Tumor specific T cell functional activities

The principal mechanisms by which T cells effect antitumor

activity are

(a)direct engagement and cytolysis of target cells

(b) production and secretion of soluble cytokines and

chemokines that directly impact tumor cells and orchestrate
a more integrated antitumor inflammatory response
 Methods for the qualitative and quantitative analysis of T cells for understanding of
the immune processes:

Qualitative assays:
Lymphoproliferative assay using 3H thymidine (PBMC or purified T cells), Cr 51 release assay /
LDH assay

Quantitative Assays:
Frequency of CTL response determination
A) Limiting dilution analysis (LDA)
* Splenocytes/PBMC stimulated ex vivo with peptide loaded cell + IL-2
* Proliferation assay
* Cr51 release assay / LDH assay
B) ELISPOT assay
C) Intracellular Cytokine assay
D) MHC TETRAMER assay
*Ex vivo PBL tetramer staining
*Tetramer based FACS sorting of antigen specific CTL
Proliferation Assay :
The proliferation of T cells in presence of various stimuli (such as exposure to mitogenic agents,
polyclonal stimuli or specific antigens) is a fundamental technique for assaying T-cell responses.

*This assay requires cells (usually PBMCs) to be incubated in the presence of

the antigenic or mitogenic stimulus for 3-7 days, before the addition of 3 H-
Thy for 6-18 hours. The total amount of the radiolabel that was incorporated
into the cells in that time period is then measured, which provides a measure of
the rate of synthesis of DNA by the entire population of cells.

*In a ‘standard’ assay, the cells are co-cultured with a range of concentrations
of stimulating antigen, and at least one other antigen to act as a control to
determine the specificity of the response.

*Results are usually expressed as a stimulation index (SI), which is the ratio
of the scintillation counts (as a result of the incorporated 3H-Thy) obtained in
the presence of the test antigen, divided by the counts obtained in the presence
of the control antigen (or culture medium alone).

[scintillation counts (as a result of the incorporated

3
H-Thy) obtained in the presence of the test antigen]
Stimulation index (SI) =
[scintillation counts obtained in the presence of the
control antigen (or culture medium alone)]
Tritiated Thymidine Assay :
Radioactively labelled (tritium) thymidine is used to measure the proliferation of lymphocytes by the
incorporation of 3H thymidine into the DNA of the dividing cell. After a fixed time period the cells are
washed and lysed, after which the incorporated radioactivity in the DNA of the cells is quantified.

*Incorporation of 3H-Thy provides a good correlate of T-cell division

* It does not necessarily reflect the overall size of the final cell population because some stimuli can
induce rapid division and yet be toxic in the longer term to many of the cells exposed to them

* There is no information about the synthesis of DNA by individual cells

* The assay is not sufficiently sensitive or quantitative to compare the relative numbers of antigen-
specific lymphocytes in a series of PBMC samples, such as might be drawn over a period of time to
follow the effect of vaccination
 Working principle of (51Cr release assay) for CTL
Detection of cell killing activity by CD8 T cells: Cytolytic assays ( 51Cr release assay)

1. Labeling 2. Co-culture 3. Lysis & release of 51Cr

51Cr released

Target cells CD8 51Cr CD8 51Cr

Cr in the form of Na2 51CrO4 offers the advantage of being taken up by live cells in its
51

hexavalent form, but is released from lysed cells in its trivalent form, which is not re-utilised.
 Method for Cytotoxicity assay 4-hr 51
Cr-release :
►Target cells were labeled with 25 μCi ml -1 of (Na2 51CrO4) for 1 h at 37°C, washed with
HBSS and pulsed with the peptide at the different concentrations for 1 h in RPMI 1640
medium.
► The washed target cells were plated with effector cells on a microtiter assay plate at different
effector : target (E : T) ratio and incubated at 37°C for 4 h.

► Radioactivity in the supernatant was then determined by γ-radiation counting.

► The specific lysis was determined by using the formula

(experimental release − spontaneous release)

× 100
(maximum release − spontaneous release)

*It has been estimated that each CTL effector cell is capable of lysing up to five target cells in the
same microtitre well, during the course of a 4-hour assay. To achieve lysis of 50% of the target cells
(of which there are usually 2000–5000 in each microtitre well, therefore, requires 200–500 CTLs).
The CTLs must be present at a frequency greater than, or equal to, approximately one per
thousand in the effector-cell population at the start of the assay in order to be detected.

