Immunology Flashcards
How are monoclonal antibodies made?
Monoclonal antibodies derive from the progeny of a single B cell that has been fused with a multiple myeloma tumor cell; the resultant hybrid line can grow forever in culture and make a specific antibody
Humira
A fully human mAb to TNF-a shown to slow the progress of rheumatoid arthritis
-omab
Murine - monoclonal antibodies made from immunized mice
-ximab
Chimeric - mAbs genetically engineered at the DNA level to have mouse VL and VH domains (25%) but human C domains (75%)
With time, patients will still make human anti-cheric antibody (HACA)
-zumab
Humanized mAbs - only the CDRs of the V domains are from the mouse (2% foreign)
Still, with time, patients will make human anti-human antibody (HAHA)
-umab
Human - SCID mice are implanted with human bone marrow, thymus, and lymph node; immunization of the mouse with antigen now produces human antibodies
Herceptin
Humanized Ab to EGF receptor HER-2
Downregulates the receptor and sensitizes the cell to killing by ADCC
Naturak Killer (NK) Cells
Large granular lymphocytes (LGLs) which make up 5-10% of blood lymphocytic cells; they are part of the innate immune system with receptors against molecules that appear on the surface of stressed or dysregulated cells; they recognize these cells and send apoptotic signals
Antibody-dependent cell mediated cytotoxicity (ADCC)
NK cells have receptors for the Fc end of IgG; NK cells can recognize therapeutic mAb coating target cells and send apoptotic signals to that cell
Chimeric Antigen Receptor (CAR)
T cells can be removed from cancer patients and transformed using a lentivirus vector to express a chimeric antigen receptor (CAR); the CAR is usually comprised of the VL/VH fragment of an antibody with high specificity against tumor-related antigens, a transmembrane region, and an intracellular T-cell signaling molecule such as CD3
This transformed CTL can then bind a tumor target with high affinity and specificity and be triggered via its normal TCR pathway to become a fully cytotoxic cell
Blood group antigens
Glycolipids found on the surface of all body cells, including RBCs; comprised of a basic “H antigen” linked by a specific glucosyl transferase to variant terminal sugars; the 3 possible alleles of the glucosyl transferase enzyme confer antigenic specificity A, B, or O
Blood group substances
Glycoproteins found in the body fluids of people who have the Secretor (Se) phenotype (80%); these glycoproteins are antigenically similar to blood group antigens
Rh antigens
Rh antigens are proteins coded for at two loci, the most important of which is the D/d locus
D is dominant over “d” which is an amorph and does not make protein
Genotypes DD or Dd type as Rh+ and 85% of US whites have this phenotype; Rh- individuals do not make isohemagglutinins against Rh unless they are immunized by Rh+ cells
ABO Isohemagglutinins
IgM antibodies made against foreign ABO blood antigens; theey appear in the blood between 3 and 6 months of age
Bombay blood type
These individuals lack the transferase gene that puts the final sugar on the core ABO antigen polysaccharide; therefore, they do not express even the basic “H” antigen and all blood is foreign to them
In a lab not looking for this the individual will type as “O”
Crossmatch procedure
Plasma from the blood recipient is mixed with red cells from the donor; agglutination means that lots of high activity antibodies against the donor’s RBCs exist in the recipient’s serum
This test is further evaluated in a medium that neutralizes the repulsive charges of RBCs, providing a more sensitive test for agglutination
Direct Antiglobulin Test Procedure
Uses antibody against human Ig to detect human Ig on the surface of RBCs
Patient’s RBCs are washed and antibody against human Ig is added; if the RBCs had some human Ig on them then the antiglobulin will cross-link the antibody-bound RBCs, leading to agglutination
Detects cells that were coated with antibody in vivo
Indirect antiglobulin test procedure
Uses antibody against human Ig to detect human Ig in plasma
Patient’s serum is added to donor RBCs; the patient’s antibody will bind the donor’s RBCs but may not be enough to agglutinate; antiglobulin is added, which cross-links the donor’s RBCs that have been bound by the patient’s Ab
Detects antibody in the circulation
Heterophile antibodies
Antibodies that are directed against one antigen but also cross-react with a different antigen
Monospot test
Antibody test for EBV (Mononucleiosis)
Patients make a serum antibody to EBV viral antigen that happens to cross-react with sheep RBCs; therefore, agglutination of sheep RBCs with addition of the patient’s serum is a positive screening test for EBV mononucleiosis
Hemolytic Disease of the Newborn - Complications
High levels of bilirubin released from lysed RBCs can cross the BBB and damage the basal ganglia, resulting in cerebral palsy or death
Jaundice
Hemolytic Disease of the Newborn - Mechanism
