This appendix presents five clinical cases illustrating various diseases involving the immune system. These cases are not meant to teach clinical skills but rather to show how the basic science of immunology contributes to our understanding of human diseases. Each case illustrates typical ways in which a disease manifests, what tests are used in diagnosis, and common modes of treatment. The appendix was compiled with the assistance of Dr. Richard Mitchell and Dr. Jon Aster, Department of Pathology, Brigham and Women’s Hospital, Boston; Dr. Robin Colgrove, Harvard Medical School, Boston; Dr. George Tsokos, Department of Medicine, Beth Israel-Deaconess Hospital, Boston; Dr. David Erle and Dr. Laurence Cheng, Department of Medicine, University of California San Francisco; and Dr. James Faix, Department of Pathology, Stanford University School of Medicine, Palo Alto.
Case 1: Lymphoma
E.B. was a 58-year-old chemical engineer who had been well all his life. One morning, he noticed a lump in his left groin while showering. It was not tender, and the overlying skin appeared normal. After a few weeks, he began to worry about it because it did not go away, and he finally made an appointment with a physician after 2 months. On physical examination, the physician noted a subcutaneous firm, movable nodule, approximately 3 cm in diameter, in the left inguinal region. The physician asked E.B. if he had recently noticed any infections of his left foot or leg; E.B. had not. E.B. did complain that he had been waking up frequently at night drenched in perspiration. The physician also found some slightly enlarged lymph nodes in E.B.’s right neck. Otherwise, the physical examination findings were normal. The physician explained that the inguinal mass probably was a lymph node that was enlarged as a result of a reaction to some infection. However, he drew blood for tests and referred E.B. to a surgeon, who performed a fine-needle aspiration of cells from the lymph node. Examination of smears prepared from aspirated cells revealed mainly small, irregular lymphocytes. Flow cytometric evaluation of these cells showed a 10-fold excess of B cells expressing λ immunoglobulin (Ig) light chain compared with B cells expressing κ Ig light chain.
Because of the suspicion of B cell lymphoma, a malignant tumor of cells of the B lymphocyte lineage, the surgeon elected to remove the entire lymph node. Histologic examination revealed an expansion of the node by follicular structures composed of mainly small- to intermediate-sized lymphocytes with irregular or “cleaved” nuclear contours mixed with smaller numbers of large lymphocytes with prominent nucleoli ( Fig. A.1 ). Flow cytometric analysis of these cells showed a predominant population of B cells expressing IgM, λ light chain, CD10, and CD20, and immunohistochemical stains performed on slides showed strong cytoplasmic staining for BCL-2. On this basis, the diagnosis of follicular lymphoma of low histologic grade was made.
Why does the presence of a B cell population in which a large majority of the cells express λ light chain indicate a neoplasm rather than a response to an infection?
If the lymph node cells were analyzed by polymerase chain reaction (PCR) to assess Ig heavy-chain rearrangements, what abnormal finding would you expect?
Normal follicular center B cells fail to express the BCL-2 protein. Why might the tumor cells express BCL-2?
E.B.’s blood tests indicated that he was anemic (low red blood cell count). He underwent staging tests to determine the extent of his lymphoma. Positron emission tomography (PET) and computed tomography (CT) scanning showed enlarged hilar and mediastinal lymph nodes, an enlarged spleen, and lesions in the liver. A bone marrow biopsy also showed presence of lymphoma. E.B. was treated with injections of a mouse/human chimeric monoclonal IgG antibody called rituximab, which is specific for human CD20. Imaging studies performed 6 months after the rituximab treatment was begun showed regression in the size of lesions, and E.B. felt well enough to continue working.
By what mechanisms would the anti-CD20 antibody help this patient?
What are the advantages of using a “humanized” antibody, such as rituximab, as a drug instead of a mouse antibody?
Answers to Questions for Case 1
During the maturation of B cells, the cells first express a rearranged μ heavy chain gene, which associates with the surrogate light chain to produce the pre–B cell receptor (see Chapter 4 ). The cells then rearrange a light chain gene: first κ, then λ. If the κ protein is produced, the λ gene does not rearrange; λ rearrangement occurs only if the κ rearrangement is unsuccessful or if the assembled Ig molecule is strongly self-reactive. So, any B cell can produce only one of the two light chains. In humans, about 50% to 60% of the mature B cells express κ and 40% to 50% express λ. In a polyclonal response to an infection or other stimulus, many B cells respond and this ratio is maintained. However, if there is a marked over-representation of one light chain (in this case, λ), it indicates that a λ-producing B cell has proliferated massively. This is characteristic of a B cell tumor (lymphoma), which arises from a single B cell.
