IMMUNOLOGY EXAM
PRACTICE: 5 VERIFIED
QUESTIONS &
DETAILED ANSWERS
FOR TOP SCORES
A COMPREHENSIVE
AND ELITE REAL EXAM
SIMULATION
75 ADVANCED
CLINICAL AND
MOLECULAR
VIGNETTES
Prepared For: Advanced Research Fellows, Clinical Immunologists, and Board Candidates
Subject: Molecular Immunology, Immunopathology, Immunogenetics, and Immunotherapy
SECTION I: MOLECULAR GENETICS,
,LYMPHOCYTE DEVELOPMENT, AND ANTIGEN
PRESENTATION
Question 1
A research team is investigating the molecular machinery of somatic recombination in murine
pro-B cells. They introduce a loss-of-function mutation in a gene encoding a specific
endonuclease. The resulting phenotype demonstrates a complete absence of mature B and T
lymphocytes (SCID). Molecular analysis reveals that while the RAG1/RAG2 complex
successfully binds to Recombination Signal Sequences (RSS) and generates double-strand
breaks, the subsequent opening of the hairpin coding ends is defective. Which of the following
proteins is most likely non-functional?
A. Terminal deoxynucleotidyl transferase (TdT) B. Artemis C. Ku70/Ku80 D. DNA Ligase IV E.
XRCC4
Answer and Detailed Analysis: The correct answer is B. Artemis.
This question addresses the precise molecular steps of V(D)J recombination, a hallmark of
vertebrate adaptive immunity that generates diverse antigen receptor repertoires. The process
occurs in the G0/G1 phase of the cell cycle in developing lymphocytes and is strictly regulated
to ensure genomic stability.
The V(D)J recombination pathway proceeds in two distinct phases: cleavage and repair.
1. Cleavage Phase: The RAG1/RAG2 recombinase complex recognizes the 12/23
Recombination Signal Sequences (RSS) flanking the V, D, and J gene segments. RAG1/2
introduces a single-strand nick between the coding segment and the RSS, leading to a
transesterification reaction that creates a closed hairpin structure at the coding end and a
blunt signal end. The formation of this hairpin is critical for preserving the coding
sequence energy but presents a barrier to fusion.
2. Repair Phase (Hairpin Opening): The critical step described in the vignette—the
opening of these hairpins—requires the endonuclease activity of Artemis. Artemis forms
a complex with DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon
phosphorylation by DNA-PKcs, Artemis acquires endonuclease activity, allowing it to
cleave the hairpin loops at the coding ends. This cleavage often occurs asymmetrically,
creating palindromic P-nucleotides which contribute to junctional diversity.
Without Artemis, the hairpins remain closed, preventing the fusion of gene segments.
Consequently, T and B cell development arrests at the pro-lymphocyte stage, resulting in a
phenotype similar to Radiosensitive Severe Combined Immunodeficiency (RS-SCID). Patients
with Artemis mutations (DCLRE1C gene) exhibit T- B- NK+ SCID but have increased
radiosensitivity due to defects in general DNA repair.
● Option A (TdT) is incorrect because TdT adds N-nucleotides to the free 3' ends of the
coding DNA during the repair process. Loss of TdT reduces junctional diversity but does
not arrest lymphocyte development or prevent hairpin opening.
● Options C (Ku70/Ku80), D (DNA Ligase IV), and E (XRCC4) are components of the
Non-Homologous End Joining (NHEJ) machinery. While they are essential for ligating the
DNA ends, the specific defect of hairpin opening is unique to Artemis. Defects in Ligase
IV or XRCC4 would prevent the final ligation step but would occur downstream of hairpin
processing.
,Question 2
A 4-year-old male presents with recurrent sinopulmonary infections. Flow cytometry reveals a
normal number of CD3+ T cells but a complete absence of HLA-DR, HLA-DP, and HLA-DQ
expression on B cells and monocytes. Genetic sequencing identifies a mutation in a
transcriptional regulator (CIITA or RFX complex). In the absence of MHC Class II expression,
which of the following molecular interactions is most directly compromised during T cell
development in the thymus?
