Question 42

CD20

Answer 42

a B-cell marker that highlights mantle zone B-cells and germinal center B-cells.

Question 43

CD3

Answer 43

a T-cell marker that highlights abundant T-cells in the paracortex and scatter T-cells in the germinal center.

Question 44

Outline of B-cell development

Answer 44

B-cells undergo maturation in the bone marrow from stem cells to pro-B, Pre-B, immature B and eventually mature naïve B-cells. The mature naïve B-cells then migrate to the peripheral organs and form the primary lymphoid follicle. The naïve B-cells undergo activation in the PALS or paracortical region by antigen primed dendritic cells and T cells. Some of the activated B-cells differentiate into 1st plasma cells that secrete IgM. Whereas, others migrate back into the lymphoid follicles and eventually differentiate into IgG secreting plasma cells and memory B-cells.

Question 45

Acute lymphoblastic leukemia –

Answer 45

immature B-cell

Question 46

Mantle cell lymphoma –

Answer 46

mantle cell


Question 47

Follicular lymphoma –

Answer 47

germinal center B-cell

Question 48

Hodgkin lymphoma

Answer 48

germinal center B-cell

Question 49

Burkitt lymphoma –

Answer 49

germinal center B-cell

Question 50

CLL/SLL –

Answer 50

mature B-cells of pre- or post-GC stage

Question 51

Plasma cell myeloma –

Answer 51

plasma cell

Question 52

CLL

Answer 52

Absolute mature lymphocytosis of ≥5 × 109/L, sustained for at least 3 months. Monoclonal B cell with mature immunophenotype: positive for CD5, weak CD20, weak surface immunoglobulin, CD23, weak CD22, and weak CD11c. Typically negative for CD10, FMC7, and CD79b. CLL is the most common leukemia in the Western world and accounts for ~ 30% of all leukemia. Most elderly patients with CLL are either asymptomatic (70% of cases) or only mildly symptomatic, whereas some patients may present with fatigue, infection, autoimmune hemolytic anemia, hepatosplenomegaly, lymphadenopathy, or extranodal involvement.

Question 53

SLL

Answer 53

Extramedullary sites of disease predominate. Diffuse infiltrate of small lymphocytes. Monoclonal B cell with mature phenotype similar to CLL. SLL accounts for ~7% of Non-Hodgkin lymphoma (NHL)

Question 54

Morphological features of SLL/CLL in peripheral blood

Answer 54

Lymphocytosis of ≥5 × 109/L. Monotonous cells, small round nuclei, highly condensed chromatin (soccer ball appearance), inconspicuous nucleoli. Scant agranular cytoplasm. Frequent smudge cells and basket cells. Prolymphocytes often present but < 55%; larger and round nucleus, more delicate chromatin, single distinct nucleolus, abundant cytoplasm

Question 55

Morphological features of SLL/CLL in lymph node

Answer 55

Architecture: effacement of the lymph node with diffuse infiltration. Cytology: predominantly small cells, slightly larger than normal lymphocytes; round nuclei with clumped chromatin. Pseudofollicles (proliferation centers): pale areas containing transformed larger cells

Question 56

Morphological features of SLL/CLL in spleen

Answer 56

miliary micronodules grossly, typically involving white pulp

Question 57

Morphological features of SLL/CLL in bone marrow

Answer 57

Nodular, interstitial, diffuse, or solid infiltration pattern. Typically lack paratrabecular infiltration

Question 58

Immunophenotype in CLL/SLL

Answer 58

Positive: CD5, CD23, CD19. Weak: CD20, surface immunoglobulin, CD22, CD11c. Negative: CD10, FMC7

Question 59

Common Genetic Findings CLL/SLL

Answer 59

Deletion of 13q14: the most common genetic alteration, in >50% of CLL, association with favorable prognosis. Trisomy chromosome 12: in < 20% of CLL, often associated with atypical morphology. Deletion of 11q22-q23: in ~ 20% of CLL. Deletion of 17p13: in 5-10% of CLL, adverse prognosis

