Immunology Team-Based Learning: Basic Concepts in Immunology · Immunology Team-Based Learning: Basic Concepts in Immunology 4 The Group Readiness Assurance Test (GRAT) was open book

Immunology Team-Based Learning: Basic Concepts in Immunology

(Number 1 of a 9 Module Series)

Wayne T. McCormack Dept. of Pathology, Immunology & Laboratory Medicine College of Medicine, Box 100275 University of Florida 1600 S.W. Archer Road Gainesville, FL 32610 Phone (352) 273-8603 E-mail [email protected] Web http://www.pathology.ufl.edu/~mccormac/

TABLE OF CONTENTS Purpose ……………………………………………………………………………………… 2 Objectives …………………………………………………………………………………… 2 Advanced Preparation Assignment …………………………………………………………. 2 Context ……………………………………………………………………………………… 3 Facilitation Schema …………………………………………………………………………. 3 Readiness Assurance Test (RAT) ………………….……………………………………….. 4 Item Analysis ………………..…………………………………………………………. 5 IRAT & GRAT Mean Scores ………………………………………………………….. 6 RAT Items ……………………………………………………………………………… 7 Group Activity Exercises …………………………………………………………………… 13 Problem 1 ………………………………………………………………………………. 14 Problem 2 ………………………………………………………………………………. 17 Problem 3 ………………………………………………………………………………. 20


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Purpose of Module By the conclusion of this module, learners will be able to understand basic principles of innate and adaptive immunity and effector mechanisms of adaptive immunity, and apply those principles in experimental settings. Specific topics introduced in this module include cell types and tissue organization, functions of B cells and T cell subsets, humoral and cell-mediated immunity, lymphocyte activation, and antigen presentation. Learners will also have an understanding of the historical development of current concepts in immunology regarding the use of antisera to cell surface molecules as markers for lymphocyte subsets, B-T lymphocyte interactions during humoral responses, and antigen recognition by B and T cells. Objectives 1. Learners will begin building a foundation of general knowledge of basic immunology concepts, such as principles of innate and adaptive immunity, and effector mechanisms of adaptive immunity. 2. Learners will be able to evaluate an immunology experimental design related to basic concepts of immunology, demonstrating the ability to:

• Identify or provide examples of appropriate controls. • Justify the use of specific methods. • Assess whether or not a given method provides a desired measure.

3. Learners will be able to interpret data provided for a given experimental protocol, including the ability to:

• Draw conclusions based on the data. • Recognize results that may not fit current understandings. • Discuss results in the context of current concepts of immunology.

4. Learners will be able to predict experimental results given an experimental protocol and relevant background information, including the ability to:

• Decide between alternative experimental outcomes based on current immunology concepts. • Justify predicted results in the context of established immunology concepts.

5. Learners will be able to design an experiment to test a hypothesis, including the ability to:

• Identify required reagents, cell lines, animal strains, etc. • Propose appropriate assays to measure experimental outcomes. • Organize a sequence of experiments. • Justify the proposed design in the context of current immunology concepts.

Advanced Preparation Assignment 1. Reading assignment: Chapter 1, “Basic Concepts in Immunology” Janeway’s Immunobiology, Eds. Kenneth M. Murphy, Paul Travers, and Mark Walport, 7th edition, Garland Science, November 2007 2. Attend relevant lectures. (e.g., Basic Principles of Immunity, Overview of Immunity, Antigens & Antibody Production, Cellular Interactions in the Immune System) 3. Review personal notes and lecture handouts.


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Context This module is the first of nine modules designed to teach experimental immunology to basic scientists and clinician scientists conducting research in immunology and/or using immunological methods. Originally developed for second-year biomedical science MS and PhD students enrolled in a semester-long immunology graduate course, this material is also appropriate for basic science courses for medical and dental students and/or for review of immunology by residents and fellows. In a graduate-level immunology course entitled “Principles of Immunology”, each TBL module has a specific reading assignment, and is preceded by a block of 3-4 lectures. An exam is administered after each set of three TBL modules. Exams consist of a combination of recall (short answer and essay) and problem-solving questions. The Group Activity Exercise problems in this module were derived from past exam questions. *TBL1: Basic Concepts in Immunology TBL2: Innate Immunity & Recognition of Antigen TBL3: Receptor Diversity & Antigen Presentation TBL4: Development of Lymphocyte Repertoires TBL5: T Cell-Mediated Immunity TBL6: Humoral Immune Response TBL7: Immunity to Infection & Host Defense Failure TBL8: Clinical Immunology TBL9: Manipulation of the Immune Response Facilitation Schema For users unfamiliar with team based learning, the following books will be helpful.

1. Team-Based Learning: A Transformative Use of Small Groups in College Teaching, Larry K. Michaelsen, Arletta Bauman Knight , L. Dee Fink, Stylus Publishing, 2004.

2. Team-Based Learning for Health Professions Education: A Guide to Using Small Groups for Improving Learning, Larry K. Michaelsen, Dean X. Parmelee, Kathryn K. McMahon, Diane M. (FRW) Billings, Stylus Publishing, 2007.