Each clone of B cells has a unique rearrangement of V, (D) and J gene segments, forming the gene that encodes V regions of heavy and light chains. B cell lymphomas are monoclonal, being composed of cells that all contain the same Ig heavy-chain and light-chain gene rearrangements. Such tumors can be reliably distinguished by the use of PCR amplification of rearranged Ig heavy-chain (IgH) gene segments. This method uses consensus PCR primers that hybridize with virtually all IgH variable (V) gene segments and joining (J) gene segments. These primers are used in the PCR to amplify essentially all the heavy-chain gene rearrangements in a sample (e.g., DNA prepared from enlarged lymph node). The size of the amplified products is then analyzed by capillary electrophoresis, which can separate PCR products that differ in size by as little as a single nucleotide. When the V, D, and J segments of IgH genes (as well as other antigen receptor genes) are joined during antigen receptor rearrangement in pre–B cells, the rearranged segments are of differing length due to the action of enzymes that remove nucleotides (nucleases) and add bases (a specialized DNA polymerase called terminal deoxyribonucleotide transferase [TdT]). Within a normal population of B cells, many PCR products of different sizes are generated, and these appear as a broad distribution of fragments of differing size. In the case of a B cell lymphoma, all the B cells have the same VDJ rearrangement, and the PCR product is of one size, appearing as a single, sharp peak.
Many lymphomas have characteristic underlying acquired chromosomal translocations or mutations that dysregulate specific oncogenes. More than 90% of follicular lymphomas have an acquired 14;18 chromosomal translocation that fuses the coding sequence of BCL2, a gene on chromosome 18 encoding a protein that inhibits programmed cell death (apoptosis), to enhance elements within the Ig heavy chain locus located on chromosome 14. As a result, BCL-2 is overexpressed in follicular lymphoma cells. Parenthetically, in most instances the chromosomal breakpoint in the IgH gene involved in the translocation is located precisely at the point where RAG proteins normally cut the DNA of B cells that are undergoing Ig gene rearrangement, suggesting that the translocation stems from a mistake that occurs during normal antigen receptor gene rearrangement. Clinically, the presence of a BCL-2/IgH fusion gene, the consequence of the t(14;18), may be determined by fluorescent in situ hybridization using probes of different colors that are specific for IgH and BCL-2. These probes are hybridized to sections prepared of tissues involved by follicular lymphoma, and spatial superimposition of the probes within the nuclei of tumor cells indicates the existence of an IgH/BCL-2 fusion gene. Alternatively, it is possible to perform PCR on DNA isolated from the tumor with primer pairs in which one primer is specific for IgH and the other specific for BCL-2. These primers will produce a product only when the IgH and BCL-2 genes are joined to one another, which is taken as indirect evidence of a t(14;18).
CD20 is expressed on most mature B cells and is also uniformly expressed by all the tumor cells in follicular lymphomas. Injected rituximab (Rituxan) will therefore bind to the lymphoma cells and facilitate their destruction, likely through similar mechanisms by which antibodies normally destroy microbes. These mechanisms involve binding of the Fc portion of rituximab to different proteins in the patient, including Fc receptors on macrophages leading to phagocytic clearance of the lymphoma cells, and to complement proteins leading to complement-mediated killing of the lymphoma cells (see Chapter 8 ). Many normal B cells will also be destroyed by rituximab, although antibody-secreting plasma cells, which do not express CD20, are not affected. The immune deficiency caused by loss of normal B cells can be corrected by administration of pooled IgG from healthy donors, a form of passive immunity.
Monoclonal antibodies (mAbs) derived from nonhuman B cells (e.g., mouse) will appear foreign to the human immune system. When injected multiple times with these mAbs, humans will mount humoral immune responses and produce antibodies specific for the injected foreign mAb. These anti-antibody responses will promote clearance of the mAb from the circulation and therefore reduce the therapeutic benefits of the mAb. Furthermore, the Fc regions of human IgG bind better than mouse IgG to human Fc receptors and complement proteins, both of which are important for the effectiveness of mAb drugs (see Answer 3). For these reasons, most recently developed mAbs used as drugs have been genetically engineered to contain mainly or all human Ig amino acid sequences. Patients will generally not react against these drugs, just as they do not respond to their own antibodies. Rituximab is a chimeric mAb, with the CD20-binding variable regions originating from mouse IgG, and the remainder of the antibody including the Fc region from human IgG. The small amount of mouse sequences in rituximab do not appear to induce anti-antibody responses in patients, perhaps because potentially responding B cells are destroyed by the drug.
