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1.6 Immunology
Expression
Name
Ligands on
APCs and
other cells
Receptors
on T cells
DCs; macrophages,
B cells
B7-1
(CD80)
C
C
V
N
N
V V
B7-2
(CD86)
C
V
V V
macrophages,
B cells, other cells
C
N
NN
DCs;
ICOS-L
(CD275)
C
C
V
N
N
NN
V V
DCs;
macrophages,
B cells; endothelial,
epithelial and tumor
cells (PD-L1 only)
PD-L1
(B7-H1,
CD274)
C
C
V
N
V
PD-L2
(B7-DC,
CD273)
C
C
V
N
N
Name
Expression
on T cells
Major
function
C CC CC C
CD28 ICOSCTLA-4 PD-1
Naive
T cells
Activation of
naive T cells;
induction of
Regulatory
T cells;
activated
T cells
Inhibition of
T cell
activation
Activated
T cells; T
follicular helper
(Tfh) cells
Generation
of T follicular
helper cells
C
Activated T cells
Inhibition of T cell
activation (mainly of
effector T cells)
immune
responses
Fig. 1.12 The major members of the B7 and CD28 families. The known B7 family ligands are expressed on antigen presenting cells (APCs) (dendritic cells [DCs],
macrophages, and B cells), and CD28 family receptors are expressed mainly on T cells. Different CD28 family members stimulate or inhibit different stages and types
of T-cell responses. (From Abbas A, Lichtman AH, Pillai S. Activation of T lymphocytes. In: Cellular and Molecular Immunology. 9th ed. Philadelphia: Elsevier; 2018.)
• Identify infected (viral) or tumor cells that have decreased
MHC I surface expression via a group of surface receptors
(CD16, NKp44/46, NKG2D, and others)
• NK cells destroy target cells via perforin/granzymes in a
manner analogous to CD81 T cells
• They also work synergistically with macrophages by
secreting IFNg that enhances macrophage phagocytic
capacity
• NK cells are activated by IL-12, IL-15, and type I interferons
• STAT4 is a key transcription factor involved in response to
IL-12 and type 1 interferon in NK and Th1 cells; SNPs in
STAT4 are a/w early-onset lupus with increased risk of
stroke and nephritis
• Tissue-resident macrophages constitute another subset
derived from hematopoietic precursors that seed the skin
during fetal life
• Derived from a common CD341 progenitor cell in the
bone marrow
• Cell markers: CD11a/b/c, CD6, Fc receptor for IgG, and
MHC II (for antigen presentation)
• Primary function is to ingest/destroy microbes, apoptotic
cells and debris
• After ingestion of these targets into phagosomes, they fuse
with lysosomes to form phagolysosomes, in which
reactive oxygen/nitrogen species and proteolytic enzymes
contribute to destruction and death
• Other functions include:
■
Mononuclear phagocytes
• Monocytes (in bloodstream) differentiate into
macrophages (in tissue)
Cytokine production → modulation of inammation
■
Tissue remodeling, wound healing (absolutely
required), and coagulation
■
Antigen presentation
25
CHAPTER 1 • Basic Science
Induction of antitumor immune response in lymph node
A
Tumor peptide-MHC
Dendritic
cell
B7
CTLA-4
CD28
TCR
CD8
T cell
+
B7
CD28
CTLA-4
Anti-CTLA-4
No costimulation
CTL-mediated killing of tumor cells
B
Tumor peptide-MHC
TCR
Costimulation
Primed CTL capable
of killing tumor cells
Activated
CTL
Tumor
cell
Inhibited
PD-L1
PD-1
CTL
PD-L1
Fig. 1.13 Checkpoint blockade. Tumor patients often mount ineffective T-cell responses to their tumors because of the upregulation of inhibitory receptors such as
CTLA-4 and PD-1 on the tumor-specic T cells, and expression of the ligand PD-L1 on the tumor cells. Blocking anti-CTLA4 antibodies (A) or anti-PD-1 or anti-PD-L1
antibodies (B) are highly effective in treating several types of advanced tumors, by releasing the inhibition of tumor-specic T cells by these molecules. Anti-CTLA-4
may work by blocking CTLA-4 on effector T cells (shown) or on Tregs. (From Abbas A, Lichtman AH, Pillai S. Immunity to tumors. In: Cellular and Molecular Immunol-
ogy. 9th ed. Philadelphia: Elsevier; 2018.)
