First Aid, Chapter 4 Laboratory Tests, Lymphocyte Function: Cell proliferation, cytokine production, and cytotoxicity Flashcards
(31 cards)
What is the cell marker on all T cells?
CD3
What is the cell marker on all CD4 T lymphoctes (helper)?
CD3 CD4
What is the cell marker on all CD8 T lymphoctes (cytotoxic)?
CD3 CD8
What is the cell marker on activated CD4 cells?
CD4 HLA-DR
What is the cell marker on activated CD8 cells?
CD8 HLA-DR
What is the cell marker on naive T lymphocytes?
CD45RA
What is the cell marker on activated T lymphocytes?
CD45RO
What is the cell marker on double-negative T lymphocytes?
CD4-CD8-
What is the marker on γδ T lymphocytes?
CD4-CD8-γδ
What is the marker on T regulatory cells (highly expressed); activated T cells
CD3 CD25
Which cells respond to recall antigens, ie antigen-specific T lymphocyte proliferation? Is prior exposure required?
Activated and memory T cells. Prior exposure is required.
Which cells respond to mitogens - ie polyclonal T lymphocyte proliferation (mitogen)? Is prior exposure required?
Naive, activated, and memory. Prior exposure is required.
What are antigens used for antigen-specific T lymphocyte proliferation assays?
CMV Tetanus VZV antigens HIV antigens gp 120, p24 Candida
What are some mitogens uised in polyclonal T lymphocyte proliferation assays?
Cell surface signaling:
Phytohemagglutinin (PHA) Pokeweed mitogen (PWM) Concanavalin A (ConA) anti-CD3 antibody
Bypass proximal signaling: Phorbol ester (PMA) Calcium ionophore (Ionomycin)
Is costimulation needed for antigen-specific T-lympohcyte proliferation (recall antigen) assays or polyclonal T lymphocyte proliferation (Mitogen) assays?
No, but for antigen-specific T lymphocyte proliferation (recall antigen) assays, antigen presenting cells are required.
Summarize how lymphocyte proliferation assays (LPA) stimulation index works? What is the equation for stimulation index?
- Cells are incubated in culture for a week. On day 3 after mitogen stimulation, and on day 6 for antigens specific T lymphocyte-specific stimulation, radioactive [3H] (tritiated) thymidine is added and is incorporated into the newly synthesized DNA of the dividing cells.
- The amount of radioactivity incorporated into the DNA of each culture well is measured in a scintillation counter and is proportional to the number of proliferating cells. In the absence of proliferation of defective lymphocytes, less radioactivity is detected.
- The readout is measured in counts per minute (cpm).
- Note that pokeweed mitogen also has B-cell proliferation activity along the Tcell proliferation capacity.
Stimulation Index (SI) = (cpm Mitogen / cpm background Unstimulated)
Net counts or cpm = (cpm Mitogen – cpm background Unstimulated)
How does in vitro T-lymphocyte proliferation assay by flow cytometry work?
Peripheral blood mononuclear cells (PBMCs) are labeled with a proliferation tracking fluorescent dye (carboxyfluorescein diacetate succinimidyl ester, CFSE) and stimulated with appropriate mitogens for 4 days. Flow cytometric evaluation of T-cell proliferation is shown in Figure 4-13.
Upon stimulation and following proliferation of dividing T cells, there is a dilution of CFSC. This will lead to its decreased concentration. In the absence of proliferation of defective lymphocytes, there will be no decrease of fluorescence detected by the machine given the absence of dilutional effect.
What are uses for delayed type hypersensitivity testing? What pathogens are those who have lack of DTH response susceptible to? What percentage of the general healthy population experiences anergy? What testing should this population undergo? What can happen with rapid repeat testing?
- Delayed-type hypersensitivity (DTH) is a cost-effective and widely available method to screen for cell-mediated immunity (antigens used may be a number of recall antigens, candida, tetanus, tricophyton, etc).
- It assess integrity of cell-mediated immunity. Those who have a lack of response are susceptible to intracellular pathogens such as viruses, protozoa, fungi, and parasites.
- DTH can montior progrssion of acquired immunodeficiency such as HIV, anergy indicated disease progression.
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Describe how the mantoux test is done and interpreted. What are the antigens used?
1) 0.1 ml antigen injected (bevel up) ID, volar aspect of arm, bleb 7-10mm, antigens 3 cm apart
2) wait 48-72 hours
3) Area of induration > 5mm? If yes, than intact cell-mediated immunity.
4) If not >5mm, then Deficient cell-mediated immunity or lack of exposure to tested antigen anergy:
- pregnancy
- extremes of age
- cancer
- surgery
- stress
- hemodialysis
- corticosteroid treatment
- anticoagulant treatment
- DM
- liver disease
- Down syndrome
Antigens used: C. albican Trichophyton Tetanus Mumps
In flow cytometry, how are B cells measured?
By their cell surface markers, CD19 and CD20.
What B cells is CD19 and CD 20 expressed on?
- CD19 is part of the CD19, CD21, CD81 coreceptor complex and functions to amplify B-lymphocyte receptor-generated signals. CD19 is expressed on early B-lymphocyte progenitors up until the B-lymphocyte plasmablast stage of differentiation.
- CD20 is expressed on pre-B lymphocytes and B-lymphocyte blasts, but are not seen in early B lymphocytes or plasma cells.
What cells are absent in XLA?
In patients with X-linked agammaglobulinemia (XLA), Bruton agammaglobulinemia tyrosine kinase (BTK) is absent in B lymphocytes and monocytes. CD19+ B lymphocytes are absent.
What cells are low or absent in CVID?
IgD+CD27+ (unswitched memory) and IgD-CD27+ (Switched memory) cells. Only B cells present are naive IgD+CD27-.
Which mitogens stimulate T cells?
Pokeweed, phytohemagglutinin, and concanavalin A