Because of scheduling constraints for the graduate course for which this TBL material was developed, we implemented what might be described as a modified form of TBL (described in more detail below), as the activity had to be adapted to a 50-minute classroom session. A manuscript describing this application of TBL in biomedical science graduate education is in preparation, and describes improved student satisfaction and performance after implementation of TBL. The author acknowledge that this is not the best way to implement TBL, and does not recommend it unless the faculty user has similar scheduling constraints. Regardless of this limitation in our implementation, this material should provide a useful resource that can be adapted to other immunology courses in a more standard TBL session. Classes were divided into 4-6 teams with 5-7 students per team. Team diversity was maximized by distributing students into different teams based on academic major, gender, and ethnic background. In our application of TBL, the Individual Readiness Assurance Test (IRAT) was closed book and consisted of only five (5) multiple choice questions answered on a bubble sheet (Scantron). This number of IRAT questions was sufficient to allow representation from the relevant lectures and elicit student comments in course evaluations about how preparing for the TBL sessions helped them keep up with the material and come to class prepared, thus fulfilling the goals of the readiness assurance process.


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The Group Readiness Assurance Test (GRAT) was open book and consisted of the same five multiple choice questions answered on an Immediate Feedback Assessment Technique (IF-AT) card (Epstein Educational Enterprises). GRAT scores were based on awarding 3 points for a correct answer on the first try, 2 points on the second try, etc. The Group Activity Exercise (GAE) was open book and consisted of a multi-part problem including 2-3 multiple choice questions and 1-3 discussion questions focused on a single problem in experimental immunology. Because of the limited amount of time available, only one problem was included in our application. Some multiple choice questions were answered by the team on the IF-AT card, which allowed students to work out partial solutions to problems in a step-wise fashion and saved some discussion time. GAE scores were based on awarding 3 points for a correct answer on the first try, 2 points on the second try, etc. If time allows, faculty user of this resource may wish to forego use of the IF-AT cards entirely and use the multiple choice questions as discussion questions as described below. Discussion questions in our application sometimes included multiple choice questions that were not answered on the IF-AT cards. Instead, in order to facilitate inter-team discussion, a set of four answer cards (A-D, printed on card stock of four different colors), was used for simultaneous reporting of team answers on multiple choice discussion questions. Discussion questions also included open-ended questions that were answered by teams that either volunteered or were called upon by the faculty member leading the class discussion. If the faculty user has scheduling constraints similar to ours, the following TBL session schedule was found to work well for a 50 minute class period: Individual Readiness Assurance Test 5-10 minutes Group Readiness Assurance Test 5-10 minutes Group Activity Exercise 10-30 minutes Faculty-Led Class Discussion 5-10 minutes In our graduate-level immunology course entitled “Principles of Immunology”, TBL constituted 25% of the students’ final grades, with each of three exams counting 25% each. The relative weights of average scores for each TBL component were determined by the faculty in 2006 and 2007 (10% IRAT, 5% GRAT, and 10% GAE), and by student vote in 2008 and 2009 (7% IRAT, 9% GRAT, and 9% GAE in 2008; 6% IRAT, 10% GRAT, and 9% GAE in 2009). Faculty Preparation Note Two books that provide very good accounts of the historical sequence of experiments that led to current concepts in immunology include “The Cellular Basis of the Immune Response” by Edwards S. Golub (2nd edition, 1981, Sinauer Associates, Inc.) and “Textbook of Immunology” by Baruj Benacerraf (Williams & Wilkins, 1984). Readiness Assurance Test A set of 15 multiple choice questions is provided. These questions have been used in sets of five questions (#1-5, #6-10, and #11-15) for the Individual Readiness Assurance Test and the Group Readiness Assurance Test. These questions are provided as a resource, and faculty users are encouraged to select any desired subsets of questions from this and/or other modules to tailor this exercise to their application. Item analysis data based on student performance on Individual Readiness Assurance Tests is provided below for all questions. Please note that not all items adhere to current guidelines of the


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National Board of Medical Examiners for multiple choice questions, and many items involve a false premise or “NOT” question. Such questions can be confusing to some students, so faculty users should feel free to modify these questions as needed for their application. Suggested possible rewording is included for some questions. Item Analysis The following item analysis based on IRAT performance is provided to help users determine the suitability of each item (Crocker L & Algina J, Introduction to classical and modern test theory, Harcourt Brace Jovanovich College Publishers, Fort Worth, 1986). Year: Year administered. N: Number of valid responses (test-takers who completed the form correctly). Difficulty Index: Percent of correct answers (best if between 40 and 80). Low values indicate a

question that is too difficult or an error with the answer key. This item set has 9 items with a difficulty index between 40 and 80.

Point Biserial: Measure of how well questions discriminate between students (best if greater than 0.1). Negative numbers indicate a question that is flawed in some way, e.g., the better examinees are getting it wrong.

Answer Frequency: Percentage of responses for answer choice (best if greater than zero). Answer Frequency: Percentage of responses for answer choice (best if greater than zero).