Question 60

Pronosis of CLL/SLL

Answer 60

Favorable: CD38-, ZAP-70-, germline IGH@ V, pre-germinal center. Unfavorable: CD38+, ZAP-70+, unmutated IGH@ V, post-germinal center

Question 61

Follicular Lymphoma (FL)

Answer 61

A lymphoma of germinal center B cells (centrocytes and centroblasts) with typically at least a partially follicular pattern

Question 62

Clinical Features of FL

Answer 62

Frequency: 40% of adult lymphomas in US, 20% worldwide. Age: mostly adults, median 60 years. Gender: male = female. Locations: mostly lymph nodes, also spleen, bone marrow, Waldeyer's ring, GI tract, skin and soft tissue. Clinical presentations: more than 80% cases have widespread stage III or IV disease at diagnosis, 40% have bone marrow involvement; however, patients often asymptomatic except for lymphadenopathy

Question 63

Patterns of follicles in FL
-

Answer 63

Closely packed, back to back, with effacement of the nodal architecture and extranodal or intramedullary involvement.

Question 64

Morphology of follicles in FL-

Answer 64

Poorly defined with scant or absence of mantle zone; Lack of polarization or starry-sky pattern; diffuse areas may be present.

Question 65

Cells in FL-

Answer 65

Two type of tumor cells, predominantly centrocytes in most cases. The other type is centroblasts.

Question 66

Centrocytes

Answer 66

small to medium size with scant cytoplasm; angulated, elongated, twisted or cleaved nuclei; inconspicuous nucleoli

Question 67

Centroblasts

Answer 67

large transformed cells with round or oval nuclei and vesicular chromatin; a narrow rim of cytoplasm; one to three peripheral nucleoli

Question 68

Immunophenotype of FL

Answer 68

B-cell markers positive: CD19+, CD20+, BCL2+ positive, Germinal center B-cell marker positive: CD10 and BCL6

Question 69

Cytogenetic Finding of FL

Answer 69

75-90% of cases have translocations of t(14;18)(q32;q21), involving the long arms of chromosomes 14 and 18, which places the BCL2 gene on chromosome 18 under the influence of the IGH promoter on chromosome 14. BCL2 is an oncogene, and overexpression of BCL2 in animal model can induce massive follicular lymphoid hyperplasia, with persistence of a mature B-cell population, but they do not develop FL. Therefore, BCL2 rearrangement by itself is not enough to result in neoplastic transformation. Additional abnormalities may be required for lymphoma development.

Question 70

Mantle Cell Lymphoma (MCL)

Answer 70

A B-cell neoplasm composed of monomorphic small to medium-sized lymphocytes with irregular nuclei that morphologically resemble centrocytes but often have slightly less irregular nuclear contours. The tumor cells typically express B-cell markers (CD19 and CD20) and CD5, and do not express CD23. This neoplasm is genetically characterized by BCL1 gene rearrangement at 11q13, leading to a constant overexpression of cyclin D1, which plays an important pathogenetic role in tumor development.

Question 71

Clinical Features of MCL

Answer 71

MCL comprise approximately 3-10% of NHL. Age: middle to older age, median age 60 years; M:F=2:1. Location: mostly lymph nodes, also spleen and bone marrow; most common extranodal sites, gastrointestinal tract and Waldeyer's ring. Clinical presentations: most patients present with stage III or IV with lymphadenopathy, hepatosplenomegaly; >50% of cases with massive splenomegaly and marrow involvement; >25% cases with peripheral blood involvement that can mimic prolymphocytic leukemia.