Table 1.16 Dermatologically Relevant Chemokines
Chemokine Chemokine Receptor Major Function and Disease Relevance
CCL2 CCR2 Mixed leukocyte recruitment; especially inflammatory monocytes (involved in wound
CCL4 CCR5 T cell, dendritic cell, monocyte, and NK recruitment
CCL5 (RANTES) CCR1, CCR3, CCR5 Mixed leukocyte recruitment, notably eosinophils; SNPs associated with atopic
CCL11 (eotaxin-1) CCR3 (preferentially expressed on eosinophils) Eosinophil, basophil, and Th2 recruitment
CCL17 CCR4 T cells; elevated in psoriasis and atopic dermatitis
CCL18 T cells; elevated in atopic dermatitis
CCL19/CCL21 CCR7 T cell and dendritic cell migration from skin to lymph nodes (CCR7 is a marker of
CCL20 CCR6 Recruitment of Th17 cells; role in psoriasis
CCL22 CCR4 NK cell, T-cell recruitment
CCL26 T cells; elevated in atopic dermatitis
CCL27 CCR10 T-cell recruitment to skin
CXCL1 CXCR2 Neutrophil recruitment
CXCL8 CRCR1. CXCR2 Neutrophil recruitment; role in psoriasis
CXCL9, CXCL10 CXCR3 Effector T-cell recruitment; especially Th1
CXCL12 CXCR4 B-cell migration into lymph nodes
CXCL13 CXCR5 B-cell migration to lymph nodes; T follicular helper cell migration into lymph node
CXCL16 CXCR6 Effector T-cell recruitment; especially Th1
CX3CL1 CX3CR1 T-cell, NK-cell and monocyte recruitment
Modied from Abbas AK, Lichtman AH, Pillai S. Leukocyte circulation and migration into tissues. In: Cellular and Molecular Immunology. 9th ed. Philadelphia:
Elsevier; 2018:39–56.
PD-1
Anti-PD-L1
healing)
dermatitis
central memory T cells)
follicles
Anti-PD-1
Dead tumor cell
26
1.6 Immunology
Langerhans cells
• Embryonically seeded tissue-resident macrophage
population residing in the epidermis
• Compared with other macrophages, they are poorly
phagocytic and instead function as professional APCs
(following antigen uptake, they cross the BMZ with help
of MMP-9, move to the lymph nodes where MHC-bound
antigen is presented to T cells, which are then activated)
• Dependent on TGF-b1 and macrophage colony-
stimulating factor receptor ligands for development and
retention in epidermis
• LCs usually not visualized during routine histologic
analysis, and on electron microscopy have rod-shaped
organelles (Birbeck granules)
• Langerin is a very sensitive and specic
immunohistochemical marker for LCs, because it stains
receptors found on Birbeck granules; CD1a is also a fairly
specic marker
• LCs are S1001, langerin (CD207)1, vimentin1, and
CD1a1; adhere to keratinocytes via E-cadherin
Dendritic cells
• Professional APCs that play a central role in initiating
T-cell response
• Activated by cytokines and direct TLR sensing of PAMPs
which increase their function
• Derived from bone marrow–derived myeloid precursors
• One subset called plasmacytoid DCs reside primarily in blood
and produce type I interferon upon activation; not found in
healthy skin, but may play a role in psoriasis and lupus
Mast cells
• Differentiate in tissues, for example, skin, from bone marrow–
derived progenitor cells expressing CD34/c-kit/CD13
■
Also stain with Giemsa, toluidine blue, and Leder
• Express high levels of c-kit receptor (CD117) and its
ligand, stem cell factor, which are critical for the
differentiation, survival, and proliferation of mast cells
• Typically located in papillary dermis
• Important in immediate-type hypersensitivity reactions
(e.g., anaphylaxis, urticaria, and angioedema)
• Express high levels of FcRI (high-afnity receptor for IgE)
• Mast cell degranulation triggers: cross-linking of FcRI by
binding to IgE, anti-FcRI antibodies, stem cell factor,
neuropeptides (e.g., substance P), drugs (opiates, aspirin,
vancomycin, curare, and polymyxin B), C5a anaphylatoxin,
and radiocontrast media
• Mast cell mediators are listed in Table 1.17
Eosinophils
• Bone marrow–derived granulocytes with important role in
defense against parasitic/helminth infections and in
allergic disease
• IL-5 produced by Th2 and type 2 ILC cells promotes
eosinophil activation and recruitment
• Weakly phagocytic, instead upon activation they release
granule contents that promote helminth killing and also
contribute to tissue damage (Table 1.18)
Table 1.17 Mast Cell Mediators
Mediator Function
Preformed
and stored in
granules
Major lipid
mediators:
newly formed
Cytokines:
newly formed
Modied from Metcalfe DD. Mastocytosis. In: Burks AW, Holgate ST,
O’Hehir RE, et al., eds. Middleton’s Allergy: Principles and Practice.
9th ed. Philadelphia: Elsevier; 2020:1216–1227.
Table 1.18 Eosinophil Mediators
Preformed and
stored in granules
Major lipid mediators:
newly formed
Cytokines: newly
formed
Modied from Abbas AK, Lichtman AH, Pillai S. Allergy. In: Cellular and
Molecular Immunology. 9th ed. Philadelphia: Elsevier; 2018:437–457.