Year N Question

Correct Answer

DifficultyIndex

Point Biserial % A % B % C % D

2006

30 1 B 43.3 0.66 33.3 43.3 10.0 13.3 31 2* A 71.0 0.71 71.0 3.2 0 25.8 31 3 A 67.7 0.73 67.7 9.7 6.5 16.1 30 4* B 53.3 0.69 16.7 53.3 3.3 26.7 31 5* C 77.4 0.80 0 0 77.4 22.6

2007

22 6* C 72.7 0.48 18.2 0 72.7 9.1 22 7 A 63.6 0.76 63.6 27.3 0 9.1 22 8* D 90.9 0.39 0 0 9.1 90.9 22 9 D 27.3 0.67 27.3 22.7 22.7 27.3 22 10 B 86.4 0.49 0 86.4 9.1 4.5

2008

31 11 B 96.8 -0.24 0 96.8 3.2 0 31 12 B 71.0 0.60 12.9 71 3.2 12.9 31 13 A 25.8 0.46 25.8 9.7 54.8 9.7 31 14 D 51.6 0.52 16.1 12.9 19.4 51.6 30 15 C 33.3 0.55 43.3 10.0 33.3 13.3

23 2* A 91.3 0.30 91.3 0 0 8.7 23 5* C 78.3 0.72 0 0 78.3 21.7

2009 23 6* C 87.0 0.63 13.0 0 87.0 0 23 8* D 91.3 0.45 4.3 0 4.3 91.3 23 4* B 47.8 0.56 30.4 47.8 8.7 13.0

*Questions re-used in 2009


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Individual and Group Readiness Test Assurance Mean Scores

IRAT Possible Points Mean Score (Range) Mean Percentage 2006 5 3.1 (1-5) 62% 2007 5 3.5 (1-5) 70% 2008 5 2.8 (1-5) 56% 2009 5 4.0 (2-5) 79%

GRAT Possible Points Mean Score (Range) Mean Percentage 2006 15 14.3 (12-15) 96% 2007 15 14.3 (13-15) 95% 2008 15 12.0 (11-13) 80% 2009 15 14.8 (14-15) 98%

Notes: Mean IRAT scores were lower in the third year (2008) due to two RAT items, numbers 13 and 15. Both of these items test the student’s ability to follow the experimental design of “classical” experiments that led to our understanding of basic concepts about antibody recognition of antigen (#13 about haptens and carriers) and T cell subsets (#15 about Ly subsets). Students also did relatively poorly on a similar item in 2007 (#9 about haptens and carriers). Although students sometimes struggled with these concepts individually, the teams are generally able to arrive at the correct answers on the GRAT. Five RAT items were re-used in 2009. Based on the item analyses above, individual performance was essentially the same in 2009 for three of the five items (#4, 5 and 8) as in previous years. Students did perform better on two of the five items (#2 and 6). The possibility that old copies of TBL materials were in circulation and helped some students on some questions can not be ruled out.


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Readiness Assurance Test Items The instructions provided to students for the IRAT were as follows: “Please circle your answer for each of the following questions. Then bubble in your response on your answer sheet. For each question, there is one BEST answer.” 1. The lymph node is divided into paracortical areas, which are mostly T cells, and primary lymphoid follicles, which are mostly B cells. Which area of the spleen corresponds to the lymph node paracortical area and is composed mostly of T cells? (A) marginal zone (B) periarteriolar lymphoid sheath (C) germinal center (D) red pulp Correct Answer: (B) The periarteriolar lymphoid sheath of lymphocytes surrounds arterioles in the

spleen, and is composed mostly of T cells. Incorrect Answers: (A) Marginal zones surround primary follicles and are rich in macrophages and B

cells. (C) Germinal centers are sites of activated B cell proliferation and differentiation into plasma cells. (D) Red pulp constitutes the majority of spleen tissue and is the site of red blood cell disposal.

2. Which of the following cell types is NOT considered to be a professional antigen-presenting cell? (A) neutrophil (B) macrophage (C) dendritic cell (D) B cell Correct Answer: (A) Neutrophils are granulocytes whose primary functions are phagocytosis of

pathogens and activation of bactericidal mechanisms. Incorrect Answers: (B-D) Macrophages, dendritic cells, and B cells are all professional antigen-

presenting cells capable of providing costimulation to T cells. 3. Which one of the following statements about antigen processing and presentation is correct? (A) MHC class I molecules present Ag derived from proteins in the cytosol, and MHC class II

molecules present extracellular Ag processed via intracellular vesicles. (B) MHC class I molecules present extracellular Ag processed via intracellular vesicles, and MHC

class II molecules present Ag derived from proteins in the cytosol. (C) B cells present antigens taken up by surface immunoglobulin receptor-mediated endocytosis to

helper T cells via the MHC class I pathway. (D) Cytotoxic T lymphocytes recognize viral antigens presented to them by virus-infected cells via

the MHC class II pathway. Correct Answer: (A) True statement. Incorrect Answers: (B) Incorrect variation of (A). (C) B cells present antigens taken up via surface Ig

receptors via the MHC class II pathway. (D) CTLs recognize viral antigens presented by virus-infected cells via the MHC class I pathway.

Suggested rewording: Which one of the following statements most accurately describes antigen processing and presentation?


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Readiness Assurance Test Items (continued) 4. Which one of the following statements about classical cellular immunology experiments is FALSE? (A) Treatment of cells with anti-Thy1 antiserum plus complement results in the elimination of all T

cells from the cell population. (B) Antibody treatment (e.g. anti-Ly1 plus complement) of lymph node cells after immunization is

a good way to determine the cell surface profile of precursor cells. (C) The graft-versus-host (GVH) reaction, mixed lymphocyte reaction (MLR) and cytotoxic T

lymphocyte (CTL) assay are all measures of cell-mediated immunity. (D) The carrier effect was revealed by secondary immunization of animals with the same hapten

but different carrier, as compared to the primary immunization, resulting in a lower antibody titer.