A. Interaction between the T cell receptor (TCR) and autoimmune regulator (AIRE) peptides B.
Interaction between CD8 and MHC Class I on cortical epithelial cells C. Interaction between
CD4 and MHC Class II on cortical epithelial cells D. Interaction between CD28 and B7-1/B7-2
on medullary dendritic cells E. Interaction between LFA-1 and ICAM-1 on thymic stroma
Answer and Detailed Analysis: The correct answer is C. Interaction between CD4 and
MHC Class II on cortical epithelial cells.
This vignette describes Bare Lymphocyte Syndrome Type II (MHC Class II deficiency). The core
of the question relies on understanding thymic positive selection.
T cell development occurs in the thymus, involving a rigorous selection process to ensure
self-MHC restriction and tolerance. CD4+CD8+ double-positive (DP) thymocytes must bind to
self-MHC molecules on thymic cortical epithelial cells (cTECs) to survive positive selection. The
"choice" of lineage is determined by which MHC molecule is engaged:
● CD4 Lineage Commitment: If a DP thymocyte's TCR interacts with MHC Class II, the
CD4 co-receptor binds to the conserved beta-2 domain of the MHC Class II molecule.
This recruits the kinase Lck (associated with the CD4 tail), initiating signaling that instructs
the cell to downregulate CD8, upregulate ThPOK, and commit to the CD4+
single-positive lineage.
● CD8 Lineage Commitment: If the interaction is with MHC Class I, the cell becomes a
CD8+ T cell.
In the absence of MHC Class II molecules on the thymic epithelium (due to defects in
transcription factors like CIITA or RFX), DP thymocytes bearing MHC II-restricted TCRs cannot
receive survival signals. Consequently, they die by neglect or fail to mature into CD4+ helper T
cells. This leads to a profound deficiency in CD4+ T cells in the periphery, severely
compromising adaptive immunity, particularly the help required for B cell antibody production
(hence the recurrent infections and hypogammaglobulinemia despite normal B cell numbers).
● Option A refers to negative selection in the medulla, which requires AIRE to express
tissue-specific antigens. This occurs after positive selection.
● Option B describes the selection of CD8+ T cells, which would be relatively preserved in
Class II deficiency.
● Option D describes co-stimulation, which is necessary for peripheral activation, not the
initial positive selection step in the cortex.
Question 3
Molecular analysis of antigen processing in a dendritic cell reveals a defect where peptides
generated by the proteasome are unable to enter the endoplasmic reticulum (ER).
Consequently, MHC Class I molecules remain unstable and are retained in the ER. Which
protein is most likely dysfunctional?
A. HLA-DM B. Invariant Chain (CD74) C. TAP (Transporter associated with Antigen Processing)
, D. Calnexin E. Cathepsin S
Answer and Detailed Analysis: The correct answer is C. TAP (Transporter associated with
Antigen Processing).
The MHC Class I antigen presentation pathway handles endogenous (intracellular) antigens
(e.g., viral proteins, tumor neoantigens). These proteins are ubiquitinated in the cytosol and
degraded by the proteasome into short peptides. For these peptides to be presented, they must
be transported from the cytosol into the lumen of the endoplasmic reticulum (ER) to be loaded
onto nascent MHC Class I heavy chain/beta-2 microglobulin complexes.
TAP (TAP1/TAP2 heterodimer) is the dedicated ATP-dependent pump responsible for this
translocation.
● Mechanism: TAP resides in the ER membrane. It actively pumps peptides (preferentially
8-16 amino acids with hydrophobic C-termini) into the ER. Once inside, these peptides
are loaded onto MHC Class I molecules with the help of the peptide-loading complex
(containing tapasin, calreticulin, and ERp57).
● Deficiency: Without TAP, the ER is starved of peptides. MHC Class I molecules are
unstable without a bound peptide; they fail to dissociate from the chaperone machinery
and are eventually retro-translocated and degraded, leading to a surface deficiency of
MHC Class I. This clinically manifests as Type I Bare Lymphocyte Syndrome,
characterized by chronic respiratory bacterial infections and necrotizing skin lesions.