Histamine Vasodilation, smooth muscle cell
Heparin Anticoagulant, controls function
Tryptase Production of C3a and
Chymase Increased mucous secretion
Cathepsin G Protease
Carboxypeptidase Protease
Prostaglandin D
Leukotrienes C
, E
D
4
4
Platelet-activating
factor
IL-3, IL-4, IL-5,
IL-6, IL-8, IL-13,
TNF-a
Mediator Function
Major basic
protein, eosinophil
cationic protein
Eosinophil
peroxidase,
lysosomal
hydroxylases,
lysophospholipase
Leukotrienes C
D4, E
4
IL-3, IL-5, IL-8, IL-10,
RANTES, MIP-1a,
eotaxin
contraction, tissue edema via
vascular permeability
of other mediators
bradykinin, increased fibroblast
proliferation
Vasodilation, bronchoconstriction,
2
leukocyte chemotaxis
,
Bronchoconstriction, dendritic
4
cell recruitment and activation
Vasodilation
See cytokine section (includes
mast cell proliferation, IgE
production, mucus secretion
and eosinophil activation)
Toxic to helminths,
bacteria, host cells
Degradation of
helminthic and
protozoan cell walls;
tissue damage/
remodeling
,
4
Bronchoconstriction,
mucus secretion,
increased vascular
permeability
See cytokine section
(includes eosinophil
production/activation,
and chemotaxis of
leukocytes)
Neutrophils
• Highly abundant; short-lived, produced in the bone
marrow
• First to arrive at sites of acute inammation (chemotactic
factors include c5a, IL-8, LTB4, kallikrein)
• Destroy microbial pathogens (phagocytosis followed by
oxidation [via ROS] → death)
• Extrude nuclear contents to form neutrophil extracellular
traps (NETosis); overactive in certain autoimmune
diseases, for example, lupus
27
CHAPTER 1 • Basic Science
• Contain four granule types, two most signicant are:
■
Primary granules (azurophilic) containing defensins,
cathelicidins, cathepsins, myeloperoxidase (along with
NADPH oxidase, creates ROS → oxidation of engulfed
organisms → death; of note, defect in NADPH oxidase
→ chronic granulomatous disease and negative
nitroblue tetrazolium test [cannot turn color from
yellow to blue])
■
Secondary granules (specic) most abundant, contain
lysozyme, elastase, collagenase
1.6.4 Major histocompatibility complex
• MHC locus in humans is known as the human leukocyte
antigen (HLA) locus
• The MHC locus is found on chromosome 6, and its key
role is to present antigen to T cells
• Divided into three classes: MHC class I, MHC class II, and
MHC class III (encodes for complement molecules)
• Typically, T cells only recognize peptides in the presence
of MHC molecules
• MHC genes are co-dominantly expressed
• During immune activation, the expression of MHC genes
is increased in response to the surrounding cytokine
milieu
• MHC class I molecules: present endogenous
antigens (peptides) to CD8
to induce apoptosis in both virus-infected and tumor
cells
■
MHC class I is expressed on all nucleated cells
■
Peptide size bound by MHC class I is 8 to 10
residues
■
Intracellular proteins are processed by proteasomes
into cytosolic peptides that are transported to the
endoplasmic reticulum, followed by binding to MHC
class I on the surface
■
Three main MHC I loci: HLA-A, HLA-B (most variable
class I), and HLA-Cw
■
Subunits encoded by these loci bind to
b
-microglobulin to produce a heterodimer
2
• MHC class II molecules present exogenous antigens
(peptides) to CD41 T cells
■
Expressed on APCs (i.e., monocytes, macrophages,
DCs, B cells, and activated T cells)
Not expressed on plasma cells
■
Peptide size bound by MHC class I is 10 to 34
residues
■
Endocytosis of extracellular antigens into vesicles
where the antigens are processed, peptides loaded
on MHC class II molecules, and expressed on the
surface
■
Three main MHC II loci:
HLA-DP: a-chain encoded by HLA-DPA1 locus,
b-chain by HLA-DPB1 locus
HLA-DQ: a-chain encoded by HLA-DQA1 locus,
b-chain by HLA-DQB1 locus
HLA-DR: a-chain encoded by HLA-DRA locus, 4
b-chains (three possible per person) encoded by
HLA-DRB1 (most variable class II locus), HLADRB3, HLA-DRB4, HLA-DRB5 loci
1
T cells and have the ability
• The old system of MHC nomenclature was based on
antibody typing where antigens were assigned letters and
numbers (e.g., HLA-DQ3); a more recent system includes
a number after the loci to provide more information
about the specic allele and loci (e.g., HLA-B*6801)
■
Where possible, both are listed below
■
MHC-associated diseases: (note prevalence of certain