Correct Answer: (B) Antibody treatment (e.g. anti-Ly1 plus complement) of lymph node cells after

immunization is a good way to determine the cell surface profile of effector cells. Incorrect Answers: (A, C, D) All true statements. 5. Hematopoietic stem cells in bone marrow give rise to leukocytes (white blood cells), including myeloid cells, lymphocytes, erythrocytes, and platelets. Which one of the following cell types does NOT differentiate from the common lymphoid progenitor? (A) B cells (B) T cells (C) monocytes (D) natural killer (NK) cells Correct Answer: (C) Monocytes differentiate from a common myeloid progenitor via the

granulocyte/macrophage progenitor. Incorrect Answers: (A, B, D) All three cell types differentiate from the common lymphoid progenitor. Suggested rewording: Which one of the following cell types differentiates from a common myeloid progenitor? 6. Which ONE of the following statements most accurately describes innate immunity? (A) Although not enough to clear an infection, an innate immune response helps keep an infection

in check until a specific immune response develops about a week later. (B) The most remarkable feature of innate immunity is the ability to respond more strongly upon

subsequent infection with the same pathogen. (C) Cell surface molecules on bacteria trigger phagocytosis and the inflammatory response, key

features of innate immunity. (D) Innate immunity involves anatomical barriers, chemical barriers, phagocytic cells, and

pathogen-specific antibody. Correct Answer: (C) True statement. Incorrect Answers: (A) Innate immunity may be sufficient to clear an infection. (B) Innate responses

do not exhibit stronger responses upon reinfection with the same pathogen. (D) Innate immunity does not include pathogen-specific antibody (specific or adaptive immunity).


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Readiness Assurance Test Items (continued) 7. Which of the following statements about lymphocyte activation is FALSE? (A) B cells require signals from both antigen via surface immunoglobulin (antibody) receptors and

costimulatory receptors on professional antigen-presenting cells. (B) T cell activation leads to the generation of effector T cells and memory T cells. (C) Signaling via two signals is required for cellular proliferation and differentiation to effector

function. (D) The antigen receptors on B cells and T cells that provide “signal 1” via antigen recognition are

antibody and T cell receptor molecules, respectively. Correct Answer: (A) B cells require signals from both antigen via surface immunoglobulin (antibody)

receptors and costimulation by a helper T cell. Incorrect Answers: (B, C, D) True statements. 8. Which experimental approach provided the best evidence for Burnet’s clonal selection hypothesis? (A) Bone marrow and thymus cell reconstitution experiments were used to demonstrate that at least

two cell types were required for antibody production. (B) As originally proposed by Ehrlich’s side chain theory, individual lymphocytes were shown to

have the capacity to produce antibody to multiple different antigens. (C) The hemolytic plaque cell assay allowed the enumeration of individual lymphocytes producing

specific antibody to multiple antigens. (D) After mice were immunized with two distinct antigens, individual lymphocytes were shown to

produce antibody to only one antigen. Correct Answer: (D) True statement about Burnet’s experiment. Incorrect Answers: (A) A true statement, but does not relate specifically to the clonal selection

hypothesis. (B) Represent’s Ehrlich’s side chain theory, but Burnet concluded that individual lymphocytes have the capacity to produce antibody to a single antigen. (C) Not the relevant assay, and same wrong conclusion as in (B).


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Readiness Assurance Test Items (continued) 9. Which ONE of the following statements does NOT accurately describe a feature of the carrier effect in an experiment demonstrating a secondary response to the hapten DNP upon immunization with DNP-EA? (DNP, dinitrophenol; EA, egg albumin; BGG, bovine gamma-globulin) (A) Adoptive transfer of EA-primed cells and DNP-BGG-primed cells allows an immune-

reconstituted mouse to mount a secondary anti-DNP antibody response. (B) Pretreatment of DNP-BGG-primed cells with anti-Thy1 antibody and complement before a

reconstitution experiment results in the removal of BGG-specific T cells. (C) The carrier effect is demonstrated by the observation that secondary immunization of a DNP-

BGG-primed mouse using DNP-EA results in a lower anti-DNP titer than a primary response. (D) The combination of DNP-specific T cells and EA-specific B cells overcomes the carrier effect,

resulting in a vigorous secondary anti-DNP response. Correct Answer: (D) The combination of carrier-specific T cells and hapten-specific B cells overcomes

the carrier effect, resulting in a vigorous secondary anti-DNP response. EA, egg albumin; DNP, dinitrophenol.