● Options A (HLA-DM), B (Invariant Chain), and E (Cathepsin S) are all exclusive to the
MHC Class II pathway. HLA-DM catalyzes peptide exchange in the endosome; Invariant
chain blocks the peptide groove of MHC II in the ER; Cathepsin S cleaves the Invariant
chain.
● Option D (Calnexin) is a chaperone that assists early folding of the MHC Class I heavy
chain but is not the transporter.
Question 4
During the formation of the MHC Class II peptide-loading complex in the late endosome (MIIC
compartment), a non-polymorphic MHC-like molecule functions to catalyze the release of the
CLIP peptide and stabilize the empty MHC II groove for binding of high-affinity exogenous
peptides. Polymorphisms in the gene encoding this chaperone are associated with autoimmune
susceptibility. Which molecule is described?
A. HLA-DO B. HLA-DM C. Tapasin D. Ubiquitin E. Calreticulin
Answer and Detailed Analysis: The correct answer is B. HLA-DM.
The MHC Class II pathway involves the vesicular transport of MHC II molecules, which are
initially blocked by the Invariant Chain (li) to prevent premature binding of ER peptides. As the
vesicle acidifies and fuses with endosomes containing exogenous antigens, the Invariant Chain
is degraded, leaving a small fragment called CLIP (Class II-associated invariant chain peptide)
in the binding groove.
HLA-DM is a non-classical MHC II molecule found in the MIIC compartment. Unlike classical
MHC molecules, it does not bind peptides itself. Instead, it acts as a peptide editor and catalyst.
● Function: HLA-DM binds to the MHC II-CLIP complex and induces a conformational
change that releases CLIP. It then stabilizes the empty MHC II molecule, preventing
aggregation, and facilitates the binding of high-affinity antigenic peptides. If a low-affinity
peptide binds, HLA-DM helps release it, ensuring that only stable, high-affinity
peptide-MHC complexes are presented to CD4+ T cells. This editing function is crucial for
immunodominance.
PRACTICE: 5 VERIFIED
QUESTIONS &
DETAILED ANSWERS
FOR TOP SCORES
A COMPREHENSIVE
AND ELITE REAL EXAM
SIMULATION
75 ADVANCED
CLINICAL AND
MOLECULAR
VIGNETTES
Prepared For: Advanced Research Fellows, Clinical Immunologists, and Board Candidates
Subject: Molecular Immunology, Immunopathology, Immunogenetics, and Immunotherapy
SECTION I: MOLECULAR GENETICS,
,LYMPHOCYTE DEVELOPMENT, AND ANTIGEN
PRESENTATION
Question 1
A research team is investigating the molecular machinery of somatic recombination in murine
pro-B cells. They introduce a loss-of-function mutation in a gene encoding a specific
endonuclease. The resulting phenotype demonstrates a complete absence of mature B and T
lymphocytes (SCID). Molecular analysis reveals that while the RAG1/RAG2 complex
successfully binds to Recombination Signal Sequences (RSS) and generates double-strand
breaks, the subsequent opening of the hairpin coding ends is defective. Which of the following
proteins is most likely non-functional?
A. Terminal deoxynucleotidyl transferase (TdT) B. Artemis C. Ku70/Ku80 D. DNA Ligase IV E.
XRCC4
Answer and Detailed Analysis: The correct answer is B. Artemis.
This question addresses the precise molecular steps of V(D)J recombination, a hallmark of
vertebrate adaptive immunity that generates diverse antigen receptor repertoires. The process
occurs in the G0/G1 phase of the cell cycle in developing lymphocytes and is strictly regulated
to ensure genomic stability.
The V(D)J recombination pathway proceeds in two distinct phases: cleavage and repair.
1. Cleavage Phase: The RAG1/RAG2 recombinase complex recognizes the 12/23
Recombination Signal Sequences (RSS) flanking the V, D, and J gene segments. RAG1/2
introduces a single-strand nick between the coding segment and the RSS, leading to a
transesterification reaction that creates a closed hairpin structure at the coding end and a
blunt signal end. The formation of this hairpin is critical for preserving the coding
sequence energy but presents a barrier to fusion.