alleles in autoimmune conditions)
Alopecia areata (AA): HLA-DQ3 (DQB1*03)
AA totalis/universalis: HLA-DQ7 (DQB1*0301) and
HLA-DR4 (DRB1*0401)
Lupus (SCLE and SLE): HLA-DR3 (DRB1*0306)
Psoriasis: HLA-Cw6 (Cw*06) especially early onset
Psoriatic and reactive arthritis: HLA-B27 (B*27)
Behçet’s disease: HLA-B51 (B*51) 80% of Asian
patients, 15% of Caucasians
Lepromatous leprosy: HLA-DQ1 (DQB1*06(11,12)),
tuberculoid leprosy: HLA-DR2 (DRB1*108),
HLA-DR3 (DRB1*0306)
Pemphigoid gestationis: HLA-DR3 (DRB1*0306)
and HLA-DR4 (DRB1*04)
Pemphigus vulgaris: HLA-DR4 (DRB1*04) and
HLA-DR8 (DQB1*0302) in Caucasians; HLA-DR14
(DRB1*14) and HLA-DQ3 (DQB1*0503) in Asians
Dermatitis herpetiformis: HLA-DQ2 (DQB1*02)
HCV-associated oral lichen planus: HLA-DR6
(DRB1*13/14)
Vitiligo: HLA-A2 (A*02); HLA-DR4 (DRB1*04)
1.7 LABORATORY TECHNIQUES
1.7.1 Tissue acquisition and processing
• Direct assays on human skin specimens are rapidly
advancing our understanding of pathogenesis of many
skin diseases
• Skin samples are usually obtained through biopsy
or excision, although other research techniques can
be performed on cells obtained via epidermal tape
stripping, skin surface swabs, or blood samples
(e.g., those looking for circulating antibodies,
genomic DNA)
• To perform these studies, proper processing of the skin
tissue is required. The rst decision point is whether the
tissue will be transferred freshly to the laboratory for
immediate processing, frozen for later processing, or
formalin xed (as for traditional pathology samples). The
rationale for each of these strategies depends on the
intended assay (Fig. 1.14).
• To investigate a specic subset of cells, isolation of the
target population is sometimes achieved by laser
microdissection (using a laser and microscope to cut a
small section of frozen/xed tissue from a slide) or
uorescence-activated cell sorting, where the whole tissue
is digested into a single-cell suspensions which is stained
with uorescent antibodies and then “sorted” for cells of
interest that express the correct markers as detected by
ow cytometry)
28
TISSUE PROCESSING
Excised tissue
1.7 Laboratory Techniques
Fresh
Tissue
culture
Electron
microscopy
Laser
microdissection
Fixed Frozen
Light
microscopy
Extraction
DNA RNA Protein
1.7.2 Specic techniques of interest
(Table 1.19)
Polymerase chain reaction (PCR)
• Used to amplify a specic piece of DNA from sample, for
example, genomic DNA, specic cell type (Fig. 1.15);
following PCR, DNA product can then be examined by gel
electrophoresis to determine its size or sent for sequencing
to determine the content of the region between the two
primers
• Applications: testing for a gene mutation (can detect a
deletion/insertion by examining PCR product size; need
mutation-specic primers; needs to be sent for sequencing
to detect SNPs/substitutions)
• Variations: quantitative PCR (qPCR) is an adaptation
designed to measure the relative copy number of a specic
segment of DNA between samples; here a special machine
can measure the amount of PCR product at the end of
each PCR cycle (i.e., as in Fig. 1.15B). The higher the
amount of that DNA type in the sample, the earlier the
cycle at which the product can be detected.
Quantitative reverse transcriptase PCR (qRT-PCR)
• In contrast to qPCR, the target starting template is RNA
rather than dsDNA. This allows one to determine the
relative level of gene expression/transcription by
measuring the amount of mRNA for that gene in a given
Flow
cytometry
and FACS
Fig. 1.14 Tissue processing. A tissue sample can be processed in
various ways for the analysis of DNA, RNA, or protein. FACS, Flowassisted cell sorting. (From Darling TN. Molecular biology. In: Bolognia
JL, Schaffer JV, Cerroni L, eds. Dermatology. 4th ed. Philadelphia:
Elsevier; 2018:66–80.)
sample or set of samples. First a polymerase and primer
set is used to generate dsDNA complementary (cDNA) to
the RNA template. Often the primers will anneal to the
poly(A) 39 end of mRNA. DNA is more stable and can
then be subjected to qPCR technique as above to measure
the relative amount of RNA in original sample.
16S ribosomal RNA (rRNA) sequencing
• Portions of the 16S rRNA gene are conserved across all
bacteria such that universal primers can be designed to
amplify this region of DNA from mixed/complex samples.