Question 72

Morphologic Features of MCL

Answer 72

Architecture: effacement of lymph node by a monomorphic lymphoid proliferation with a vaguely nodular, diffuse or mantle zone growth pattern; Cytology: small to medium size, slightly larger than the normal lymphocytes; slightly irregular nuclear contours, resembling centrocytes but often have slightly less irregular nuclear contours; occasional round nuclei, resembling B-CLL/SLL; the nuclei have moderately dispersed chromatin with inconspicuous nucleoli; No neoplastic transformed cells resembling centroblasts, immunoblasts, or paraimmunoblasts; no pseudofollicles; Hyalinized small vessels commonly present; Many cases have scattered single epithelioid histiocytes with a "starry sky" appearance;

Question 73

Immunophenotype of MCL

Answer 73

B-cell markers positive: CD19+, CD20+ | , CD5 positive, CD23 negative, Cyclin D1 (BCL1) positive, Germinal center B-cell marker positive: CD10 and BCL6

Question 74

Cytogenetic Finding of MCL

Answer 74

Characteristic t(11;14)(q13;q32), involving BCL1 gene on 11q13 and IGH gene on 14q32

Question 75

Clinical Behavior of MCL

Answer 75

In contrast to other small B-cell lymphoma that are mostly indolent, MCL is usually moderately aggressive

Question 76

Burkitt ’s lymphoma (BL)

Answer 76

A highly aggressive B-cell lymphoma, often presenting at extranodal sites or as a leukemic form. The tumor is typically composed of monomorphic, medium-sized B cells with basophilic cytoplasm and a high mitotic rate. Translocations involving the MYC oncogene on chromosome 8 at band 8q24 are a constant feature. May present in blood and diffuse marrow involvement at diagnosis

Question 77

Epidemiology of BL

Answer 77

Endemic BL: typically in the malaria belt of equatorial Africa, as the most common childhood malignancy in this region, peak 4-7 years of age, involves jaw or abdomen, 95% EBV positive. Sporadic BL: mostly in children or young adults. 30% of childhood lymphomas. Immunodeficiency-associated BL: primarily in HIV patients

Question 78

Locations of BL

Answer 78

Endemic: jaw and other facial bones in 50% cases, also distal ileum, cecum and omentum. Sporadic: mostly in ileocecal area. Immunodeficiency-associated: HIV

Question 79

Morphologic Features of BL

Answer 79

Diffuse infiltration of monomorphic, medium-sized cells with starry sky pattern. Tumor cells: Abundant basophilic cytoplasm. Some times "squared off" boarders. Non-cleaved round nuclei with finely clumped and dispersed chromatin. Multiple basophilic medium sized, paracentrally situated nucleoli. Imprints demonstrate deeply basophilic cytoplasm with lipid vacuoles

Question 80

Immunophenotype of BL

Answer 80

B-cell markers positive: CD19+, CD20+. High proliferation index, nearly 100% by Ki-67 staining. EBV mostly positive in endemic BL, and ~30% positive in sporadic and immunodeficiency-associated BL. Negative: BCL2, CD5, CD23, TdT

Question 81

Cytogenetic Finding of BL

Answer 81

Most cases have a characteristic translocation, t(8;14)(q24;q32), which juxtaposes the MYC gene at 8q24 next to IGH@ at 14q32

Question 82

Clinical Behavior of BL

Answer 82

Highly aggressive but potentially curable. Curable with aggressive therapy in ~60% cases.

Question 83

Plasma cell neoplasms

Answer 83

A clonal proliferation of immunoglobulin-producing plasma cells that secrete a single class of immunoglobulin or a polypeptide subunit of a single immunoglobulin, which is usually detectable as a monoclonal protein (M protein) on serum or urine protein electrophoresis. Most plasma cell neoplasms originate as bone marrow tumors, but they occasionally present in extramedullary sites.