2. Repair Phase (Hairpin Opening): The critical step described in the vignette—the
opening of these hairpins—requires the endonuclease activity of Artemis. Artemis forms
a complex with DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon
phosphorylation by DNA-PKcs, Artemis acquires endonuclease activity, allowing it to
cleave the hairpin loops at the coding ends. This cleavage often occurs asymmetrically,
creating palindromic P-nucleotides which contribute to junctional diversity.
Without Artemis, the hairpins remain closed, preventing the fusion of gene segments.
Consequently, T and B cell development arrests at the pro-lymphocyte stage, resulting in a
phenotype similar to Radiosensitive Severe Combined Immunodeficiency (RS-SCID). Patients
with Artemis mutations (DCLRE1C gene) exhibit T- B- NK+ SCID but have increased
radiosensitivity due to defects in general DNA repair.
● Option A (TdT) is incorrect because TdT adds N-nucleotides to the free 3' ends of the
coding DNA during the repair process. Loss of TdT reduces junctional diversity but does
not arrest lymphocyte development or prevent hairpin opening.
● Options C (Ku70/Ku80), D (DNA Ligase IV), and E (XRCC4) are components of the
Non-Homologous End Joining (NHEJ) machinery. While they are essential for ligating the
DNA ends, the specific defect of hairpin opening is unique to Artemis. Defects in Ligase
IV or XRCC4 would prevent the final ligation step but would occur downstream of hairpin
processing.
,Question 2
A 4-year-old male presents with recurrent sinopulmonary infections. Flow cytometry reveals a
normal number of CD3+ T cells but a complete absence of HLA-DR, HLA-DP, and HLA-DQ
expression on B cells and monocytes. Genetic sequencing identifies a mutation in a
transcriptional regulator (CIITA or RFX complex). In the absence of MHC Class II expression,
which of the following molecular interactions is most directly compromised during T cell
development in the thymus?
A. Interaction between the T cell receptor (TCR) and autoimmune regulator (AIRE) peptides B.
Interaction between CD8 and MHC Class I on cortical epithelial cells C. Interaction between
CD4 and MHC Class II on cortical epithelial cells D. Interaction between CD28 and B7-1/B7-2
on medullary dendritic cells E. Interaction between LFA-1 and ICAM-1 on thymic stroma
Answer and Detailed Analysis: The correct answer is C. Interaction between CD4 and
MHC Class II on cortical epithelial cells.
This vignette describes Bare Lymphocyte Syndrome Type II (MHC Class II deficiency). The core
of the question relies on understanding thymic positive selection.
T cell development occurs in the thymus, involving a rigorous selection process to ensure
self-MHC restriction and tolerance. CD4+CD8+ double-positive (DP) thymocytes must bind to
self-MHC molecules on thymic cortical epithelial cells (cTECs) to survive positive selection. The
"choice" of lineage is determined by which MHC molecule is engaged:
● CD4 Lineage Commitment: If a DP thymocyte's TCR interacts with MHC Class II, the
CD4 co-receptor binds to the conserved beta-2 domain of the MHC Class II molecule.
This recruits the kinase Lck (associated with the CD4 tail), initiating signaling that instructs
the cell to downregulate CD8, upregulate ThPOK, and commit to the CD4+
single-positive lineage.
● CD8 Lineage Commitment: If the interaction is with MHC Class I, the cell becomes a
CD8+ T cell.
In the absence of MHC Class II molecules on the thymic epithelium (due to defects in
transcription factors like CIITA or RFX), DP thymocytes bearing MHC II-restricted TCRs cannot
receive survival signals. Consequently, they die by neglect or fail to mature into CD4+ helper T
cells. This leads to a profound deficiency in CD4+ T cells in the periphery, severely
compromising adaptive immunity, particularly the help required for B cell antibody production
(hence the recurrent infections and hypogammaglobulinemia despite normal B cell numbers).