When an RhD- mom is pregnant with an RhD+ baby, any event (labor, abortion, miscarriage) that allows escape of RBCs from placental to maternal circulation can sensitize the mother to produce IgG against RhD
In a subsesquent pregnancy with an RhD- fetus these anti-RhD IgG can cross the placenta and gain access to fetal circulation where they opsonize fetal RBCs for destruction by the RE system, causing massive hemolysis
Rh-immune globulin (RhoGAM)
RhoGAM is IgG antibody to RhD; these antibodies combine with fetal red cells in maternal circulation, opsonizing them for destruction before they can immunize her against RhD
Should be administered at 28 weeks and also each time there is a chance of being immunized by RhD cells, including delivery, abortions, fetal manipulations, amniocentesis, etc.
ABO Hemolytic Disease of the Newborn
Rarely, some type O mothers can make IgG isohemagglutinins against the fetal ABO antigens; these IgG can cross the placenta and cause hemolytic disease of the wrong-type fetus
Serum protein electrophoresis
Serum is applied to an agar plate and voltage is applied across the plate causing proteins to separate by size; proteins can be stained and the results are given as a trace with the following peaks:
Albumin, alpha1, alpha2, B, and gamma
Immunoglobulins generally segregate into the gamma peak
Bence Jones Protein
Free Ig light chains seen in the urine of patients with multiple myeloma; diagnosed by protein electrophoresis
Single radial immunodiffusion
Gel containing rabbit anti-serum to human Ig is plated and human serum is added to a well in the middle of the plate
Ig within the patient’s serum will precipitate out with the anti-Ig in the agar and the size of the precipitated ring gives information about how much Ig was present in the serum
This technique can be used to measure any multivalent antigen that can form a precipitate with the appropriate antibody, as long as you have the specific antiserum
Test for antinuclear antibodies
Patient serum is added to human cells grown on a slide and fixed with an agent that makes the cells’ plasma membranes permeable to antibodies; fluorescent-labeled anti-human IgG is added and the pattern of fluorescences allows detection of antibodies attached to nuclear antigens
Test for immune complexes in the serum
Immune complexes in serum are insoluble in the cold; if a Type III process is suspected, a sample of patient serum is refrigerated and examined after 1-7 days for precipitate called mixed cryoglobulin
Passive agglutination
Ex: Rheumatoid factor (IgM anti-IgG) is detected by its ability to agglutinate latex beads that have been coated with IgG; this test is more sensitive due to the larger effective size of the antigen
Agglutination titer
The reciprocal of the weakest dilution of a patient’s serum that will agglutinate against antigen
Ex: If a 1:64 dilution of serum agglutinates against antigen but a 1:128 dilution does not then the agglutination titer is 64
Simple ELISA
Antigen is coupled to a plate and test serum is added; if there is antibody to the antigen then it will bind and can be identified using a secondary, enzyme-coupled antiglobulin
Capture ELISA
Detects divalent antigens in a patient sample
An antibody that binds a specific epitope on the antigen of interest is plated and patient serum is added; then a second antibody that is specific to a different epitope on the antigen of interest is added and sticks to the epitope on the antigen in proportion to how much antigen is present; the second antibody is coupled to an enzyme (usually peroxidase) that produces a colored product with an intensity that is proportional to the amount of antigen present in the patient’s sample
Rapid strep test
A throat swab is extracted in a tube of buffer and the extract is passed through a membrane to which a dot of anti-strep antibody has been coupled; strep antigens in the patient sample will stick to this dot; a different antibody is passed through the membrane and binds where the antigen has been trapped by the dot; this second antibody is linked to liposomes (fat droples with a water interior containing a dye); detergent is added to pop the liposomes and the spot turns color if strep antigen was present in the swab
Flow Cytometry
Takes cells in suspension and pumps them through an orifice so small that they emerge in a single file stream; lasers illuminate the cells and light emitted or scattered by each cell (or by fluorescently labeled antibodies attached to cell surface markers) gives information about cell type
DTH Test
Skin test with common antigens to which most people exhibit DTH
Failure to produce a DTH reaction suggests incompetent T cell response
Challenge DTH Test
Dinitrofluorobenzene painted on the skin will sensitize 98% of individuals within 10 days to exhibit a DTH response
Failure to produce a DTH response on second exposure suggests an incompetent T cell response
Mitogen test
T cells are added to mitogens in culture and observed for production of IL-2, IL-4 and INFy
HIV - Structure
HIV-1 is an enveloped, non-transforming RNA retrovirus
How does HIV gain entry to the body?