If the frequency of CTL is less than one per thousand cells the direct 51Cr-release assay will not
show any result. Therefore the diluted CTL cells should be to grow sufficient number by which
it can reach the detectable frequency of CTL cells.
Limiting-Dilution Analysis (LDA)

It provides quantitative estimates of the number or frequency of precursor T cells present in a

given PBMC population that are specific for a particular antigen.

Positive results in this assay (as function of proliferation, cytokine production or cytotoxicity),
indicate the presence of antigen-specific precursor cells in the PBMC population at the start, which have
become activated and have subsequently divided during the period of cell culture.

*In LDAs, many micro-lymphoproliferation assays are prepared in vitro, using a range of dilutions
of the cell population under investigation, with at least 24 replicate cultures at each dilution.

*Other factors (such as growth factors, antigen and APCs) need to be added to the microtitre wells
in excess, so that the only parameter that is limiting is the number of responding antigen-specific
cells that are present in the cell population at the start.

*Under these conditions, and assuming that ‘single-hit’ kinetics apply, the number of non-
responding cultures follows the Poisson distribution.

*A semi-log plot of the percentage of non-responding cultures negative [ln (fraction of

negative cultures)] plotted against the number of input cells per culture should produce a
straight line, and the input number of cells at the start that contained on average one specific
precursor cell can be calculated from the zero term of the Poisson distribution.
 Principle of limiting dilution analysis:
Let us first take, as a hypothetical example, a suspension of cells containing 1000 specific cells in 1000
ml of tissue culture medium.
*The average number of cells per milliliter, a value we shall designate m, (in this example it is 1).
*Withdraw a series of 1 ml samples and place these in individual culture wells.
*Expected to get exactly one cell at each attempt.
*But in fact although sometimes get one cell, occasionally get more than one, and often come up
without cell.
The frequency for each of these outcomes follows a Poisson distribution.
Expect to withdraw one cell and without any cell at the same frequency, 0.368 or roughly 37% of the
time.
The frequencies for the various outcomes are.
1 0.368
2 0.184
3 0.061 F0 = In our hypothetical example, m = 1 and, as we found, (frequency) F0 =
0.368.
4 0.015
m = −ln F0 = -ln( 0.368) = 0.99
5 0.003
>5 0.001

Suppose one dilution of a cell suspension gave 10% negative cultures (F0 = 0.1), the average number of
cells (m) in each culture would be 2.3. If a greater dilution of the cell suspension gave 20% negative
cultures, m becomes 1.6.
*Whichever dilution of the cell suspension gives 37% negative cultures, m=1, and we have established
the frequency of our cells in the suspension.
For example, if 37% of the cultures established with 4000 cells per culture are negative, then the
frequency of our cells is 1 in 4000.
 Limiting-dilution analysis and split-well analysis of human cytotoxic T
lymphocytes (CTLs)
(a) Peripheral blood mononuclear
cells (PBMCs) are obtained from the
original peripheral (venous) blood
sample by differential centrifugation
over a sucrose-based cushion.

(b) An additional purification step

[such as selection of CD8+ T
lymphocytes (T cells) using magnetic
beads] can be performed [the specific
precursor cells that are to be detected
are indicated in (darker) purple].

(c) A fixed concentration of antigen (Ag), extraneous growth factors and (d) an excess of irradiated
antigen-presenting cells (APCs) are added to each of the 96 wells of a microtitre cell culture plate.

(e) Dilutions of the population of responder cells are made, and added to the microtitre wells. At least 24
replicates for each cell concentration are set up. As the input number of ‘responder’ cells increases,
then, on average, the number of specific cytotoxic T-lymphocyte precursor cells (CTLp) in each well also
increases.

(f) The cells are cultured for 9-14 days to allow differentiation and expansion.
 Limiting-dilution analysis and split-well analysis of human cytotoxic T
lymphocytes (CTLs)
(f) The cells are cultured for 9–14 days (g) Cells are expanded to sufficient
with growth factors
number for cytotoxicity assay

(h) To assay the samples for cytotoxicity,

the content of each well is equally
divided (split) for testing against a panel
of target cells that are labelled with 51
chromium (typically, 2–5 different types
of target cells). The number of
microtitre-well cultures that fail to lyse
the specific target cells is then recorded
for each set of replicates with the same
cell input number.

(i) A plot of the log (base 10) of negative

cultures against the number of cells that
were started with allows determination of
the concentration of cells in a well that
would, on average, have contained a
single, specific precursor cell.

