204_ImmtutIGRA_TUTOR_120516 (2).docx

Module 204 Immunology Tutorial 2 The immunology of infection This second immunology tutorial considers immune responses to infection using the example of Mycobacterium tuberculosis to link with T cell immunology and diagnostic immunology. As preparation for this tutorial, you should have already vie...

Module 204 Immunology Tutorial 2 The immunology of infection This second immunology tutorial considers immune responses to infection using the example of Mycobacterium tuberculosis to link with T cell immunology and diagnostic immunology. As preparation for this tutorial, you should have already viewed https://youtu.be/lkHmyC5C84A Your tutor will give you about 30 minutes to complete the exercises, before discussing your answers for the remaining time. You may work individually, in groups or as directed by your tutor. We suggest that you don’t access learning resources to answer the questions: there simply isn’t enough time, and you will learn more by working out the answers together. Aims This tutorial will cover the following: Phagocytosis Antigen processing by the class II pathway CD4 T cell help to macrophages Latent TB Principle of Interferon-γ release assay (IGRA) Principle of ELISA Learning outcomes You should be able to: Draw/ describe phagocytosis and the associated processes for intracellular killing Draw/ describe antigen presentation to CD4 T cells after phagocytosis Explain how CD4 T cells interact with phagocytes to control TB infection Describe the principles of IGRA for the diagnosis of latent TB Draw a diagram of an ELISA system A chid is exposed to mycobacterium tuberculosis by the respiratory route. Which immune cells initially recognise and deal with the infection and how? Phagocytosis: Lung macrophages ingest the MTB organisms into phagosomes. These then fuse with lysosomes. Killing: The acidic environment, activation of digestive enzymes and oxidative burst aim to destroy the TB organism. Other effects: The macrophages will also release cytokines such as TNF-alpha that cause local inflammation, attracting further inflammatory cells to the site..,……..However, the MTB organisms are adapted to arrest phagosome maturation and inhibit phagosome/ lysosome fusion, some strains can also live and divide in macrophage phagolysosomes and are difficult to eliminate. How will CD4 T helper cells be activated during this infection? (you may wish to draw a diagram) Macrophages will migrate to local lymph nodes. MTB antigens will be processed via the class II pathway and presented as peptides to CD4 T cells. The MTB antigens induce the macrophage to secrete IL-12, which in turn elicits a strong Th1 response; lFN-γ and other Th1 cytokines, in turn activate the macrophage, in an attempt to eliminate the MTB. For example lFN-γ promotes phagosome maturation and phagosome/lysosome fusion. Suggest tutors use whiteboards to take students through the steps involved. The child did not become sick. A routine chest radiograph was taken many years later with the following appearances. What is the name of the lesion at the right hilum, and what immunological process has produced it? The lesion is known as a Ghon focus, and represents a site of contained primary MTB infection. It has fibrosed and calcified over time, allowing it to become visible on CXR (see T cell lecture module 102 and vaccine lecture module 204). If removed at the right stage in its development, the lesion would show granuloma, probably with central necrosis [due to hypoxia]. The underlying immunology is: A blood sample is taken to assess for possible (latent) TB infection by Interferon-γ release assay (also known as IGRA). The principles of the test are: The patient’s blood is incubated in tubes with the TB antigens CFP10 and ESAT-6 for 16 hours The tube is then centrifuged, so that the cells form a pellet at the bottom of the tube The fluid that the cells were suspended in (the ‘supernatant’) is removed by pipetting, leaving the cells at the bottom of the tube The supernatant is then analysed for IFN-γ content using ELISA The ELISA system is shown below. Complete the labels A = anti-interferon-γ  B = interferon gamma [from blood] supernatant C = labelled detection antibody The results are shown below. Comment on the technical validity of the results. What is the interpretation and why? What might the patient be offered? Tube Interferon-γ (IU/ ml) Ranges (IU/ ml) Negative control (unstimulated) <0.35 0-1.20 Positive control (PHA) 23.1 12.5-35.6 Stimulated with TB antigens 9.4 <1.75 negative 1.75-3 indeterminate >3 positive No significant IFN production in negative control well, copious amounts in PHA well – ie test is technically valid The patient’s T cells have produced large amounts of interferon-γ  after stimulation with TB antigens. This shows the patient has circulating effector memory Th1 cells specific for TB antigens, consistent with latent TB infection It would be important to understand that the antigens used in the QFT Gold test (ESAT-6, CFP-10, fragment of TB7.7) are not contained in the BCG vaccine, since only this will allow the clinician to discriminate between vaccine-induced and exposure-induced immunity. Be careful to explain that this is not a good test for active TB. This patient may warrant treatment with chemoprophylaxis to reduce the risk of progression to active TB. For more discussion, see: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5905a1.htm