HIV enters the body and adheres to a lectin on DCs called DC-SIGN; this allows it to use the DC as a Trojan horse to get into the lymph nodes where it has access to CD4 helper T cells, it’s main target
HIV - Binding & Cellular Entry
HIV envelope protein gp120 binds to the CD4 molecule on the surface of the Th cell; this binding induces a conformational change in gp120 which allows it to now bind a co-receptor CCR5 on the host cell; binding of CCR5 leads to a conformational change in the gp41 envelope-associated protein, exposing a hydrophobic fusion peptide region that inserts into the T cell membrane
CCR5 mutation
10% of Caucasians have a mutant allele of CCR5 (delta 32); homozygotes do not express any surface CCR5 on their T cells and so while they can become chronically infected with HIV the infection remains sequestered in DCs and macrophages and the patients never develop AIDS
HIV Genome
Encodes 9 genes: gag, pol, env, and 6 genes that regulate latency and virulence
Can make 16 distinct polypeptides by virtue of several mechanisms for generating genetic diversity, including:
Using all 3 reading frames
Alternative RNA splicing
Protease-mediated cleavage of large precursor proteins
Formation of syncytium in HIV
As the HIV virus replicates it produces gp120/gp41 early on in the cycle; these proteins become inserted into the host cell plasma membrane, allowing fusion of the infected cell with a nearby, uninfected CD4 cell; this creates a syncytium allowing the virus to spread without an extracellular phase; as viruses bud en mass from the infected cell they create holes in the cell membrane, causing cell death
Common opportunistic infections of HIV
Candida albicans
Tuberculosis
Kaposi Sarcoma
Burkitt Lymphoma
Common Viral Infections of HIV
Cytomegalovirus Hepatitis HSV VZV HSV - 8: Kaposi Sarcoma Virus
Common fungal infections of HIV
Candida albicans
Pneumocystis jirovecii
Common protozoan infections of HIV
Toxoplasma
Cryptosporidium
Isospora
Elite Controllers
People who harbor HIV but retain normal immune function for many years
65% are HLA-B57 - this allele has higher affinity for HIV peptides, allowing APCs to better present HIV to the immune system which makes a better CTL response against HIV
Laboratory diagnosis of HIV
Antibody to HIV measured by ELISA and confirmed by Western Blot in which standardized viral proteins are separated by electrophoresis and “stained” with patient serum; patient’s antibodies must bind to gp120 and gp41 for the test to be considered positive
PCR can be done as a follow-up to detect viral load
Oraquick - takes a sample of serum transudate from the gingival crevice; detects anti-HIV IgG in this fluid
Broadly neutralizing antibody to HIV
Rare antibodies that can be isolated from the serum of HIV+ patients and have the ability to bind any variation of HIV, regardless of antigenic variation, within the CD4 binding site
Ex: B12 antibody
HIV Reverse Transcriptase Inhibitors
Nucleosides - Competitive inhibitors of reverse transcriptase; terminate the DNA chain
Non-nucleoside inhibitors - Bind a hydrophobic pocket on the enzyme that changes the conformation of the active site, inhibiting its activity
HIV Protease Inhibitors
Inhibit the viral protease that cleaves the single gag-pol-env polyprotein into its active constituents
HIV Fusion Inhibitor
Binds to gp41 so that it cannot change conformation and cause fusion of the viral and host cell membranes
CCR5 Antagonist
Blocks viral entry into CD4 cells by binding to CCR5 and causing changes in the conformation of the external receptor so that it no longer binds gp120
Integrase Inhibitor
Blocks the enzyme integrase, necessary for insertion of viral DNA into the host genome
Immunoediting - Elimination
