Exam 13: Failures of Host Defense Mechanisms

In the late 1990s, a group of individuals was discovered that remained uninfected with HIV, in spite of multiple exposures to the virus. Analysis indicated that these HIV-resistant individuals had a homozygous deficiency caused by a 32-bp deletion in a single gene, and furthermore, that this mutation was present at a frequency of ~10% (in heterozygous form) in individuals of European descent. These data provided clear evidence indicating that:

Viruses utilize many different strategies to evade the immune response after infection. One strategy, illustrated in Figure , is an effect on virus-infected cells. The virus used in this experiment is most likely:

Patients with X-linked lymphoproliferative syndrome type 1 (XLP1) have mutations in the SLAM-family adapter protein, SAP. These patients suffer from an inability to control infections of the herpesvirus Epstein-Barr virus (EBV) that infects B cells. Studies have been done using peripheral blood T cells from these patients to examine their response to EBV in comparison to the responses of normal individuals (EBV-infected) as controls. These data were obtained by ELISPOT analysis, to quantify the numbers of IFN- $\gamma$ -producing CD8 T cells per million peripheral blood leukocytes in response to several EBV peptides, indicated as peptide 1, 2, 3, or 4 (note: the patients and controls were matched for HLA class I molecules). These results indicate that:

Nearly all immune deficiency diseases that result in impaired T_H17 and ILC3 function lead to chronic mucocutaneous candidiasis (CMC), usually accompanied by increased susceptibility to pyogenic bacterial infections. However, a subset of these patients also show increased susceptibility to intracellular bacterial infections, such as those caused by Mycobacteria species. What is the explanation for why some, but not all, of these patients have increased susceptibility to intracellular bacteria and which patients fall into this category?

Yersinia pestis, the causative agent of the bubonic plague, has multiple mechanisms of immune evasion. This Gram-negative bacterium is transmitted from fleas (body temperature, 26°C) to humans (body temperature, 37°C) by flea bites. Studies have shown that the lipopolysaccharide (LPS) produced by Y. pestis grown at 37°C is about 10-fold less potent at stimulating TLR4 signaling than is the Y. pestis LPS from bacteria grown at 26°C. When these two forms of Y. pestis LPS are compared for their abilities to induce responses from human macrophages, one would expect that the 26°C Y. pestis LPS would result in:

Herpesviruses are a class of viruses that establish life-long infections in human hosts. Estimates suggest that >90% of the population is infected with several of these viruses, including EBV, CMV, and herpes zoster, the cause of chicken pox. One herpesvirus, Herpes Simplex virus, causes recurrent cold sores in infected individuals. Thus, in spite of a robust anti-viral CD8 T cell response, these individuals still suffer from periodic virus-induced cold sores following exposure to UV light or certain hormones or other stressors. This anti-viral CD8 T cell response:

Patients with Wiskott-Aldrich syndrome show severely impaired responses to vaccines such as the tetanus vaccine, which is composed of the inactivated tetanus toxin (i.e., tetanus toxoid). Yet, these patients can generate normal antibody responses to bacterial polysaccharide antigens. This selective defect in antibody responses in Wiskott-Aldrich syndrome patients is due to:

The current view in the field of immunology is that dendritic cells are the primary antigen-presenting cells for stimulating naive T cells. One piece of evidence supporting this conclusion is the observation that IRF8-deficient individuals, which retain their tissue-resident macrophages, are susceptible to a range of severe opportunistic infections caused by intracellular bacteria, viruses, and fungi. Explain the reasoning behind this argument.

Important information can be learned by studying the immune system of female carriers of the XLA disease gene. These individuals, identified as mothers of boys with XLA, show non-random X chromosome inactivation in their B cells, but random X chromosome inactivation in all of their other cells, including their T cells and macrophages. This finding indicates that:

The Borrelia hermsii spirochete causes a relapsing fever, characterized by multiple periods of high fever followed by asymptomatic intervals. The major outer membrane protein of the bacterium is known as VMP, for variable membrane protein, as >50 variants of this protein are found among B. hermsii isolates. Interestingly, each bacterium contains genes encoding all of these variants, and during bacterial replication, ~1 in 10³-10⁴ bacteria undergo VMP switching per generation, thereby changing the VMP protein variant expressed on their surface. How does the VMP system explaining the relapsing/remitting fever caused by this pathogen?

Some forms of SCID are due to defects in common 'housekeeping' enzymes, such as enzymes involved in nucleotide biosynthesis pathways, that are present in all cells of the body. These genetic deficiencies cause SCID because:

HIV encodes several proteins that function to promote viral replication, packaging, and budding from host CD4 T cells. In addition to inhibiting cell-intrinsic antiviral mechanisms and down-regulating surface expression of MHC class I and class II molecules, these proteins function to:

A variety of genetic defects can result in patients exhibiting SCID. For many of these diseases, infants with the disease are given bone marrow transplants from healthy donors to restore normal immune function. Yet, some diseases causing T cell deficiencies cannot be treated by bone marrow transplantation. Which of the diseases below is treatable by giving patients hematopoietic stem cells from a healthy donor?

Bone marrow transplantation is currently used to treat many immunodeficiency diseases resulting from defects in hematopoietic cells, such as lymphocytes or myeloid cells. Often, the donor of the bone marrow is a healthy relative of the patient, such as a sibling or a parent . Which of the individual relatives above would not be a potential donor for the patient's bone marrow transplant?

One important feature of retroviruses such as HIV is their generation of a provirus, a copy of the viral genome that is inserted into the host cell chromosome. In addition to providing a template for viral mRNA transcription, the proviral genome:

Infants with RS-SCID generally require treatment by bone marrow transplantation from a healthy donor. In this treatment, hematopoietic stem cells from the donor will give rise to normal B and T lymphocytes, thereby restoring normal immune function. However, the bone marrow transplantation treatment does not alter the high incidence of cancer in these patients. Why not?