Incorrect Answers: (A-C) All true statements. Note: This is one of several questions involving classical experimental immunological approaches that

individual students have difficulty with, but groups are able to work through. 10. Which one of the following is NOT a good assay for a cell-mediated immune function? (A) cytotoxic T lymphocyte (CTL) assay (B) radial immunodiffusion (Ouchterlony) (C) graft-versus-host (GVH) reaction (D) mixed lymphocyte reaction (MLR) Correct Answer: (B) Radial immunodiffusion is an antibody assay. Incorrect Answers: (A, C, D) All good assays for cell-mediated immune functions. Suggested rewording: Which one of the following is a good assay for an antibody-mediated immune function? 11. Which ONE of the following statements accurately describes tissue organization of the immune system? (A) Central lymphoid organs are sites where primary immune responses occur. (B) Peripheral lymphoid tissues are organized to facilitate interaction of APCs with T cells and T

cells with B cells. (C) One of the shared features of lymph node, spleen, and Peyer’s patch tissue organization is the

presence of M cells. (D) A histological sign of an active immune response is the presence of germinal centers in the T

cell-rich paracortical areas of lymph nodes. Correct Answer: (B) True statement. Incorrect Answers: (A) Central lymphoid organs are sites where lymphocytes are generated. (C) M

cells are present only in Peyer’s patches. (D) Germinal centers are located in B cell-rich follicles in the outer cortex of the lymph node.


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Readiness Assurance Test Items (continued) 12. Which ONE of the following statements about T cell subsets is TRUE? (A) The primary function of TH1 cells is the help B cells produce antibody. (B) The end result of CTL activation is killing of the antigen-presenting target cell. (C) TH1 cells recognize antigen presented by MHC class I molecules via TCR and CD4 co-

receptors. (D) Cytotoxic T lymphocytes (CTL) recognize antigen presented by MHC class II molecules via

TCR and CD8 co-receptors. Correct Answer: (B) True statement. Incorrect Answers: (A) The primary function of TH1 cells is to activate macrophages infected with

intracellular bacteria, in addition to stimulating antibody production by B cells. (C) TH1 cells recognize antigen presented by MHC class II molecules. (D) CTLs recognize antigen presented by MHC class I molecules.

13. Which row of data BEST predicts the results of an immunodiffusion experiment in which antiserum raised against meta-azobenzenesulfonate coupled to chicken egg albumin (anti-ABS-CEA) is reacted with a variety of test antigens? Antiserum: + − + + + + + +

Antigen: − ABS-CEA ABS-CEA ABS-HSP ABA-HSP CEA HSP DEA A − − +++ ++ ± ++ − + B − − +++ ++ − ++ − − C − − +++ + − + − + D + − +++ + + + + +

Abbreviations: ABS, meta-azobenzenesulfonate; ABA, meta-azobenzenearsonate CEA, chicken egg albumin; DEA, duck egg albumin; HSP, horse serum protein Correct Answer: (A) Indicates a vigorous antibody response to the specific hapten (ABS) and carrier

(CEA), and probable cross-reactions to both a structurally related hapten (ABA) and an evolutionarily related carrier (DEA), but not to an unrelated carrier protein (HSP).

Incorrect Answers: (B) Ignores antibody cross-reactions. (C) Does not recognize good reaction to immunizing hapten coupled to different carrier. (D) Should not recognize unrelated carrier (HSP).

Note: This is one of several questions involving classical experimental immunological approaches that individual students have difficulty with, but groups are able to work through.


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Readiness Assurance Test Items (continued) 14. A mouse is immunized with DNP-BGG. Which experimental result BEST illustrates the carrier effect? (A) Boosting with BGG results in an unchanged anti-DNP titer. (B) Boosting with DNP-EA results in a higher anti-DNP titer. (C) Boosting with DNP-BGG results in a higher anti-DNP titer. (D) Boosting with DNP-EA results in a lower anti-DNP titer. Correct Answer: (D) True statement that best illustrates the carrier effect. Incorrect Answers: (A) Anti-DNP titer might be unchanged or lower, but does not illustrate the carrier

effect. (B) Boosting with DNP-EA results in a lower anti-DNP titer. (C) True statement, but does not illustrate the carrier effect.

15. A “classic” series of experiments performed in the 1970’s used a variety of antisera to determine cell surface markers (e.g., Ly1, Ly2, and Ly3) on lymphocytes. Which one of the following statements about their experimental design is FALSE? (A) Treatment of lymphocytes with antiserum plus complement before antigen exposure revealed

the Ly profile of precursor cells. (B) Treatment of lymphocytes with antiserum plus complement after antigen exposure revealed the

Ly profile of effector cells. (C) Mitomycin-treated cells provided convenient target cells for both MLR and CTL assays. (D) Sheep red blood cells (SRBC) provided a convenient antigen for both in vivo and in vitro

immunizations in studies of antibody formation. Correct Answer: (C) Mitomycin-treated cells were often used as stimulator cells for CTL assays, and

X-irradiated cells were commonly used as stimulator cells for MLR assays. Incorrect Answers: (A, B, D) True statements. Note: This is one of several questions involving classical experimental immunological approaches that

individual students have difficulty with, but groups are able to work through.


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Group Activity Exercises A set of 3 group application exercises is provided, however only one problem set should be used in a single 50-minute TBL session. Each exercise requires learners to understand and evaluate an experimental design, interpret data, predict experimental results, and/or design an experiment to test a hypothesis. Answers to these questions can not be found in the assigned textbook, and can only be answered by a team discussing, debating, and reaching a consensus on a single best answer. Each problem has multiple parts, including 3-4 multiple choice questions that may be answered on an IF-AT card for immediate feedback, plus 1-3 discussion questions. The group activity exercises are based on classical experiments in immunology. They were designed for graduate students to introduce basic concepts. For medical immunology courses in which little or no time is devoted to some of these historic concepts, these exercises may be less useful for medical student applications. Sample Instructions to Learners: For numbered multiple-choice questions only, please use the IF-AT card for feedback, after your team has discussed the question and arrived at a group consensus. This activity is “open book”, but you should attempt to answer the question first based on what you and your team members know. Simply looking up the answer is often not possible, and may waste time. Keep in mind that part of your grade depends on the success of your team. Be sure to discuss the pros and cons of each answer, and be prepared to justify your team answers in the class discussion.