This PCR product can then be sequenced and variability
in specic regions of the 16S gene used to identify the
types of bacteria present in the sample, that is, taxonomic
classication (Fig. 1.16).
• Benets: can be performed non-invasively from skin
swabs or sebum samples; provides signicant information
about the types of bacteria present in a clinical sample;
does not require cultivation/culturing of microbes
• Limitations: does not distinguish live from dead bacteria
(both are detected), can determine relative amounts of
bacteria between samples but not absolute quantities,
reference databases for making taxonomic assignments
based on 16S sequence are still incomplete and
depending on the region of gene amplied there can be
misassignments or failure to assign, cannot distinguish
cause from effect in terms of relationship to disease
• Adaptations: 18S sequencing, analogous analysis for
fungal communities
29
CHAPTER 1 • Basic Science
Table 1.19 Specic Laboratory Techniques of Interest
Method Purpose Benefits Limitations Applications
Polymerase chain
reaction (PCR)
Quantitative reverse
transcriptase PCR
(qRT-PCR)
16S rRNA sequencing Determine types of bacteria
Sanger DNA
sequencing
Next-generation DNA
sequencing
RNA sequencing Determine level of
RNA microarray Determine relative level of
Fluorescence in situ
hybridization (FISH)
Comparative genomic
hybridization (CGH)
T-cell receptor (TCR)
gene rearrangement
Immunofluorescence
staining (direct)
Immunofluorescence
staining (indirect)
Immunohistochemistry
(IHC)
Amplify a specific piece
of DNA
Determine relative gene
expression by measuring
the amount of mRNA for
a specific gene between
samples
present in a sample
Determine oligonucleotide
sequence of a DNA
sample
Determine oligonucleotide
sequence of a DNA
sample
expression of many genes
in parallel
expression of many genes
in parallel
Visualize large
chromosomal
abnormalities
Visualize large
chromosomal
abnormalities
Detection of clonal
populations of T cells
using DNA extraction,
PCR of TCR gene(s) and
size detection on a gel
Localize specific antigens
using fluorescent primary
antibodies
Localize specific antigens
using fluorescent
secondary antibodies
Localize specific antigens
in tissue sections
Rapid, sensitive, inexpensive Contamination by small amount of
Rapid, sensitive, inexpensive Contamination of cDNA with genomic
Can be performed on non-
invasive samples, e.g., skin
swabs; does not require
culturing of microbes
Fairly rapid; can contiguously
sequence several hundred
DNA bases
Cheaper and higher
throughput
Looks broadly at
transcriptional landscape
within a tissue sample; no
prior knowledge of gene
sequence required
Looks broadly at
transcriptional landscape
within a tissue sample
Can be performed on
formalin-fixed tissue, can
select specific areas of
tissue to test (i.e., tumor vs.
normal skin), requires only
20–30 visualized cells
Typically assays a larger
genomic region than does
FISH
Can detect clones of
malignant T cells that lack
variability present in mixed
healthy cell populations
Very helpful for diagnosis
of autoimmune skin
conditions
Can be used to detect
antibodies and other
factors circulating in serum
Can be performed on FFPE
tissue, wide variety of
different target antigens
that can be detected and
studied
foreign DNA can confound results;
need to know enough about DNA
target to design specific primers
DNA can lead to false detection
of genes that are not actually
expressed/transcribed; isolation of
high-quality RNA requires fresh or
specifically stored tissue
Does not distinguish live vs. dead
bacteria; not highly quantitative;
does not examine content of
microbial genomes
Still somewhat expensive on a per
sample basis; requires high-quality
DNA; difficulty with G/C-rich
regions of DNA; requires primers
complementary to DNA template
Difficulty with highly repetitive regions
of DNA, large genome assembly
from shorter sequences fragments
can introduce errors
Somewhat costly, mostly still used
for research rather than diagnostic
purposes; fresh or specially frozen
tissue is usually required; lowly
expressed genes may not be
detected depending on depth of
sequencing
Looks at many (100s–1000s) but
not all genes; requires lots of
high-quality RNA to ensure good
hybridization; lowly expressed
genes may not be detected, assays
relative vs. absolute level of gene
expression
Probe design requires knowledge
of likely abnormalities, processing/
interpretation not standardized
across labs; technical issues
may arise such as incomplete
hybridization or non-specific binding
Assays a population of cells so
changes in a subset (,30–50%)
may be not be detected; more
expensive and longer turnaround
time than FISH because cells are
microdissected
Can have false-negative and false-
positive results; good adjunct assay
but cannot be used alone for basis
of a diagnosis
Requires fresh tissue; qualitative
rather than quantitative
Not widely available, not very
standardized across labs, not highly
quantitative
Non-standardized across
laboratories, not all antigens equally
preserved during fixation/processing
steps, less sensitive and specific
than PCR-based diagnostics
Amplification of a specific
region (gene) in genome;
other methods can then be
used to identify alterations
in intervening sequence
Determine relative
expression of a gene of
interest across a set of
samples, e.g., IL-17 from
skin biopsies from different
diseases
Determination of relative
abundance of bacteria
present on healthy vs.