Question 84

Classification of plasma cell neoplasms

Answer 84

Monoclonal gammopathy of undetermined significance (MGUS). Plasma cell myeloma (Asymptomatic (smoldering) myeloma, Nonsecretory myeloma, and Plasma cell leukemia). Plasmacytoma (Solitary plasmacytoma of bone and Extraosseous (extramedullary) plasmacytoma ). Immunoglobulin deposition diseases (Primary amyloidosis and Systemic light- and heavy-chain deposition diseases). Osteosclerotic myeloma (POEMS syndrome)

Question 85

Plasma Cell Myeloma (PCM, or Multiple Myeloma)

Answer 85

a bone marrow–based, multifocal plasma cell neoplasm associated with an M protein in serum or urine. PCM originates from the marrow with disseminated marrow involvement in most cases. Other organs may be secondarily involved. The diagnosis of myeloma is made by a combination of clinical, morphologic, immunologic, and radiographic information. The disease spans a clinical spectrum from asymptomatic to highly aggressive disease.

Question 86

Diagnostic Criteria for Symptomatic Plasma Cell Myeloma

Answer 86

M protein in serum or urine: No minimal level of M protein is included, but in most cases it is >30g/L of IgG, >25g/L of IgA, or >1g/24 hours of urine light chain. Bone marrow clonal plasma cells or plasmacytoma: no minimal level is designated, but in most cases the monoclonal plasma cells usually > 10% of nucleated cells in the marrow. Related organ or tissue impairment: hypercalcemia, renal insufficiency, anemia, bone lesions (CRAB).

Question 87

Clinical Features of plasma cell myeloma

Answer 87

~ 4 cases per 100,000 persons per year in the United States. Rare in adults younger than 35 years, ~90% of patients > 50 years of age, median age at diagnosis ~ 68 years. Most frequent symptom at presentation: bone pain in the back or extremities due to lytic lesions or osteoporosis. Weakness and tiredness, often related to anemia, are common complaints.

Question 88

Laboratory findings in plasma cell myeloma

Answer 88

Serum/urine protein electrophoresis: serum and urine immunofixation electrophoresis is the “gold standard” for characterizing heavy and light chains and for detecting small quantities of M protein, plasmacytoma, heavy-chain disease, and light-chain deposition disease and following treatment of myeloma. IgG M protein is found in ~ 55% of patients with myeloma, IgA ~20%, light chain only 20%, IgD/IgE/IgM/biclonal myeloma all rare, <3% nonsecretory. Anemia is present in about two thirds of patients at diagnosis o Hypercalcemia is present in ~1/5 of patients, creatinine elevated in 1/5-1/3. Radiologic findings: lytic lesions, osteoporosis or fractures seen in 70% to 85% of myeloma patients at diagnosis. The vertebra, pelvis, skull, rib, femur, and proximal humerus are most often affected.

Question 89

Blood Smear Findings with plasma cell myeloma

Answer 89

Rouleaux formation is often present when the M protein is markedly elevated. Plasma cells are found on blood smears in ~15% of cases, usually in small numbers.

Question 90

Bone Marrow Biopsy with plasma cell myeloma

Answer 90

the bone marrow aspirate smear and core/clot biopsies are nearly always required to confirm the diagnosis of myeloma, to evaluate the cytology of tumor cells and degree of marrow involvement, and to obtain specimen for necessary genetic/molecular studies.

Question 91

Monoclonal Gammopathy of Undetermined Significance (MGUS)

Answer 91

MGUS refers to the presence of a monoclonal immunoglobulin in the serum or urine of a patient with no evidence of plasma cell myeloma, amyloidosis, Waldenström's macroglobulinemia, or other lymphoproliferative disorder or any other disease known to produce monoclonal immunoglobulins. MGUS is the most common monoclonal gammopathy and is found in about 3% of persons older than 50 years and in more than 5% of those older than 70. MGUS is a precursor lesion of PCM, and the actuarial probability for malignant transformation of MGUS is approximately 1.5% a year.

Question 92

Diagnostic criteria for MGUS

Answer 92

M component less than myeloma levels. Marrow plasmacytosis <10%. No lytic bone lesions. No myeloma-related symptoms

Question 93

Solitary plasmacytoma of bone

Answer 93

solitary plasmacytoma of bone is a localized tumor of the bone, which is composed of clonal plasma cells that are cytologically, immunophenotypically, and genetically similar to those of plasma cell myeloma.