● Option A refers to negative selection in the medulla, which requires AIRE to express
tissue-specific antigens. This occurs after positive selection.
● Option B describes the selection of CD8+ T cells, which would be relatively preserved in
Class II deficiency.
● Option D describes co-stimulation, which is necessary for peripheral activation, not the
initial positive selection step in the cortex.
Question 3
Molecular analysis of antigen processing in a dendritic cell reveals a defect where peptides
generated by the proteasome are unable to enter the endoplasmic reticulum (ER).
Consequently, MHC Class I molecules remain unstable and are retained in the ER. Which
protein is most likely dysfunctional?
A. HLA-DM B. Invariant Chain (CD74) C. TAP (Transporter associated with Antigen Processing)
, D. Calnexin E. Cathepsin S
Answer and Detailed Analysis: The correct answer is C. TAP (Transporter associated with
Antigen Processing).
The MHC Class I antigen presentation pathway handles endogenous (intracellular) antigens
(e.g., viral proteins, tumor neoantigens). These proteins are ubiquitinated in the cytosol and
degraded by the proteasome into short peptides. For these peptides to be presented, they must
be transported from the cytosol into the lumen of the endoplasmic reticulum (ER) to be loaded
onto nascent MHC Class I heavy chain/beta-2 microglobulin complexes.
TAP (TAP1/TAP2 heterodimer) is the dedicated ATP-dependent pump responsible for this
translocation.
● Mechanism: TAP resides in the ER membrane. It actively pumps peptides (preferentially
8-16 amino acids with hydrophobic C-termini) into the ER. Once inside, these peptides
are loaded onto MHC Class I molecules with the help of the peptide-loading complex
(containing tapasin, calreticulin, and ERp57).
● Deficiency: Without TAP, the ER is starved of peptides. MHC Class I molecules are
unstable without a bound peptide; they fail to dissociate from the chaperone machinery
and are eventually retro-translocated and degraded, leading to a surface deficiency of
MHC Class I. This clinically manifests as Type I Bare Lymphocyte Syndrome,
characterized by chronic respiratory bacterial infections and necrotizing skin lesions.
● Options A (HLA-DM), B (Invariant Chain), and E (Cathepsin S) are all exclusive to the
MHC Class II pathway. HLA-DM catalyzes peptide exchange in the endosome; Invariant
chain blocks the peptide groove of MHC II in the ER; Cathepsin S cleaves the Invariant
chain.
● Option D (Calnexin) is a chaperone that assists early folding of the MHC Class I heavy
chain but is not the transporter.
Question 4
During the formation of the MHC Class II peptide-loading complex in the late endosome (MIIC
compartment), a non-polymorphic MHC-like molecule functions to catalyze the release of the
CLIP peptide and stabilize the empty MHC II groove for binding of high-affinity exogenous
peptides. Polymorphisms in the gene encoding this chaperone are associated with autoimmune
susceptibility. Which molecule is described?
A. HLA-DO B. HLA-DM C. Tapasin D. Ubiquitin E. Calreticulin
Answer and Detailed Analysis: The correct answer is B. HLA-DM.
The MHC Class II pathway involves the vesicular transport of MHC II molecules, which are
initially blocked by the Invariant Chain (li) to prevent premature binding of ER peptides. As the
vesicle acidifies and fuses with endosomes containing exogenous antigens, the Invariant Chain
is degraded, leaving a small fragment called CLIP (Class II-associated invariant chain peptide)
in the binding groove.
HLA-DM is a non-classical MHC II molecule found in the MIIC compartment. Unlike classical
MHC molecules, it does not bind peptides itself. Instead, it acts as a peptide editor and catalyst.
● Function: HLA-DM binds to the MHC II-CLIP complex and induces a conformational
change that releases CLIP. It then stabilizes the empty MHC II molecule, preventing
aggregation, and facilitates the binding of high-affinity antigenic peptides. If a low-affinity
peptide binds, HLA-DM helps release it, ensuring that only stable, high-affinity
peptide-MHC complexes are presented to CD4+ T cells. This editing function is crucial for
immunodominance.
