Page 1305 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPter 93 Assessment of Functional Immune Responses in Lymphocytes 1267

KeY COnCePtS Phosphorylation of H2AX appears normal, as it is also phos-
Assessment of DNA Repair Pathways phorylated by ATR; however, the magnitude of phosphorylation
is significantly decreased (Fig. 93.14B). The kinetics of phos-
by Phosphoflow phorylation indicates that maximal phosphorylation occurs 1 hr
• DNA double-strand break (DSB) repair defects are relevant to VDJ after induction of DSB and that there is dephosphorylation by
recombination, isotype class switching, and lymphocyte maturation. 24 hr postirradiation (not shown). This flow-based assay enables
• Defects in this process can lead to immunodeficiency with susceptibility a rapid assessment of radiation sensitivity as well as the ability
to infection and malignancy. to visualize the function of multiple proteins of the DNA repair
• Homologous recombination (HR), which is error-free, involves RAD50, pathway. It can also be used to characterize the DNA repair
RAD51, RAD52, and Mre11, while nonhomologous end-joining (NHEJ) function of known and unknown genetic defects, and it can
is error-prone and utilizes Ku70/80, DNA-PKcs, Artemis, DNA Ligase
IV, XRCC4, XLF/Cernunnos. identify functional phenotypes in patients with atypical presenta-
• ATM is a key regulator of cell-cycle checkpoints following irradiation- tions that may be missed if only genetic information is used.
induced DSB and coordinates timing of phosphorylation of checkpoint
proteins. ASSESSMENT OF B-CELL FUNCTION
• Cell cycle analysis can help identify checkpoint defects and can be
easily assessed by flow cytometry. While T cells and NK cells form the foundation of the cellular
• Phosphorylation of H2AX (γH2AX) is a useful marker of DNA damage immune response, B cells are the main driver of humoral immu-
related to irradiation-induced DSB.
• However, several kinases phosphorylate H2AX, which can confound nity. B cells are multifaceted in their function; they produce
identification of specific defects; therefore assessing multiple proteins antibodies via differentiation into plasma cells, they act as APCs
in the pathway allows identification of a broader group of DNA repair for T cells, and they secrete potent immunomodulatory cytokines
defects. while downregulating immune responses via IL-10 production.
• Measurement of 53BP1 phosphorylation is a useful surrogate marker B cells can proliferate in response to polyclonal mitogenic
of the integrity of the chromatin ubiquitin ligase pathway.
stimuli such as PWM (Fig. 93.15), albeit with a much weaker
response than seen with T cells stimulated with PHA (Fig. 93.3).
Clearly, the starting point for any evaluation of B-cell function
to cross-link the BCR (for 3 minutes), because Y551 phosphoryla- involves measuring serum immunoglobulin levels followed by
tion could not be detected in the time interval the assay was in vivo antibody responses to vaccination with both protein
performed. In addition to PBMC, Ramos cell line (a B-cell line (e.g., tetanus toxoid) and carbohydrate (e.g., Pneumovax 23)
derived from a patient with Burkitt lymphoma) is used as a antigens. 47
control for B cells, while pervanadate (complex of vanadate with A more recent area of focus in laboratory evaluation of B
hydrogen peroxide), an irreversible protein tyrosine-phosphatase cells has been the regulatory function of B cells, specifically with
inhibitor, is used in this assay as a positive control (Fig. 93.13). regard to the subset classified as regulatory B (Breg) cells. B cells
In the Ramos cell line, it is possible to visualize the Y551 phos- that secrete IL-10 have been described as Breg cells and are now
phorylation (Fig. 93.13) with pervanadate treatment. recognized to be an important component of the host immune
A number of genetic disorders, collectively classified as XCIND response that protects against autoimmunity and also limits
(x-ray [irradiation] sensitivity, cancer susceptibility, immuno- inflammatory damage. 48,49 In humans, the phenotype of Breg
+
−
+
hi
hi
deficiency, neurological involvement, and double-strand DNA cells has been described as CD19 CD27 CD24 CD38 CD5 CD
hi
breakage), cause impairment in cellular ability to repair DNA 1d . However, the ability to produce IL-10 appears to be the
double-strand breaks (DSB). These defects have a significant defining feature rather than any particular constellation of cell-
50
impact on the ability of cells to grow, differentiate, and function surface markers. Also, unlike Treg cells, no exclusive transcription
normally; they include ataxia telangiectasia (AT) due to factor equivalent to FOXP3 is expressed in Breg cells. In general,
+
ATM gene mutations and radiosensitive severe combined IL-10 Breg cells in blood and spleen appear to be enriched in
+
+
hi
immunodeficiencies (rs-SCID) due to mutations in the DCLRE1C, the CD19 CD27 CD24 memory B-cell subset. IL-10 exerts potent
LIG4, NHEJ1, and PRKDC genes. These disorders make it very antiinflammatory effects and enhances survival, proliferation,
desirable to have an assay capable of rapidly assessing DNA differentiation, and isotype class-switching of B cells. Both naïve
repair in lymphocytes in response to radiation damage. A flow and memory human B cells have the ability to produce IL-10
cytometry assay has been developed that is capable of measuring in response to stimulation via TLR9 and the BCR, but only
the function of several proteins in the DNA repair pathway after ~15% of B cells can produce IL-10 in response to stimulation.
induction of DSBs via irradiation (Fig. 93.14A). The assay includes Stimulation through CD40L or IL-21 also promotes differentiation
+
hi
hi
assessment of nonphosphorylated ATM protein and 53BP1 44,45 of IL-10–producing Breg cells. CD19 CD24 CD38 Breg cells
without irradiation to determine the amount of native protein play critical regulatory roles in autoimmune disease, chronic
present. Following low-dose irradiation, the function of ATM GvHD, and transplant tolerance. 51-53
and ATR (ATM-Rad3–related kinase) pathways are assessed by In the diagnostic laboratory, Breg cells can be assessed by
analyzing the autophosphorylation of ATM at serine 1981. This isolating PBMC from blood and culturing with CpG-B (TLR9
step is required for ATM activation and is followed by phos- stimulation) or CpG-B plus recombinant CD40L for 3 days in
phorylation of downstream targets SMC1, H2AX, 53BP1, and vitro, followed by the addition of PMA, ionomycin, and brefeldin
CHK1. H2AX is a histone that belongs to the H2A family and A (BFA) for the last 5 hours of culture. Cells can then be harvested,
is a component of the histone octamer in nucleosomes. It is washed, and stained with CD19 and IL-10 antibodies (the latter
phosphorylated by both ATM and ATR and is the first step in requires intracellular staining) and then analyzed with a flow
46
the recruitment and localization of DNA repair proteins. In cytometer. To assess the presence of Breg cells in blood without
patients with AT, there is a complete absence of phosphorylation in vitro differentiation, blood or PBMC can be analyzed for
+
hi
hi
of ATM, SMC1, and 53BP1 after irradiation-induced DSBs. CD19 CD24 CD38 B cells that are positive for intracellular