diseased skin
Determine genetic
sequence, i.e., of a specific
gene to determine if there
is a mutation
Screen for many gene-
associated mutations at
once; search for genetic
basis for poorly understood
disease
Broadly profile level of
expression of all genes in
healthy vs. diseased skin
Broadly profile level of
expression of many genes
in healthy vs. diseased skin
Assist in distinguishing
melanocytic nevi with
atypical features from
melanoma
Assist in distinguishing
melanocytic nevi with
atypical features from
melanoma
Detection of malignant
clones in CTCL
DIF for IgG in pemphigus
or lupus
Detection of circulating auto-
antibodies in cicatricial
pemphigoid pemphigus
or lupus
Identification of plasmacytoid
dendritic cells (stain
positive for CD123)
in tissue sections of
cutaneous lupus
30
1.7 Laboratory Techniques
Table 1.19 Specic Laboratory Techniques of Interest—cont'd
Method Purpose Benefits Limitations Applications
Enzyme-linked
immunosorbent
assay (ELISA)
Western blot Detects and measures size
Mass spectrometry Analysis of all proteins
Data from Bolognia JL, Schaffer JV, Cerroni L, eds. Dermatology. 4th ed. Philadelphia: Elsevier; 2018; Jo JH, Kennedy EA, Kong HH. Research techniques
made simple: bacterial 16S ribosomal RNA gene sequencing in cutaneous research. J Invest Dermatol. 2016;136(3):e23–27; Grada A, Weibrecht K. Next-generation sequencing: methodology and application. J Invest Dermatol . 2013;133(8):e11; Chen AYY, Chen A. Fluorescence in situ hybridization. J Invest Dermatol
2013;133(5):e8; Schacht V, Kern JS. Basics of immunochemistry. J Invest Dermatol. 2015;135(3):1–4; Odell ID, Cook D. Immunouorescence techniques. J In-
vest Dermatol. 2013;133(1):e4; Chitgopeker P, Sahni D. T-cell receptor gene rearrangment detection in suspected cases of cutaneous T-cell lymphoma. J Invest
Dermatol. 2014;134(4):1–5.
Technique to detect and
quantify peptides, protein
or antibodies in serum
and amount of a protein
present in a sample
Fast, can be somewhat
standardized, more
quantitative than IIF, may
be cheaper and easier than
western blot
Can detect amount of a
given protein in a sample,
determine its size and
conformation
Highly sensitive Very expensive and technically
Requires knowledge of substance to
be detected.
Requires a specific and sensitive
antibody to the protein of interest,
proteins may be degraded during
extraction
difficult and time consuming, more
qualitative than quantitative
Detection and quantification
of circulating anti-dsg3
antibodies in patients with
pemphigus vulgaris
Measurement of epidermal
proteins and their
enzymatic processing
May have future diagnostic
application where ability to
detect small amounts of a
peptide is required
• Alternative approaches: whole-genome shotgun
sequencing broadly examines the sequence of all DNA in
a sample, both microbial and host; advantages here are
that functional genes in microbial genomes are assays
which might provide insight into potential function;
limitations include both cost and depth of sequencing
required to adequately assay microbial DNA which is a
small percent (, 1%) of most skin samples
DNA sequencing
• Determination of a DNA sequence can help inform the
likelihood of disease based on presence or absence of
specic gene mutations or variants
• In rst generation, chain termination (Sanger) sequencing
an oligonucleotide primer (as used in PCR above)
hybridizes to the target DNA and a polymerase starts to
synthesize a complementary strand until a uorescent
nucleotide analog (e.g., ddATP instead of dATP) is instead
5’
5’
Denaturation
(94°C–96°C)
5’ 3’
3’
incorporated and terminates the chain extension; gel
electrophoresis is then used to separate the synthesized
strands of varying length and a detector is used to
determine the order of the sequence (Fig. 1.17)
• In next-generation sequencing, DNA is sheared into
smaller pieces which are then ligated to adapter sequences
which help anchor the DNA in the machine and can be
used to identify samples later during data download and
analysis; these “DNA libraries” are then amplied and
sequenced by synthesis, meaning that the existing DNA
fragments serve as a template and complementary
nucleotides are incorporated by introducing these
sequentially one by one into the machine; identity and
order of the nucleotides incorporated is recorded either by
uorescent signal or pH change (Fig. 1.18)
• Although sequencing is most often still applied in the
realm of research rather than routine diagnosis, its rapidly
decreasing cost is leading to its increasing use for clinical
purposes
5’
Annealing (45°C–65°C)
5’
3’
3’
Extension (72°C)
5’
3’
3’
Fig. 1.15 First cycle of polymerase chain reaction. During the
denaturation step, DNA is heated to above 90°C and the two
strands of the DNA target sequence separate. The temperature
of the reaction is then cooled to 45°C–65°C and primers anneal
5’
to their complementary sequence in the template DNA. In the
extension step, the reaction is heated to 72°C to allow the DNA
polymerase to synthesize a new DNA strand complementary to
the template strand. (From Jalali M, Zaborowska J, Jalali M. The
polymerase chain reaction: PCR, qPCR, and RT-PCR. In: Jalali
M, Saldanha FYL, Jalali M. Basic Science Methods for Clinical
5’
Researchers. Philadelphia: Elsevier; 2017:1–18.)