The first drug treatment for HIV licensed in the US was zidovudine (AZT), a reverse transcriptase inhibitor. However, AZT has now been completely replaced by HAART as the recommended treatment for HIV-infected individuals. The use of HAART, rather than AZT, is preferred because:

Common variable immunodeficiency (CVID) is a rather mild immunodeficiency disease that generally appears in young adults, with a peak onset between ages 20-30. Approximately 25% of the cases of CVID are familial, indicating a genetic basis for the disease. CVID patients suffer from recurrent infections of the respiratory tract, the ears, and the gastrointestinal tract. Common infections in these patients are caused by Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus. Analysis of their peripheral blood cells shows normal numbers and subsets of T cells. B cell data from peripheral blood are shown in Figure : a) In general, what is the immune mechanism most likely defective in patients with CVID? Explain your reasoning. One particular family is chosen for further study. In this family, two of four children have CVID, as shown in Figure Given this pedigree, the mother’s peripheral blood leukocytes are examined for their patterns of X-chromosome inactivation. The results show that all of her T cells, B cells, and monocytes show random (50:50) inactivation of each of her X chromosomes. b) What do these data indicate about the gene defect causing CVID in this family? In a second series of studies, peripheral blood cells from one CVID patient and one healthy child in this family were collected, and T cell and B cell populations were separately isolated. These cell populations were mixed in different combinations, and then cultured for one week in the cytokines IL-4, IL-21, and IL-6. At the end of one week, the concentrations of secreted antibody in the culture supernatants were measured c) What do these data indicate about the gene defect causing CVID in this family? In a final set of experiments, the isolated T cells and B cells from the study above were used in a transwell assay. In this assay, shown in Figure , cell populations are separated by a semi-permeable membrane. This allows soluble mediators to equilibrate between the top and bottom chambers, but prevents direct cell–cell contact. As a control, the cell populations were mixed together in the bottom chamber, a result that would replicate the findings from the study above. In addition, in this study, all wells contained IL-4, IL-6, and IL-21. After 7 days, culture supernatants were analyzed for antibody concentrations.The results of the transwell experiments are shown in Table . d) What do these data indicate about the function of the gene that is defective in the CVID patients from this family?

A mouse model of an immunodeficiency disease affecting the innate immune response was generated by knocking out a single gene. Studies of these mice, performed in the 1990s, showed that they were highly susceptible to infections of pyogenic bacteria, such as Staphylococcus and Lactobacillus species, and to infections of fungal pathogens, such as Paecilomyces. In fact, without deliberately infecting the mice, the researchers performing these studies found that the knockout mice were spontaneously succumbing to these infections while housed in their vivarium (animal facility), as shown in Figure. To determine the potential mechanism leading to the immune deficiency, a series of additional studies were performed. In one study, peripheral blood leukocytes from the mice were mixed with Staphylococcus aureus bacteria, and the ability of the cells to kill the bacteria was assessed. In a second study, the compound thioglycolate was injected into the peritoneal cavity of the mice, and five hours later the numbers of white blood cells were measured. This compound elicits a robust inflammatory response in the peritoneum, including the production of several inflammatory cytokines, such as TNFα, IL-1, and IL-6, as well as a number of chemokines known to recruit neutrophils and monocytes from the blood to the site of inflammation. The results of these studies are shown in Figure . Given these data, a likely candidate for the gene that was targeted in these knockout mice is:

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium. Infection with L. monocytogenes is the leading cause of food-borne illness. After uptake by phagocytic cells (e.g., macrophages) in the infected host, L. monocytogenes replicates to high levels in the macrophage cytosol, and then travels to neighboring macrophages by polymerizing cytosolic actin, and using these 'actin rockets' to traverse into an adjacent cell. A mutant strain of L. monocytogenes (Lm-mutant) has been identified that shows greatly reduced virulence in animal models, and is impaired in its ability to establish a focus of bacterial replication in isolated macrophages in culture, when compared to wild-type L. monocytogenes (wild-type Lm). Additional data indicated that wild-type Lm and Lm-mutant strains were equally efficiently phagocytosed by macrophages, indicating no defect in Lm-mutant bacteria in gaining access to their target host cell. The graphs in Figure show: (top) % macrophages with large numbers of replicating bacteria after in vitro infection; (middle and bottom) CFU per liver or per spleen following infection of mice with non-lethal doses of bacteria. The table shows the LD₅₀, a measure of the dose of bacteria that will kill 50% of the mice following infection. a) Based on these data, name two possible functions of the gene missing in the Lm-mutant strain. To assess the function of the defective gene in the Lm-mutant strain, wild-type Lm and Lm-mutant bacteria were used to infect isolated macrophages and the subcellular localization of individual bacteria was examined by electron microscopy. Representative images of a timecourse post-infection are shown in Figure b) What is the most likely function of the gene that is defective in the Lm-mutant strain and what is the fate of the Lm-mutant bacteria? L. monocytogenes infection triggers a broad range of immune responses. Early after infection, L. monocytogenes induces innate immune responses consisting of the production of several pro-inflammatory cytokines and other mediators. This results in elimination of most of the bacteria. However, complete clearance of the L. monocytogenes infection requires an effective T cell response targeting the bacterial antigens. Thus, mice that lack T cells become chronic carriers of L. monocytogenes after infection. c) Which subset of T cells is most likely to provide the protective immunity to wild-type L. monocytogenes? d) Would you expect to see an altered T cell response to macrophages infected with Lm-mutant bacteria compared to those infected with wild-type Lm? Explain your reasoning