Group Activity Exercise - Problem 1 For numbered multiple-choice questions only, please use the IF-AT card for feedback, after your team has discussed the question and arrived at a group consensus. This activity is “open book”, but you should attempt to answer the question first based on what you and your team members know. Simply looking up the answer is often not possible, and may waste time. Keep in mind that part of your grade depends on the success of your team. Be sure to discuss the pros and cons of each answer, and be prepared to justify your team answers in the class discussion. The experiment illustrated to the right explores which cell

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types are involved in an in vitro antibody response. Lymph nodes and spleen are especially good tissue sources for lymphocytes. The peritoneal cavity of mice is a particularly good source of macrophages, which are usually harvested a few days after being elicited by the injection of thioglycolate broth. Anti-Ig is short for anti-immunoglobulin antibody, e.g., rabbit antiserum that recognizes mouse Ig. C′ stands for complement. The mice used in this experiment have not been previously immunized. Where columns of plastic beads are indicated, cells that flowed through were used. 1. What population of cells is enriched in tube A? (A) plasma cells (B) B cells (C) macrophages (D) T cells 2. What population of cells is enriched in tube B? (A) plasma cells (B) B cells (C) macrophages (D) T cells 3. What population of cells is enriched in tube D? (A) plasma cells (B) B cells (C) macrophages (D) T cells Be sure everyone on your team understands how the cells in each tube were enriched. 4. This experimental protocol was used to determine which cell populations are needed to generate an effector cell population. Note that cells from tubes A and B are incubated with an antigen for 4 days in tube C. What population of effector cells is being generated during this time in tube C? Hint: The function of these cells is tested in tube E. (A) helper T cells (B) cytotoxic T lymphocytes (C) plasma cells (D) activated macrophages

antigen Tube A Tube B

Tube C Tube D

+ anti-Thy1 + C´

lymph node cells (LN) peritoneal

exudate cells (PEC)

antigen

Tube E

pfc assay

+ anti-Ig + C´

plastic beads

cells

spleen cells (SC)

+ anti-T 1 + C´ hy

plastic beads


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Group Activity Exercise - Problem 1 (continued) Discussion Questions A. In order to measure the relative activity of the effector cells generated in tube C after the 4-day culture period, antigen and cells from tube D are added for a second culture period (labeled as tube E). After this second culture, cells in tube E are assayed for “pfc”. Briefly describe what is meant by the “pfc assay”, how the assay works, and what cells are being enumerated. B. Using this protocol to investigate the cell types required for the generation of the effector cells in tube C, the data below were obtained.

Cells from Tube A Cells from Tube B pfc response in Tube E − + 7 + − 3 + + 273

How do you interpret these results? Be sure to name the cell types involved. C. Which one of the following statements best describes the results of this experiment? (A) Helper T cells (tube A) stimulate macrophages (tube B) to become antigen-presenting cells in

tube C. The activity of these antigen-presenting cells is measured by their ability to present antigen to B cells (from tube D), which become antibody-forming cells in tube E, as measured by the pfc assay.

(B) Naïve T cells (tube B) activate naïve B cells (tube A) to become plasma cells in tube C. The

activity of these plasma cells is measured by their ability to be stimulated in a secondary response to T cell help (from tube D) and become antibody-forming cells in tube E, as measured by the pfc assay.

(C) Macrophages (tube B) act as antigen-presenting cells to activate naïve T cells (tube A) to

become helper T cells in tube C. The activity of these helper T cells is measured by their ability to help naïve B cells (from tube D) become antibody-forming cells in tube E, as measured by the pfc assay.

(D) Macrophages (tube B) act as antigen-presenting cells to activate naïve T cells (tube A) to

become helper T cells in tube C. Because this is a second exposure to antigen (tube C followed by tube E), the activity of these helper T cells is measured by their ability to help a secondary antibody response by B cells (from tube D), as measured by the pfc assay.