Question 94

Diagnostic criteria of solitary plasmacytoma of bone

Answer 94

Single bone lesion consisting of monoclonal plasma cells. No evidence of other bone lesions by radiographic examination, which includes magnetic resonance imaging. Absence of renal failure, hypercalcemia, and anemia that could be attributable to myeloma. Absent or low serum or urine M protein. Normal levels of uninvolved polyclonal immunoglobulins

Question 95

Extraosseous Plasmacytoma

Answer 95

Extraosseous or extramedullary plasmacytomas are localized plasma cell tumors that arise in tissues outside of the bone marrow. They appear to be biologically distinct from solitary plasmacytoma of bone and plasma cell myeloma. Extraosseous plasmacytomas constitute less than 5% of plasma cell neoplasms. The median age at diagnosis is about 55 years, and two thirds of patients are male. Extraosseous plasmacytomas present as localized mass lesions. About 75% of them occur in the upper respiratory tract, including nasal passages, sinuses, oropharynx, and larynx. The histologic features are similar to those of solitary plasmacytoma of bone.

Question 96

Classification of Hodgkin’s Lymphoma

Answer 96

the WHO classification has recognized two major types of Hodgkin lymphoma, the nodular lymphocyte-predominant subtype of Hodgkin's lymphoma (NLPHL) and the classical Hodgkin's lymphomas (CHLs), which is further classified into four subtypes. Nodular lymphocyte-predominant Hodgkin's lymphoma and Classical Hodgkin's lymphoma (Nodular sclerosis classical Hodgkin's lymphoma, Lymphocyte-rich classical Hodgkin's lymphoma, Mixed cellularity classical Hodgkin's lymphoma, Lymphocyte-depleted classical Hodgkin's lymphoma). Both NLPHL and CHLs are of germinal center B-cell origin; however, there are clear and consistent histologic, epidemiologic, immunologic, and genetic differences between these two separate entities.

Question 97

Nodular lymphocyte-predominant subtype of Hodgkin's lymphoma (NLPHL)

Answer 97

NLPHL is an indolent malignancy, representing a nodular proliferation comprising a minority of large neoplastic centroblasts with multilobated nuclei, the so-called popcorn or lymphocyte-predominant (LP) cells (formerly called L & H cells, for lymphocytic and/or histiocytic Reed-Sternberg cell variants), and a majority of reactive lymphocytes and histiocytes.

Question 98

Classical Hodgkin's lymphomas (CHLs)

Answer 98

In CHL, the malignant cells usually represent only a small minority, between 0.1% and 2%, of the total cellular population of involved tissues. A histopathologic diagnosis of CHL is based on the identification of diagnostic Reed-Sternberg (RS) cells in an appropriate inflammatory background. The classic RS cell is large (up to 100 μm) and contains multiple nuclei or a large lobated nucleus. The nuclei show an accentuated membrane, pale chromatin, and a single large, eosinophilic, viral inclusion–like nucleolus. The cytoplasm is ample and amphophilic. Mononuclear variants are called Hodgkin cells. The degenerated RS and Hodgkin cells are called mummified cells with condensed, darkly stained cytoplasm and condensed nuclear chromatin. RS cells and variants express the CD30 and CD15 antigens in the majority of cases, lack the common leukocyte antigen CD45.

