Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S5. Advantages and disadvantages of different assay approaches for
delineating T cell immunity. Page 1 of 6
Supplementary File S5: Advantages and disadvantages of different assay
approaches for delineating T cell immunity. For specific details and references,
please refer to Supplementary File S1.
T cell assay
Advantages
Disadvantages
approach
Whole blood


Smaller blood volumes are
Incubation with antigens is
required, compared with
on a per-volume basis; T
PBMC-based assays.
cell depletion may
Assessment of a whole
therefore result in loss of
immunological
sensitivity and may require
compartment is
adjustment of blood
accomplished. The
volumes.
measured T cell response



Whole blood may be used
will reflect cellular and
undiluted when incubation
soluble components that
is ≤18 hours, but longer
affect antigen presentation
term incubation requires
and T cell activation.
dilution with RPMI, and
The stimulation phase of
therefore more resources.
this approach requires

The measured response
relatively few resources;
reflects not only T cells,
assessment may often be
but other peripheral blood
completed in 2 phases:
components also. For
first immediate incubation
example, when cytokines
with antigen, followed by
levels are determined in
cryopreservation of
plasma obtained from
plasma, fixed white cells or
whole blood,
Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S5. Advantages and disadvantages of different assay approaches for
delineating T cell immunity. Page 2 of 6
RNA for later analysis,
measurements may reflect
e.g., soluble, cell-
contributions of
associated or mRNA
neutrophils and
expression analysis of
monocytes.
cytokine in the respective

samples.
The need for immediate
processing of collected
blood limits versatility in
what may be measured.
Freshly isolated

PBMC
PBMC-based assays are
PBMC isolation requires
incubated on a per-cell
more resources, compared
basis, resulting in
with whole blood
increased sensitivity in the
stimulation.
face of T cell depletion,



PBMC isolation is
such as in HIV infection,
associated with
compared with whole blood
considerable cellular loss,
assays.
requiring larger blood
PBMC isolation may allow
volumes.
for a description of T cell

Incubation of PBMC often
responses without the
involves introduction of
influence of other whole
foreign material, such as
blood components such as
foetal calf or unrelated
neutrophils and plasma.
human serum; however,
serum-free media may
also be used.

In short term assays, the
cellular activation as a
Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S5. Advantages and disadvantages of different assay approaches for
delineating T cell immunity. Page 3 of 6
result of PBMC isolation
may result in higher
backgrounds, compared
with similar incubation of
whole blood.
Cryopreserved

PBMC

PBMC are available for

The measured response in
later assaying in batches,
assays using
which is of particular
cryopreserved PBMC is
importance for reducing
invariably lower than when
variability in longitudinal
fresh PBMC or whole blood
vaccination studies.
is used.
PBMC are available at a

Optimal biobanking
later date for application of
procedures are critical for
scientific advances not
long-term storage of
known when blood was
PBMC, and require
collected. This is very
significant resources, such
important in tuberculosis
as LN2.
vaccination studies, as the

The specific protocol used
immune correlates of
for PBMC cryopreservation
protection against
may significantly impact
tuberculosis are not yet
on yield of viable,
known.
functional cells after
thawing.

Assays may produce more
optimal results when
Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S5. Advantages and disadvantages of different assay approaches for
delineating T cell immunity. Page 4 of 6
thawed PBMC are “rested”
in medium for at least 6
hours, prior to incubation
with antigens.
Short term

These assays are often

Whole blood should ideally
(<24hr, e.g.,
called “directly ex vivo”
be incubated and PBMC
ELISPOT assays,
assays, meaning that
isolated and incubated as
intracellular
results should reflect the in
soon as possible after
cytokine assays)
vivo state of circulating T
collection, to avoid loss of
cells. Effector function of
sensitivity. This appears to
specific cells is assessed
be of particular concern
without a prolonged period
when protein or whole
of cell activation, not
bacterial or viral antigens
allowing time for cellular
need to be processed, and
division, and considered to
may be less important for
result in more quantitative
peptide antigens.
outcomes. For example,
the frequency of antigenspecific T cells may be
defined by cell-associated
cytokine production or
activation marker
expression, as these T cells
would not have divided
during the short period of
incubation.
Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S5. Advantages and disadvantages of different assay approaches for
delineating T cell immunity. Page 5 of 6

Whole blood may be used
undiluted.
Intermediate and

Expansion of cells may
longer term assays
increase sensitivity, i.e.,
(1-7 days, e.g., 48
ability to measure a
hour ELIPOT,
response not measurable
lymphoproliferation
with other systems.
assays)


Increased risk of
contamination.

Requires more resources,
including CO2 incubators.

Results are necessarily
Both the presence of
semi-quantitative,
specific T cells and their
reflecting phenotype and
capacity to proliferate and
function of expanded cells
produce cytokines or other
– these phenotypes may
functional markers may be
not have been the same at
assessed. The expansion
the onset of the assay.
over the culture period
may result in increased
sensitivity, as “resting” T
cells, or T cells that require
more than just a short
period of antigenpresentation to become
activated, may expand. It
has therefore been
proposed that central
memory cells may be more
optimally measured with
longer term assays,
Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S5. Advantages and disadvantages of different assay approaches for
delineating T cell immunity. Page 6 of 6
compared with shorter
term assays; however, the
latter hypothesis remains
unproven. Measures of the
central memory response
are currently thought to be
critical for determining
long-term vaccine efficacy.

It appears that the time to
incubation or PBMC
isolation, after blood
collection, is not as
important for longer term
T cell assays. The assays
are also less affected by
the manipulation at PBMC
isolation and
cryopreservation.