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Group Activity Exercise - Problem 1 A concept that some students sometimes find difficult to understand in this problem is using anti-Ig to remove B cells, i.e., antibody (Ab) that recognizes Ab. Students sometimes must recognize that there are species-specific differences in Ig that can be recognized by a reagent such as rabbit anti-mouse Ig, and that Ig is expressed not only as a secreted effector molecule but also as a cell-surface receptor. ANSWER KEY 1. (D) Lymph node cells include B cells, T cells and macrophages. Anti-Ig plus complement (C′) removes B cells. Passage plastic bead column removes macrophages, leaving mostly T cells in tube A. 2. (C) Peritoneal exudate cells are mostly macrophages. Contaminating T cells are removed by treatment with anti-Thy1 antiserum plus C′, so the remaining cells in tube B are macrophages. 3. (B) Spleen cells include B cells, T cells and macrophages. Anti-Thy1 plus C′ removes T cells. Passage over a plastic bead column removes macrophages, leaving mostly B cells in tube D. 4. (A) T cells and macrophages are cultured with antigen (Ag). The effector cells generated in tube C are then added to B cells from tube D and cultured with Ag. Ab production is measured using a plaque-forming cell (pfc) assay. The effector cells required for the antibody response, and generated in tube C, are antigen-specific helper T cells (TH). This problem was used in 2006 with 12 possible points. Five of six teams had perfect scores and one team had a score of 10. The average team score was 11.2 (93%). Discussion Questions A. The pfc assay measures “plaque-forming cells”, or Ab-secreting cells, and is a convenient assay when using sheep red blood cells (SRBC) as the antigen in in vitro assays of Ab production. A lymphocyte suspension is incorporated into low melting point agarose containing SRBC and poured into a Petri dish. After incubation, a solution containing C′ is added to the plate, and incubated to promote lysis of SRBC coated by Ab secreted by an adjacent B cell. Zones of SRBC lysis (“plaques”) appear around individual B cells producing anti-SRBC Ab, allowing one to directly count the number of Ab-secreting cells. A direct pfc assay measures IgM-producing B cells, due to the superior ability of IgM to fix C′. IgG antibody-producing B cells may be counted in an indirect pfc assay by incubating with an anti-IgG-specific antibody prior to incubation with C′. B. In the absence of T cells or macrophages (first 2 lines of data), no TH cells are generated in tube C, therefore virtually no Ab response is seen in the pfc assay results from cells in tube E. If T cells and macrophages are present (third data line), antigen-specific TH cells are generated in tube C, which, when added to B cells from tube D, can help B cells make Ab, resulting in a strong Ab response. C. Statement (C) is correct. Statement (A) is incorrect because macrophages do not present Ag to B cells. Statement (B) is incorrect for several reasons: tube B contains macrophages (not T cells) and tube A contains T cells (not B cells); naïve B and T cells alone would not support the differentiation of plasma cells in tube C; and B cells only see Ag once in this protocol, so it is not a secondary response. Statement (D) is incorrect because the B cells are seeing Ag for the first time in tube E, therefore it is a primary Ab response, not a secondary Ab response.


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Group Activity Exercise - Problem 2 Please do NOT look at the back side of this page until you are instructed to do so! For numbered multiple-choice questions only, please use the IF-AT card for feedback, after your team has discussed the question and arrived at a group consensus. This activity is “open book”, but you should attempt to answer the question first based on what you and your team members know. Simply looking up the answer is often not possible, and may waste time. Keep in mind that part of your grade depends on the success of your team. Be sure to discuss the pros and cons of each answer, and be prepared to justify your team answers in the class discussion.

Your microbiologist colleague has identified a novel pathogenic bacterium called Escherichia gatorii, and has asked you to help design a vaccine. In order to do so, you decide to determine first whether immunity to E. gatorii is mediated by T cells and/or B cells. You have been provided with a virulent strain of E. gatorii and have already determined the lethal dose for BALB/c mice. Design an experiment using cellular immunology methods similar to those discussed so far in class to determine whether protective immunity to E. gatorii requires T cells, B cells, or both T and B cells. Be sure to indicate expected results for all three possible outcomes. Be sure to mention appropriate controls.

1. Note that the word “protective” is underlined above. Which of the following is therefore NOT an important feature of your experimental design? (A) Consider how to generate separate populations of immune B and T cells. (B) Use a lethal dose of E. gatorii in a final in vivo assay for protection from infection. (C) Measure antibody titers to E. gatorii after infection with a sublethal dose. (D) Use a sublethal dose of E. gatorii to generate immune B cells and immune T cells. Sketch your experiment in the space below. Need a hint? Give it a good try on your own first! If your team gets stuck, look at the back.


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Group Activity Exercise - Problem 2 (continued) Your first clues should have come from question #1. Here are a few more clues. What can you use as sources of immune B & T cells and naïve (non-immune) B & T cells? How might you use antibody reagents plus complement to prepare populations of B cells and T cells?

Would an immune reconstitution experiment work? 2. Based on your current understanding of immunology, which of the following is the best interpretation for the data shown below? B cells T cells % survival naive naive 5 (A) only T cells are required naive immune 8 (B) only B cells are required immune naive 12 (C) both T and B cells are required immune immune 82 (D) not enough information 3. Which of the following is an appropriate control for this kind of experiment? (A) reconstitute with naïve spleen cells → infect with lethal dose → observe poor survival (B) reconstitute with immune spleen cells → infect with lethal dose → observe good survival (C) use normal mouse serum (NMS) plus complement as an antibody treatment cell sample (D) all of the above Work on your experiment some more. Be sure to indicate expected results for all three possible outcomes, i.e. that protective immunity to E. gatorii requires T cells, B cells, or both T and B cells.


Group Activity Exercise - Problem 2 ANSWER KEY This problem emphasizes the importance of protective immunity, i.e., an immune response that protects the host from death due to infection. 1. (C) is not important. This question is intended to provide hints about one possible experimental design. Emphasis is placed on the word “protective” to encourage learners to focus on the functional role of the immune response rather than measures of specific effector functions, e.g. antibody titers, which may or may not provide actual protection from the pathogen. All three other answers are useful elements of an experimental design. Hints: What can you use as sources of immune B & T cells and naïve (non-immune) B & T cells? Immune

cells may be obtained from mice infected with a sublethal dose. Naïve cells may be obtained from uninfected mice.