When a cellular clone becomes malignant it may begin to express DAMPs which activate the innate immune system; antigen presentation of these DAMPs on DCs activate T cells, causing macrophages and CTLs to infiltrate the tumor
Immunoediting - Equilibrium
T lymphocytes infiltrate the tumor but do not fully destroy it; the tumor and lymphocytes exist in equilibrium as long as the immune response is strong enough to keep tumor progression at bay
Immunoediting - Escape
Some tumors produce blocking factors that specifically block the killing of cancer cells by tumor-specific T cells
Blocking factors include:
Shed tumor antigen
Antigen-MHC complexes
Antibodies to tumor antigens that shield it from T cells without harming the tumor cell
Tumor Associated Antigens (TAAs)
Antigens found on tumor cells which are either not found on normal cells or are found in uch lower quantities; TAAs are overexpressed or abnormally expressed by the tumor
Ex: HER-2 in breast cancer, PSA in prostate cancer
Carcinoembryonic antigen (CEA)
An antigen made in normal fetus colon tissue but not found in normal adult tissue; may be re-expressed in a colon tumor
Screening tests for CEA are used in the setting of high suspicion for colon cancer, when colon cancer has been removed to confirm complete excision, or to monitor for colon cancer recurrence
Role of CD8 in tumor immunogenesis
TAAs are presented on MHC-I by DCs in the lymph node, activating naive CD8 cells; following activation, T cells undergo clonal expansion, leave the lymph node, and migrate to the tumor where they kill tumor cells by inducing apoptosis
CTLs also secrete IFN-y, stimulating and activating M1 macrophages
PD-1 role in tumor pathogenesis
CTLs have a surface molecule PD-1 which inactivates the CTL as part of normal regulatory mechanisms; many tumors up-regulate PD-1 ligands to suppress CTLs
Role of CD4 in tumor immunogenesis
CD4 T cells recognize tumor antigens, make lymphokines, and attract M1 macrophages; they also help CTL become activated
Tumor-infiltrating Lymphocytes (TIL)
Specific, anti-tumor T cells can be isolated from within a patient’s tumor; these cells can be expanded in culture and the patient’s immune system partially destroyed by irradiation to “make room” for the reintroduction of the expanded, anti-tumor clone
Hellstrom Experiments
- T cells from blood of regressors added in culture to melanoma cells = T cells killed tumor cells
- T cells from progressors added in culture to melanoma cells = T cells killed tumor cells
- T cells from either regressors or progressors added to melanoma in the presence of serum from progressor patients = T cells did not kill tumor cells
Conclusion: Serum of progressor patients contains blocking factors that block the killing of tumor cells by CTLs
BCG-mediated tumor regression
BCG (Bovine Tuberculosis) vaccine is injected directly into the tumor; a DTH type reaction to BCG ensues and the tumor cells are killed by innocent bystander tissue damage
Mechanisms for use of mAbs in tumor immunotherapy
- Antibodies to TAAs activate complement, causing lysis of phagocytosis of the tumor
- Antibodies tagged with toxin or radioisotope can deliver the agent straight to the tumor
- Antibodies to growth factor or growth factor receptors can inhibit autocrine (self-stimulating) tumors
- Antibody against tumor-specific ligands
Nivolumab
Human mAb against PD-1, binds and blocks CTL inactivation by tumors that express PD-1 ligands
Role of CD8 in tumor immunogenesis
TAAs are presented on MHC-I by DCs in the lymph node, activating naive CD8 cells; following activation, T cells undergo clonal expansion, leave the lymph node, and migrate to the tumor where they kill tumor cells by inducing apoptosis
CTLs also secrete IFN-y, stimulating and activating M1 macrophages
Role of CD4 in tumor immunogenesis
CD4 T cells recognize tumor antigens, make lymphokines, and attract M1 macrophages; they also help CTL become activated