Question 99

Nodular sclerosis CHL (NSHL)

Answer 99

Most frequent subtype, accounts for 50-80% of all CHLs. Predominates in young adults, especially in females. Typically occurs above the diaphragm. Morphology: Thickened lymph node capsule with broad bands of collagen. Nodular areas surrounded by broad bands of collagen. Lacunar cells: a type of HRS cells with polypoid nuclei and small nucleoli. Perinuclear clearing due to formalin fixation artifact which causes the cytoplasm to retract. Background of small round lymphocytes (predominantly T-cells), eosinophils, plasma cells, and some neutrophils. EBV+, 10-25%

Question 100

Mixed cellularity CHL

Answer 100

This subtype accounts for 20 to 25% of HL. Its cellular composition is similar to that of NSHL. However MCHL. Second most frequent subtype, 20-30% of CHLs. High percentage of this subtype in children and older patientsFrequently in stages III and IV and with B symptoms, more often below or on both sides of the diaphragm. Lack of broad bands of collagen seen in nodular sclerosis CHL. EBV+, ~ 75%

Question 101

Lymphocyte rich CHL

Answer 101

Accounts for around 5% of all CHLs. Usually nodular growth pattern, with residual germinal centers or remnants of residual germinal centers. Classic HRS cells are present although they are rare

Question 102

Lymphocyte depleted CHL

Answer 102

Least frequently subtype, ~1% of CHL. Relatively paucity of lymphocytes in this subtype. Numerous RS cells, some of which may appear very anaplastic and bizarre (sarcomatous). Usually EBV+

Question 103

Explain the basic principles for the WHO Classification of lymphomas.

Answer 103

on several criteria, including morphology, immunophenotype, genetic findings, location, and age.

Question 104

Describe the factors that regulate the differentiation of Th0 cells in the Peyer’s Patches to Th1, Th2, or Th17 versus into Treg cells.

Answer 104

There is normally a lot of the cytokine TGFβ in the submucosal Peyer’s Patches, and that favors the differentiation of Th0 cells into Treg. The resident dendritic cells here make IL-10, and that also favors Treg development. Thus these sites are rich in Treg cells, which is desirable considering the constant exposure to with bacteria- and food-derived, non-pathogenic, potential immunogens coming through the M cells of the gut epithelium. If a peptide comes in unaccompanied by damage or inflammation, you probably don’t want to make an immune response to it, so it’s good to make Tregs. Also very common in Peyer’s Patches are Tfh that specifically drive B cells towards making IgA, so that the mucus layer nearest the epithelial cells that line the gut is, surprisingly, almost sterile. More than one immunologist has suggested the Tregs can differentiate easily into such Tfh, and vice versa, as they’d both prevent harmful responses and help protective ones.

Question 105

TGFβ and IL-6

Answer 105

However, the combination of TGFβ and IL-6 has been shown to downregulate Treg and upregulate Th1, Th2, and Th17 (the CD4+ Th that makes IL-17 and is expanded by IL-23; both these cytokines are also common in areas of inflammation.) IL-6 is produced by epithelial and other cells in response to stress or damage. This model links a lot of disparate observations. Normal commensal gut organisms have evolved to live in the lumen and not try to invade; the immune response to them, taking place in an environment dominated by TGFβ, is mostly by Treg at a steady level. When the innate response indicates a threat, it makes stress cytokines like IL-6 and the response switches from Treg production to defensive Th1, Th17, or Th2.

Question 106

The recognition of normal vs. abnormal organisms in the gut

Answer 106

is doubtless mostly carried out by innate immunity via PRR that bind various PAMPs. These include the TLRs we discussed early on, and several other PRR systems, including one called NOD2. NOD2 detects muramyl dipeptide, a component of bacterial cell walls, and triggers cytokine production by activating NF-κB. There are also complex PAMP-recognizing assemblies called “inflammasomes.”

Question 107

INFLAMMATORY BOWEL DISEASE, IBD.

Answer 107

includes Crohn Disease (CD) and ulcerative colitis (UC). CD affects the large and small intestine, especially the terminal ileum. There are microabcesses in the wall of the intestine, generalized inflammation throughout the wall (so that fistulas can develop between the lumen and the peritoneum), and the disease process is ‘patchy’ with affected areas interspersed with healthy ones. The abscesses eventually become granulomas. UC is usually more superficial in the large intestine, and can erode the surface leading to bleeding. Both are thought to involve dysregulated immune responses, perhaps to commensal bacteria.