How might you use antibody reagents plus complement to prepare populations of B cells and T cells? Antibody reagents such as anti-Ig and anti-Thy1 may be used with complement to deplete cell populations of B cells and T cells, respectively.

Would an immune reconstitution experiment work? Mice could be irradiated to destroy their own mature lymphocytes and reconstituted with different combinations of naïve and immune B and T cells to test which arm of the immune system is providing protective immunity.

2. (C) Although learners might suggest that either T cells or B cells alone provide some protection (nearly two-fold higher % survival), clearly the best protection (16-fold higher % survival) is provided in the presence of both immune T and immune B cells. 3. (D) All three answers are appropriate controls for this experiment. This problem was used in 2007, with 9 possible points. One of four teams had a perfect score (range 7-9). The average team score was 8.0 (89%). Diagram of experiment Other answers are possible. This experimental design is suggested by the hints and questions in this exercise.

Infect with sublethal dose

of E. gatorii

spleen cells spleen

cells

19

Immune Immune

good survival

+ anti-Ig + C´

Immune Naïve

poor survival

+ anti-Thy1 +C´

Naïve B cells

Naïve T cells

+ anti-Ig + C´ + anti-Thy1

+C´

Immune B cells Immune

T cells

Naïve Immune

poor survival

Naïve Naïve

poor survival

B cells: T cells:

Irradiated recipient mice

Challenge with lethal dose of E. gatorii


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Group Activity Exercise - Problem 3 For numbered multiple-choice questions only, please use the IF-AT card for feedback, after your team has discussed the question and arrived at a group consensus. This activity is “open book”, but you should attempt to answer the question first based on what you and your team members know. Simply looking up the answer is often not possible, and may waste time. Keep in mind that part of your grade depends on the success of your team. Be sure to discuss the pros and cons of each answer, and be prepared to justify your team answers in the class discussion. The “Raff experiment”, published in 1970, used anti-Thy1 antisera in an adoptive transfer protocol to demonstrate that carrier-primed cells are T cells. In this assignment you will design an experiment using a similar strategy to prove that hapten-primed cells are B cells. The following materials are available to you for use as antigens. DNP, dinitrophenol ABS, m-azobenzenesulfonate HSP, horse serum protein EA, egg albumin 1. Which one of the following combinations of mouse immunizations is the most appropriate for this experiment? Mouse 1 Mouse 2 (A) DNP-HSP EA (B) DNP-HSP ABS-HSP (C) DNP-HSP HSP (D) DNP DNP-EA 2. Which antiserum will be the most helpful to prove that hapten-primed cells are B cells? (A) anti-Thy1 (B) anti-Ly1 (C) anti-immunoglobulin (D) anti-MHC class II How would you raise such an antiserum? 3. Given your answers above, which antibody response is the most appropriate one to measure in this experiment? (A) anti-ABS (B) anti-EA (C) anti-HSP (D) anti-DNP


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Group Activity Exercise - Problem 3 (continued) In the space below, diagram an experiment similar to the “Raff experiment” to prove that hapten-primed cells are B cells. Use symbols and diagrams similar to those shown in class. Be sure to show the expected results.


Group Activity Exercise - Problem 3 The problem is based on a classic experiment that demonstrated that carrier-primed cells are T cells (Raff MC. Role of thymus-derived lymphocytes in the secondary humoral immune response in mice. Nature 226:1257–1258, 1970). Learners must understand the difference between haptens and carriers and the basics of B-T cell cooperation in order to interpret the experiment. ANSWER KEY 1. (A) In the Raff experiment, one donor mouse was immunized with a hapten-carrier and as second donor mouse was immunized with a different carrier (no hapten). The same protocol can be used, and the best match is answer A. Learners must recognize that DNP and ABS are haptens, and HSP and EA are carriers. 2. (C) Immunoglobulin is the only target cell surface marker listed that is specific to B cells. One could raise such an antiserum by immunizing a different species (e.g. rabbit) with purified mouse immunoglobulin. 3. (D) Given answer A in question #1, the most appropriate antibody response would be against the hapten DNP. This problem was used in 2008, with 9 possible points. One of five teams had a perfect score. The range of scores was 6-9, and the mean was 7.2 (80%). Diagram of experiment

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The simplest answer is to repeat the Raff experiment with the single change of using anti-Ig to remove B cells, rather than anti-Thy1 to remove T cells, as in the original experiment. Expected results should show a poor anti-hapten antibody response when B cells are removed from the cells derived from the hapten-primed donor.

spleen cells spleen

cells

EA

Irradiated recipient

DNP-HSP

DNP-EA

Anti-hapten response

+

+ anti-Ig + C´

spleen cells spleen

cells + normal mouse serum + C´

DNP-HSP EA

+ normal mouse serum + C´ + anti-Ig

+ C´

Irradiated recipient DNP-EA

Anti-hapten response

++++

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Immunology Team-Based Learning: Basic Concepts in Immunology · Immunology Team-Based Learning: Basic Concepts in Immunology 4 The Group Readiness Assurance Test (GRAT) was open book