31
CHAPTER 1 • Basic Science
Collection of
skin microbes
High-throughtput sequencing
of amplified 16S rRNA genes
Data processing, quality control
and analysis using bioinformatic tools
DNA isolation
from sample
PCR amplification
of bacterial 16S rRNA gene
Kingdom
Phylum
Class
Order
Family
Genus
Species
(Reference-based approach)
(Diversity-based approach)
Fig. 1.16 Schematic illustration of basic workow for skin 16S rRNA gene-based sequencing. (From Jo JH, Kennedy EA, Kong HH. Research techniques made
simple: bacterial 16S ribosomal RNA gene sequencing in cutaneous research. J Invest Dermatol . 2016;136(3):e23–27; Adapted and modied with permission from
Kong HH: Skin microbiome: genomics-based insights into the diversity and role of skin microbes. Trends Mol Med. 2011;17(6):320–328.)
• Variations: whole-exome sequencing helps to further
reduce the costs of sequencing by focusing only 1%–2%
of the genome that encodes for protein, where mutations
disproportionately contribute to disease. Note: this will
miss mutations in non-coding regions and enhancers/
promoters/RNAs that also contribute to disease in some
instances
cDNA library for mRNA. This is then sequenced by nextgeneration methods as described above.
• Variations/alternatives: prior to next-generation
sequencing, RNA microarrays were more commonly used
for a similar purpose. This method is based instead on
designing a panel of probes to genes of interest on a chip/
array and then visualizing the extent to which nucleic
acids within a sample bind to these probes.
RNA sequencing
• RNA sequencing (RNAseq) is a method of broadly
assaying the number and identication of mRNAs in a
sample to better understand which genes are actively
being transcribed and thus (likely) expressed at the
protein level. Just as DNA sequencing expands the breath
of focus from one gene (as in a single qPCR reaction) to
all potential genes, RNAseq does the same for mRNA. As
with qRT-PCR, the rst steps involve generation of a
Fluorescence in situ hybridization (FISH)
• FISH is designed to visualize the location of specic genetic
sequences. The method involves hybridization of labeled
probes to DNA in a tissue sample, that is, on a sectioned
slide. Each bound probe (appearing as a dot) identies a
single copy of the target DNA sequence. Nuclei with two
copies will have two dots. It is best used to identify large
chromosomal changes (copy number variations), that is,
32
Fixed end
CA C C G A A T ACA T C T G
1.7 Laboratory Techniques
DNA SEQUENCING
A T C
A reaction
dATP
dTTP
dCTP
dGTP
ddATP
CA
CACCGA
CACCGAA
CACCGAATA
CACCGAATACA
G
3'
T
C
T
A
C
A
T
A
A
G
C
C
A
C
5'
Sequencing gel
Fig. 1.17 Sanger DNA sequencing. An oligonucleotide primer hybridizes to the DNA to be sequenced and DNA polymerase synthesizes a second complementary
strand. The synthesis of the second strand is interrupted randomly by the incorporation of uorescent nucleotide analogs (ddATP, ddGTP, ddCTP, ddTTP). The DNA
fragments containing this nal nucleotide analog can be identied because each of the four ddNTPs is labeled with a different color uorochrome. Gel electrophoresis
is used to separate the different sizes of DNA fragments. The different-length DNA strands terminating with different uorochrome-labeled nucleotide analogs pass a
uorescence detector and indicate the order of the DNA sequence. (From Darling TN. Molecular biology. In: Bolognia JL, Schaffer JV, Cerroni L, eds. Dermatology
4th ed. Philadelphia: Elsevier; 2018:66–80.)