Question 108

Genetics of IBS

Answer 108

Genome-wide association studies (GWAS) have identified 163 loci associated with significant risk in IBD. 30 are specific for CD, and 23 for UC. A hundred and ten loci are in common between the two conditions. So there is a strong genetic component; but the environment and ‘bad luck’ also play important roles, since concordance in monozygotic twins is only 30-35% for CD, and 10-15% for UC. In some IBD patients, an early (genetic?) event is an increase in gut permeability so that certain secreted defensins, made by gut lining cells, are able to penetrate back into the tissues. There, acting as DAMPs, they stimulate macrophages to produce cytokines, including IL-6.

Question 109

CELIAC DISEASE.

Answer 109

Also called gluten-sensitive enteropathy, this condition affects almost 1% of the world’s population. In infants it presents as malabsorption, diarrhea, and failure to thrive; in adults it can be so nonspecific as to defy clinical diagnosis, with a variety of symptoms (osteoporosis, anemia, rash) secondary to malabsorption as the villi in the gut atrophy. The diagnostic hallmark is a small intestinal biopsy. Also useful in diagnosis is antibody to the gut endomysium, the lining that supports the smooth muscle layer; the specific antigen is tissue transglutaminase 2 (TG2). This enzyme makes protein crosslinks through glutamines, and in some people may, if it couples to but can’t release digestion-resistant, glutamine-rich gliadin (wheat) peptides, inadvertently turn itself into a B-cell autoantigen by the ‘foreign + self hybrid antigen’ help mechanism. Note, though, that it is T cell immunity to gliadin peptides that is responsible for the chronic inflammation. Ninety percent of people with this condition are HLA-DQ2, and most of the rest are HLA-DQ8; but most HLA- DQ2 or 8 people don’t get celiac disease, implicating other genetic and environmental factors. This is another example of a frustrated immune response: the body has decided that gluten is dangerous and must be destroyed, so the gut becomes the battleground on which this endless struggle is waged. However, unlike IBD, there is a fix available: if the patient avoids gluten (wheat, rye, and barley) the symptoms will fade and the gut can revert to normal.

Question 110

Herpetiformis.

Answer 110

Some patients with poorly-controlled celiac disease will develop a skin condition called dermatitis herpetiformis. Biopsy shows that there is autoantibody in the skin to skin-specific transglutaminase 3 (TG3). There is evidence that this antibody actually causes the skin lesions. Perhaps not surprisingly, the antibody is IgA, which probably arose from the anti-TG2 of celiac by epitope spreading.

Question 111

CHRONIC BERYLLIUM DISEASE.

Answer 111

This is a pulmonary inflammatory and fibrotic disease caused by exposure to inhaled beryllium dust. It is seen in miners and machinists, especially in the nuclear industry where Be alloys find many uses. Perhaps a million people have been exposed, and 15% of them are symptomatic. Inhaled Be can become covalently linked to various peptides and it is thought that this creates novel epitopes to which a Th1 (Th17 also?) response is made, and later a scarring Th2 response as well. Since the Be cannot be removed effectively by macrophages, the condition can become established and chronic even after a single inhalation exposure. It is strongly linked to HLA-DP alleles that have a glutamic acid at position 69 of the β chain (DPβE69). This creates a negatively charged pocket which could bind a Be+ coupled peptide.

Question 112

PSORIASIS.

Answer 112

There is some evidence that this chronic inflammatory condition of skin also involves an inappropriate, unregulated T cell response to normal skin organisms. It is associated with the allele HLA-Cw*06:02 (a class I gene, which in this case may be in linkage disequilibrium with a pathogenic Class II allele.) Interestingly, this allele is high in African Americans, who are in most studies at greater risk for psoriasis than Caucasians—but in sub- Saharan Africa the allele is high among Black people but prevalence of psoriasis is low, clearly suggesting environmental factors play a role. Genome-wide association studies implicate HLA, a gene that affects skin cell differentiation, and IL-23 (a Th17 cytokine).

