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Primary Immunodeficiency Diseases
The control of killer cell activation  |  The case of Nicholas Nickleby  |  X-Linked Lymphoproliferative Disease (XLP)

X-Linked Lymphoproliferative Syndrome

The control of killer cell activation

Viruses pose a constant challenge to our immune system. Unable to reproduce on their own, they have evolved as parasites, capable of residing within living cells whose biosynthetic machinery they subvert for their own reproduction. The only effective immune response to these hidden invaders is to kill the host cells within which they reside. Both innate and adaptive immune strategies have evolved to control viral infections. In the innate immune response, natural killer (NK) cells are constantly on surveillance for cells with telltale markers of viral infection. This response does not require any previous immunological experience with the virus and is particularly important in the first encounter with a virus. In the adaptive immune response, virus-specific effector cytotoxic T lymphocytes are generated during the primary immune response to the virus and establish a pool of virus-specific memory cytotoxic T cells. In the event of reexposure to the same virus, by either reinfection from the environment or reactivation of a latent virus persisting in the body, these cytotoxic T cells rapidly recognize and kill infected host cells displaying viral antigens.

The induction of effective cytotoxic T cell and NK cell responses requires the coordinated action of multiple independent signals. NK cells express two classes of receptors, which activate or inhibit the NK cell's killer activity, respectively. The fate of potential target cells is determined by the balance of activating and inhibitory signals they deliver to NK cells through these receptors. The activating receptors include NKR-P1 family, which recognize carbohydrate structures, and FcγRIII, which binds IgG and can mediate antibody-dependent cell -mediated cytotoxicity. A third activating NK receptor is 2B4, a member of a subfamily of the immunoglobulin superfamily. 2B4 interacts with CD48, another member of the same subfamily, on target cells. Several inhibitory receptors have been characterized, including killer inhibitory receptors (KIRs) and CD94/JKG2A, which both interact with MHC class I molecule.

Virus infection often interferes with normal host cell functions, including protein synthesis and thus the synthesis of MHC molecules. In addition, some viruses alter the glycosylation of cellular proteins, or induce the transcription of cellular genes, giving rise to the expression of novel cell-surface structures. CD48, a target for the activating NK receptor 2B4, was first recognized on B cells infected with Epstein- Barr virus (EBV). Thus, the combined effects of viral infection include increased expression of NK-cell-activating signals, including CD48, with a concomitant decrease in KIR-mediated NK cell inhibition on recognition of MHC class I molecules; these signals act in concert to trigger NK cell-mediated killing.

The adaptive immune response to viruses relies upon activation of virus-specific CD8 T cells to their effector status as cytotoxic cells, which is also tightly regulated by several receptors. Them most critical of these is the T-cell antigen receptor, which interacts with a complex of antigen-derived peptide and MHC class I molecule at the surface of a cell presenting viral antigens. For naïve CD8 T cells, however, engagement of T-cell receptors although necessary, is not sufficient for activation. In fact, when T-cell receptor ligation occurs in isolation, it can act as a negative signal and induce cellular energy, a state in which T cells are resistant to activation following subsequent encounters with the same antigen. Second signals, generated by the interaction of accessory molecules expressed on T cells and targets, are required to support T-cell receptor-triggered activation of the helper T cells, which are necessary for the functional activation of the cytotoxic T cells. A number of receptor-ligand interactions can provide such co-stimulatory signals, including CD40-CD40 ligand (CD154) and B7-CD28 interactions.

Recently, the signaling lymphocytic activation molecule SLAM (CD150) has been characterized as a potent T-cell co-activator, and has also been found to be a receptor for measles viruses. SLAM is rapidly induced on T cells following their activation and is a powerful 'self-ligand' which is stimulated by the binding of identical SLAM molecules on another cell, such as a B cell or monocyte. The cytoplasmic portions of 2B4 and SLAM have similar structures, which include tyrosine residues that provide potential docking sites for intracellular signaling proteins that contain Src homology 2 (SH2) domains. Once such associated protein has been designated SLAM-associated protein (SAP).