T reaction
dATP
dTTP
dCTP
dGTP
ddTTP
CACCGAAT
CACCGAATACAT
CACCGAATACATCT
CACCGAATACATCTG
CACCGAATACATCT
CACCGAATACATC
CACCGAATACAT
CACCGAATACA
CACCGAATAC
CACCGAATA
CACCGAAT
CACCGAA
CACCGA
CACCG
CACC
CAC
CA
C
C reaction
dATP
dTTP
dCTP
dGTP
ddCTP
C
CAC
CACC
CACCGAATAC
CACCGAATACATC
CACCGA ATA CA T CTG
80 390
Automated fluorescent
G
G reaction
dATP
dTTP
dCTP
dGTP
ddGTP
CACCG
CACCGAATACATCTG
sequencing scan
deletions, amplications, translocations. While classically
used for prenatal diagnosis, its central role in dermatology
is in identifying genomic aberrations diagnostic of certain
skin malignancies, most often melanoma. A commercially
available four-probe FISH assay reportedly has . 85%
sensitivity and . 95% specicity in distinguishing
melanoma from melanocytic nevi. More recently,
researchers designed an 11-probe panel to assist in the
diagnosis of cutaneous T cell lymphoma (CTCL) with
leukemic involvement, by examination of peripheral blood.
• FISH is best used as a supplementary diagnostic tool to
traditional histopathology
• Variations/alternatives: microarray-based comparative
genomic hybridization which can more broadly
interrogate copy number aberrations across the whole
genome but has other limitations
Immunouorescence (IF)
• In IF, uorescently labeled antibodies are incubated with a
sample (tissue biopsy or serum) to allow binding to
specic antigens and binding is then assayed
microscopically
• Direct IF (DIF) detects and localizes antigens in the skin.
In dermatology, DIF is most often used to detect
autoantibody-antigen complexes deposited in the skin as
a result of autoimmune skin disease. Here the
uorescently labeled antibodies are designed to bind to
patient’s own autoantibodies (IgA, IgG, IgM) or
complement (C3). DIF must be performed on fresh tissue
that is transported in ammonium sulfate–containing
media to preserve autoantibody-antigen complexes. Other
applications of DIF include detection of infectious
organisms by incubating with labeled primary antibodies
against the suspected microbe
• Indirect IF (IIF) is used to detect circulating autoantibodies
in a patient’s serum. First, the sample is incubated with an
unlabeled primary antibody designed to bind the target
molecule, then it is incubated with a labeled secondary
antibody directed against the Fc portion of the primary
antibody. This two-step process makes IIF more complicated
and time-consuming but also more sensitive than some
33
CHAPTER 1 • Basic Science
TEMPLATE PREPARATION
Genomic DNA or cDNA
Library preparation
Fragmentation of DNA
Library amplification
Emulsion PCR
DNA is amplified onto microbeads
SEQUENCING AND IMAGING
Ion torrent PGM MiSeq
A T A G T C A G C T G
T A T
C
Fig. 1.18 Next-generation sequencing methodology. Although specic processes vary based on the sequencing platform, this gure depicts the major steps involved
in next-generation sequencing. (From Grada A, Weibrecht K. Next-generation sequencing: methodology and application. J Invest Dermatol. 2013;133(8):e11.)
pH change
DATA ANALYSIS
Adapter ligation
Cluster generation
DNA is bridge amplified onto a flow cell
A T A G T C A G C T G
T A T
C
Fluorescence
other assays. In dermatology, we more commonly think of a
variation of IIF used to detect circulating autoantibody.
Here, serum is incubated with slide sections of a foreign
tissue known to consistently bind the antibody of interest
(e.g., monkey esophagus for anti-desmoglein antibodies), a
labeled secondary antibody directed against the Fc portion
of the autoantibody is then added and the uorescence
pattern interpreted by microscopy.
Immunohistochemistry (IHC)
• IHC is used to localize specic molecules/antigens in
formalin-xed, parafn-embedded (FFPE) tissue based on
binding of a specic antibody to that target and
visualizing where and to what extent that binding occurs.
Because formalin xation reversibly compromises
antigenicity of epitopes to a certain extent, after tissue
sectioning these are “retrieved” or “unmasked” usually by
heating the slide in a buffered solution. The tissue section
is then incubated with the antibody/ies of interest and
these patterns are then visualized (Fig. 1.19) and
interpreted by the pathologist. IHC is used extensively in
34
dermatopathology to identify cell types within a tissue
section.
• IHC is also commonly used on fresh frozen tissue during
Mohs surgery for melanoma (Melan-A), poorly-
differentiated keratinocyte carcinomas (CK5/6), and
EMPD (CK7).
Enzyme-linked immunosorbent assay (ELISA)
• ELISA is a test to detect and measure levels of peptides,
protein, or antibodies from a sample in the form of a
liquid suspension (i.e., cannot be performed on whole
tissue without further processing). It is most often used
to detect levels of circulating proteins or antibodies. When
detecting antibodies, the target antigen is bound to the
bottom of wells in a plate and the sample incubated so
that if antibodies to that antigen are present they will
bind to the antigen. A secondary antibody directed at the
Fc portion of the autoantibody bound to an enzyme is
then added. Finally, a substrate for the enzyme is added
(often resulting in generation of a colored product that