Question 113

PERIODONTAL DISEASE.

Answer 113

This chronic inflammatory condition is the major cause of tooth loss; prevalence in the US is about 8% in the young, 16% in the elderly. It is strongly associated with several bacterial species. The gingival crevice between the gum and the tooth root is not easily cleared by saliva, and yet cell- mediated immunity cannot reach there because it’s outside the body; so it’s a great place for bacteria to live, if it’s not kept scrupulously clean. It has many of the characteristics of inflammatory bowel disease, including a shift from a TGFβ milieu to one with IL-6 and TGFβ. There is an association with rheumatoid arthritis (antibodies to citrullinated peptides are seen in both conditions, and they are linked to smoking and environmental pollution.) Immunosuppressive therapy improves periodontitis, though it’s rarely prescribed for it. Tocilizumab, a blocking monoclonal antibody to the IL-6 receptor, has been reported to be effective in severe periodontal disease, but the side effects were worrisome.

Question 114

HYGIENE HYPOTHESIS.

Answer 114

Broadly, there has been less of an increase in: poor countries as compared to rich ones; equatorial versus northern countries; rural populations as opposed to urban; slums as opposed to rich neighborhoods; children of large families as opposed to only children. All of this suggested that exposure to environmental dirt and infections helped the immune system mature normally, while lack of such exposure might leave a child in an infantile state. There is good evidence that newborns start out with a Th2-dominated system which gradually balances out with Th1. This might explain the increase in Th2- (and the Th2-like Tfh that drive B cells to switch to IgE) mechanism diseases. It’s an appealing idea, but it ran into some trouble because the same clean rich people who should, by this explanation, be Th2-dominated are also at increased risk of Th1 diseases like ulcerative colitis and Crohn disease, multiple sclerosis and juvenile diabetes. How can the same group be Th2- and Th1-dominated at the same time? The model was too simple.

Question 115

Old Friends Hypothesis.

Answer 115

It says that certain harmless microorganisms—notably non-tuberculosis Mycobacteria, lactobacilli, and helminth worms—have been in humans so long that we rely on their presence to instruct our immune systems not to overreact against commensals or low-grade pathogens. Specifically, if you have adequate exposure to these old friends, you develop a balance between activation and regulation, driven by the right number of Treg. But if you have been old-friendless most of your life, you may have too few Treg and be too ready to make a strong Th1 or Th2 or even Th17 response to some organism that really isn’t much of a threat (gut flora) or is no threat at all (pollen), especially if you already have allelic variants of genes that predispose you to do so.

Question 116

WHIPWORMS

Answer 116

in Crohn Disease (CD) and Ulcerative Colitis (UC), Th1 are bad and Th2 might, by opposing Th1, be good. It was thought that the important event was Th1-Th2 rivalry. (Th1 do, to an extent, suppress Th2 development, and vice versa, as they perhaps compete for limiting growth factors.) How to effect a switch? Parasite responses are strongly Th2-dominated. So they recruited a group of quite ill CD patients and fed them some drinks of fresh pig whipworm ova. This was safe because the worms will only live a few days in the human gut. In a short, open-label study, the improvement in their patients’ symptom scores was remarkable. Subsequent work has shown that the mechanism of the effect was not Th2 suppressing Th1, but rather an impressive increase in Treg in the gut, which can suppress Th1, Th17, and Th2 responses. It is fascinating to think that this could still take place in adults, and we are fortunate that although Treg are stimulated by recognizing their specific epitopes as are any other T cell, the effect of their suppression is not antigen-specific, so that many nearby activated T cells are down-regulated or do not differentiate into effectors. The FDA has approved several trials of worm ova recently. This therapy is not harmless; too strong a Treg response can suppress needed responses to viruses and bacteria. It is all about balance.