EBV is a very prevalent virus that is usually well controlled by NK and cytotoxic T-cell responses. EBV infects most people by the age of 15, and primary EBV infection triggers activation and cell division in B cells infected by the virus. The infected B cells express CD48, an activating ligand for NK cells, and a number if viral antigens that are targets for specific cytotoxic T cell responses. These cell-surface proteins together drive powerful NK and cytotoxic T-cell responses, which rapidly control the proliferation of infected B cells. In most people, EBV infection remains asymptomatic, but in a minority of cases (a subset of patients who first encounter the virus in adolescence) it gives rise to acute infectious mononucleosis. Following resolution of the acute infection, the virus persists in a latent form in B cells, salivary glands, and epithelial cells of the most and throat, and can be shed in saliva. Occasional reactivation of virus replication later in life is rapidly brought under control by NK cells along with EBV-specific memory cytotoxic T cells. This cellular immune surveillance is critical in maintaining the balance between host and virus. Primary and acquired deficiencies of T-cell function are associated with a marked susceptibility to lethal EBV infection.

In very rare instances, acute EBV infection in boys is not contained, and results in a failure to eliminate the virus that is accompanied by a massive lymphoproliferation, cytokine production, tissue destruction, and often death. Such susceptibility to overwhelming infection can be inherited through unaffected females, and the condition has thus been designated X-linked lymphoproliferative syndrome (XLP). As this case illustrates, a defect in the gene encoding the signaling protein SAP has effects on both NK cells and T cells that render them unable to kill target cells and control the infection.

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The case of Nicholas Nickleby: inefficient killing of EBV-infected B cells by cytotoxic lymphocytes.

Nicholas was brought to the pediatrician at 5 years old because of several days of fever (38-39oC). He had no cough, runny nose, rash, diarrhea or any other symptom of infection. The physical examination revealed only some mildly enlarged lymph nodes in his neck and his neck and his parents were advised to treat the fever with acetaminophen. Over the following weeks, the fevers persisted and Nicholas seemed less energetic than usual. He was brought to the doctor several times but the only consistent finding was persistent enlarged, nontender lymph nodes. Finally, after six weeks of illness, Nicholas complained of abdominal pain and was referred to the Children's Hospital.

The past medical history was significant, revealing problems with persistent and recurrent middle ear infections (otitis media) as well as several episodes of bacterial pneumonia between the ages of two and three. Imunological evaluation at the time had revealed decreased blood levels of IgG of 314 mg dl-1 (normal 600-1500 mg dl-1). And normal IgA and IgM. Nicholas was briefly treated with prophylactic antibodies with a good response. These were discontinued at the age of four and he had no further infections or follow-up tests before his admission to Children's Hospital. The family history was notable for the presence of a maternal uncle with persistent unexplained low blood platelet count (thrombocytopenia). Nicholas's maternal grandfather had recurrent lymphomas.

On admission to hospital, Nicholas appeared tired but not acutely ill. His temperature was 38.5oC and heart rate, respiration, and blood pressure were all normal. His height and weight were both in the 25th percentile for age. A few scattered small skin hemorrhages (petechiae) were noted over the lower extremities. Several lymph nodes were palpable in his neck and these appeared significantly larger than on previous examinations. Superclavicular, axillary, or inguinal lymph nodes were not enlarged. The tonsils were moderately enlarged but were not red, and there was no evidence of inflammation. The heart sounds were normal. The abdomen was moderately distended but soft, and slightly tender in the right upper quadrant. The liver was enlarged and its edge was palpable 4 cm below the right coastal margin.

Laboratory evaluation showed a mild anemia with a hematocrit of 28% (normal 35-40%). The white blood cell count was 6400 μl-1 (normal 5-10,000 μl-1) and the platelet count was decreased at 47,000 μl-1 (normal 150-200,000 μl-1). The count of different types of white blood cell was remarkable for the very high proportion (22%) of atypical lymphocytes (normal less than 2%). Liver function tests indicated liver damage. Tests for antibodies to hepatitis A, B and C viruses were negative. The titer of IgM antibody against EBV viral capsid antigen (VCA) was positive at greater than 1:40. Anti-VCA IgG antibody was 1:320 and antibodies to Epstein-Barr nuclear antigen (EBNA) and early antigen (EA) were undetectable, consistent with an acute EBV infection. Circulating EBV genome was detected in Nicholas's blood cells by the polymerase chain reaction (PCR). A chest X-ray showed clear lungs and a normal-sized heart, but the lymph nodes in the mediastinum were enlarged. Ultrasound examination of the abdomen revealed a significant amount of free fluid in the abdominal cavity (ascites) and an enlarged liver. An abdominal CT scan revealed marked enlargement of lymph nodes in the retroperitoneum.