Case 2: Heart Transplantation Complicated by Allograft Rejection
C.M., a computer software salesman, was 48 years old when he came to his primary care physician because of fatigue and shortness of breath. He had not seen a doctor on a regular basis before this visit and felt well until 1 year ago, when he began experiencing difficulty climbing stairs or playing basketball with his children. Over the past 6 months he has had trouble breathing when he was recumbent. He did not remember ever experiencing significant chest pain and had no family history of heart disease. He did recall that approximately 18 months ago he had to take 2 days off from work because of a severe flu-like illness.
On examination, he had a pulse of 105 beats per minute, a respiratory rate of 32 breaths per minute, and a blood pressure of 100/60 mm Hg and was afebrile. His physician heard rales (evidence of abnormal fluid accumulation) in the bases of both lungs. His feet and ankles were swollen. A chest x-ray showed pulmonary edema and pleural effusions and a significantly enlarged left ventricle. These findings were consistent with right and left ventricular congestive heart failure, which is a reduced capacity of the heart to pump normal volumes of blood, resulting in fluid accumulation in various tissues. C.M. was admitted to the cardiology service of the University Hospital. On the basis of further tests, including coronary angiography and echocardiography, C.M. was given the diagnosis of dilated cardiomyopathy (a progressive and fatal form of heart failure in which the heart chambers become dilated and inefficient at pumping blood). His physicians told him he may benefit from aggressive medical management, including drugs that enhance heart muscle contraction, reduce the workload of the heart, and enhance excretion of accumulated fluid, but if his underlying heart disease continued to progress, the best long-term option would be to receive a heart transplant. Unfortunately, despite optimal medical management, his symptoms of congestive heart failure continued to worsen until he was no longer able to manage even routine activities of daily living, and he was listed for heart transplantation.
A panel-reactive antibody (PRA) test was performed on C.M.’s serum to determine whether he had been previously sensitized to alloantigens. This test (performed monthly) showed the patient had no circulating antibodies against human leukocyte antigens (HLAs), and there was no further immunologic testing done at that time. Two weeks later in a nearby city, a donor heart was removed from a victim of a construction site accident. The donor had the same ABO blood group type as C.M. The transplant surgery, performed 4 hours after the donor heart was removed, went well, and the allograft was functioning properly postoperatively.
What problems might arise if the transplant recipient and the donor have different blood types or if the recipient has high levels of anti-HLA antibodies?
C.M. was placed on immunosuppressive therapy the day after transplantation, which included daily doses of tacrolimus, mycophenolic acid, and prednisone. Endomyocardial biopsy was performed 1 week after surgery and showed no evidence of myocardial injury or inflammatory cells. He was sent home 10 days after surgery, and within a month he was able to do light exercise without problems. A routinely scheduled endomyocardial biopsy performed within the first 3 months after transplantation was normal, but a biopsy performed 14 weeks after surgery showed the presence of numerous lymphocytes within the myocardium and a few necrotic muscle fibers ( Fig. A.2 ). The findings were interpreted as evidence of acute allograft rejection.
What was the patient’s immune system responding to, and what were the effector mechanisms in the acute rejection episode?
C.M.’s serum creatinine level, an indicator of renal function, was high (2.2 mg/dL; normal, <1.5 mg/dL). His physicians therefore did not want to increase his tacrolimus dose because this drug can be toxic to the kidneys. He was given three additional doses of methyl prednisolone (a steroid drug) over 18 hours, and a repeat endomyocardial biopsy 1 week later showed only a few scattered macrophages and a small focus of healing tissue. C.M. went home feeling well, and he was able to live a relatively normal life, taking tacrolimus, mycophenolic acid, and prednisone daily.
What is the goal of the immunosuppressive drug therapy?
Coronary angiograms performed yearly since the transplant showed a gradual diffuse narrowing of the lumens of the coronary arteries. In the sixth year after transplantation, C.M. began experiencing shortness of breath after mild exercise and showed left ventricular dilation on radiographic examination. An intravascular ultrasound examination demonstrated significant diffuse thickening of the coronary arterial walls with luminal narrowing ( Fig. A.3 ). An endomyocardial biopsy showed areas of microscopic subendocardial infarction, as well as evidence of sublethal ischemia (myocyte vacuolization). C.M. and his physicians are now considering the possibility of a second cardiac transplant.