In light of the family history and laboratory evidence of acute EBV infection, a diagnosis was made of X-linked lymphoproliferative syndrome with fulminant infectious mononucleosis. Nicholas was kept in hospital and initially treated with an antiviral agent, acyclovir, and intravenous immune globulin (IVIG) in an attempt to control the EBV infection. However, the fever persisted and his liver dysfunction and ascites rapidly worsened. The glucocorticoid dexamethasone was added to his therapy. Despite these aggressive interventions, Nicholas developed sever shock symptoms, resembling those following blood stream infection (sepsis), with diffuse vascular leakage, a fall in blood pressure (hypotension), poor circulation, and multiorgan failure. All cultures for bacterial pathogens were negative. He died 10 days after admission to the hospital.

At post mortem, the liver was markedly enlarged. Fluid had accumulated in the abdomen and around the lungs (pleural effusions). EBV was identified by culture and PCR in the liver and bone marrow. There was a striking infiltration of the liver, spleen, and lymph nodes by a mixed population of mononuclear cells including small lymphcytes, plasma cells, and lymphoblasts. In the liver, these infiltrates were associated with extensive tissue death (necrosis). Examination of the bone marrow revealed a decreased number of erythoid, megakaryocytic, and myeloid cells along with increased numbers of histiocytic cells, lymphocytes, and plasma cells.

Analysis of SH2D1A gene function by Northern blotting revealed the complete absence of SAP (SH2D1A) mRNA. None of the four exons encoding SAP could be isolated by polymerase chain reaction (PCR), consistent with complete deletion of the gene. Such complete deletions have now been shown to be common in XPL.

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X-Linked Lymphoproliferative Disease (XLP)

The gene responsible for many, if not all, cases of familial XLP has been mapped to the X chromosome at position Xq25, and has been identified as SH2D1A, which encodes the intracellular signaling protein SAP. Patients with this defect sustain uncontrolled T-Cell activation, especially in response to an EBV infection, and a reduced capacity to kill EBV-infected B-cells. In a minority of cases, symptoms of XLP occur without evident past or current EBV infection. Boys presenting with EBV-induced fulminant infectious mononucleosis, and who have a family history of affected male relatives, have XLP as a result of mutations in the SAP gene. The fulminant infectious mononucleosis following their initial encounter with Epstein-Barr virus commonly proves lethal; among 161 boys with XPL, 57% died of culminant infectious mononucleosis. Of those who survived, half developed lymphomas, ad did Nicholas' maternal grandfather, and the other half became agammaglobulinemic as a result of the destruction of their B cells. In rare instances, the bone marrow of affected males may be destroyed, resulting in the fatal disease of aplastic anemia or in thrombocytopenia, as in Nicholas' maternal uncle.

In normal individuals, EBV-infected B cells are attractive targets for killing by both NK cells and virus-specific effector cytotoxic T cells. The expression of MHC class I molecules is reduced on infected B cells, whereas CD48 is induced at high levels. Thus, on encountering NK cells, the inhibitory signal via KIR is minimal and the activating stimulus via 2B4 is maximal, making the infected B cells highly susceptible to lysis by NK cells. Signaling via 2B4 is critical in driving NK killing of EBV-infected target calls. 2B4 contains two tyrosines in its cytoplasmic tail, which after the receptor is activated, become phosphorylated and constitute docking sites for cytosolic proteins containing SH2 domains. Under normal conditions, the SH2-containing SLAM-associated protein SAP binds to these cytoplasmic tyrosines, and allows propagation of the activating signal onward. The interaction of SAP with the cytoplasmic tyrosines of 2B4 inhibits the binding of other cytosolic SH-2 containing molecules.

In the absence of SAP, the phosphorylated tyrosines of 2B4 become available for interaction with other cytosolic SH2-containing proteins. One such, SHP-1, has been shown to bind to the cytosolic domain of 2B4. SHP-1 is typrosine phosphatase and can thus dephosphorylate the tyrosines and inhibit cellular activation pathways involving tyrosine phosphorylaton. Thus, in the case of patients lacking SAP, the interaction of CD48 on EBV-infected targets with 2B4 on patrolling NK cells has a dramatic inhibitory effect on their killing activity, undermining the innate immune response to the virus.

Elimination of EBV-infected targets by cytotoxic T cells is also hindered in the case of SAP deficiency. Under normal conditions, the interaction of the T-cell receptor with MHC molecules complexed with EBV-derived peptides is rapidly followed by marked increase in surface expression of SLAM on the T cells to enhance T-cell activation. Indeed, experiments in which SLAM on the T cells is stimulated with anti-SLAM antibodies show markedly enhanced antigen-triggered T-cell activation whereas SLAM antagonists inhibit signaling. Like 2B4, the cytosolic domains of SLAM contain tyrosines that become phosphorylated when SLAM molecules bind each other, and which can then interact with SAP. In the absence of SAP, they can interact with the SH2-domain-containing tyrosine phosphatase, SHP-2, which has an inhibitory effect on T-cell activation. T cells also express 2B4. The binding of CD48 on EBV-infected targets to 2B4 on cytotoxic T cells acts via SAP to augment activation of cytolytic function, as in NK cells. So in the absence of SAP, in XLP, this normally co-activating signal is converted to an inhibitory one.

In individuals with SAP mutations, the combined defects in cytolytic function of NK cells and cytotoxic T cells, the two pillars of antiviral immunity, is a devastating blow to the immune response to EBV infection. B cells harboring the virus are not eliminated and EBV persists. Although SAP deficiency impairs the execution of an effective cytotoxic response, it does not inhibit other aspects of cellular immune activation, possible because the SLAM/SAP pathway is not necessary for cytokine gene expression and cytokine secretion. In fact, the persistent heavy viral burden drives a massive T-cell response characterized by lymphoproliferation and cytokine secretion in the absence of effective generation of cytotoxic T cells. Plasma levels of a number of T-cell-derived cytokines, including interferon (IFN)-γ, interleukin (IL)-2, a tumor necrosis factor (TNF)-α, are all markedly elevated. The same cytokines are present at much lower (or undetectable levels) in normal EBV-infected individuals, even those with infectious mononucleosis. The end result of this ineffective response is bystander injury to normal tissues and eventual lethal organ damage. In patients with fulminant infectious mononucleosis, the uncontrolled lymphocyte proliferation and cytokine secretion leads to a syndrome of severe inflammation of the liver (hepatitis), destruction of bone marrow cells, and systemic shock.

Activated T cells drive this pathology in several ways. T-cell derived cytokines, particularly IFN- γ, trigger activation of monocytes/macrophages. The activated T cells and macrophages infiltrate tissues, including liver and bone marrow, displacing the normal resident cells and destroying functional organ architecture. The activated macrophages engage in indiscriminate phagocytosis of surrounding cells. Histologic analysis of tissues from patients with fulminant infectious mononucleosis often reveals 'erythrophagocytosis', a phenomenon in which macrophages appear to engulf entire red blood cells and others are heavily laden with cellular debris. Erythrocyte phagocytosis is further enhanced because the polyclonal B-cell activation induced by EBV infection produces complement-fixing antibodies that interact with red cell antigens.

In a second potential mechanism of tissue injury, particularly in the liver, T-cell derived cytokines may promote the expression of Fas on hepatocytes. The interaction of this apoptosis-inducing receptor with its ligand, FasL, on the surface of activated T cells can induce hepatocyte death.

Further aggravating the cell death induced by cellular invasion and FAS activation is the general hypoperfusion associated with shock. T-cell derived cytokines, particularly TNF-α as monocyte-derived IL-1, result in enhanced vascular permeability and loss of intravascular volume. This is analogous to the clinical scenario observed in toxic shock syndrome in which uncontrolled T-cell activation and cytokine secretion can also lead to multiorgan damage and a clinical picture resembling sepsis.

We would like to thank Dr. Fred Rosen and Dr. Raif Geha for their contribution of the above information from their book, "Case Studies in Immunology 3."

If you or your physician would like additional information on this disease please click here.

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Disclaimer: The information contained on these pages is not intended to provide specific medical advice; rather it is intended for informational purposes only, in order to provide a better understanding of these diseases. Please consult with a qualified physician for diagnosis and answers to your questions.

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