(j) Split-well analysis; cells in wells that were seeded at a cell concentration lower than that of the
precursor frequency, can be assumed to be clonal in origin. Cells in these wells can be tested for
cytotoxicity to a range of antigens.
 Example of experimental microwell plates used for CTL analysis in LDA

The number of microtitre-well cultures that fail to lyse the specific target cells ( ) and ln fraction of
negative (-)ve well ( not responding target cell killing activity) plotted with cells per well. The frequency
of tumor cell specific CTL is 1 in 5966.

ln (fraction) of negative
Summary of Limiting Dilution Analysis of CTL (51Cr)
*The limiting dilution analysis (LDA) is a modified CTL assay that gives a
quantitative measure of the number of precursor CTLs.
*Antigen-specific T cells are first serially diluted and then cultured until the
cells have multiplied to a sufficient number to perform CTL lysis assays.
*The number of precursor CTLs can be estimated from the lowest dilution
at which CTL activity can no longer be detected (LDA )

Drawback of LDA analysis:

LDA only detects the portion of cells that are able to proliferate and kill
targets after in vitro culture, it may result in a significant underestimation of
the total number of antigen-specific CTLs (which are not able to multiply)
 ELISPOT Assay

*The ELISPOT assay is useful to measure both clonal size and effector function of low-
frequency antigen-specific T-cell populations (T cell epitope mapping) directly ex vivo
*ELISPOT assay is performed with a freshly obtained sample (or cryopreserved sample)
using less than 24 h of culture
*ELISPOT assay allows for the simultaneous analysis of hundreds of variables in parallel
from a single tissue specimen
*ELISPOT assay has found widespread use in the context of direct ex vivo immune
diagnostic monitoring in humans
In ELISPOT assays : The measurement of the production of cytokine or
any other released effector molecule (e.g., granzyme B )
*Cells are plated in the absence of antigen and the constitutive production of secreted
molecule forms the background against which antigen-induced responses are
compared.
* Experimental wells contain test antigens. The presence of the antigen stimulates
production of cytokine (or any secreted molecule). As these molecules are secreted,
they are captured in the proximity of the secreting cell by capture antibodies on the
plate surface.
*The secreted molecule is captured before dilution in the culture supernatant,
before binding to receptors on other cells, and before becoming degraded.
*In this way, each secreting cell leaves a trace of its secretory activity. When the
cytokine assay is optimized, each spot reflects the activity of a single cell .
*The spot size and intensity reflect the amount of cytokine secreted per cell.
*A typical ELISPOT assay is performed with freshly obtained sample (or
cryopreserved sample) using less than 24 h of culture. When performed in this
manner, ELISPOT assays reflect cell secreting clonal size and differentiation.
 ELISPOT assay to quantify secretion of cytokines by T lymphocytes (T cells)

(a) The T lymphocytes (T cells) are activated in

vitro by being co-cultured with antigen.
(b) The wells of the ELISPOT plate are coated
with antibody (immunoglobulin; Ig) that is
specific for the cytokine that is being assayed for.
(c) The activated T cells are transferred to the
ELISPOT plate,
(d) Cytokines are released during the incubation
period.
(e) Those cytokines that are released locally
around each T cell bind to, and are therefore
‘captured’ by, the specific antibody.
(f) The cells and any excess cytokines are washed
off.
(g) A second antibody that is also specific for the
cytokine of interest is added; this antibody is
coupled to an enzyme that is capable of
converting a substrate into an insoluble coloured
product.
(h) The plates are washed once more, and the
enzyme substrate is added.
(i) The substrate is converted into the insoluble product, forming spots of colour that represent the areas of
captured cytokines that were secreted by adjacent T cells.
(j) The coloured spots are counted using a microscope or digital-imaging system.
MHC TETRAMER
*The number of T cells producing a given cytokine in the presence of specific peptides can
also be measured by flow cytometry, ELISPOT.
*These techniques give information about both specificity and function, but both are limited
because they detect only the CTLs releasing a particular cytokine.
*Furthermore, the results from these methods have failed to answer some questions, such as
whether individual T cell clones of the same specificity have identical cell surface
phenotypes and cytokine-producing profiles.

Why MHC tetramer ?

The MHC class I tetrameric complex technology provides an ideal tool to address these
issues and can be used to follow the course of T cell response to the challenge of a
particular antigen.
MHC TETRAMER (Principle)
*The MHC class I tetrameric complex technique was introduced by Altman and Davis in Stanford, USA, and
their collaborators in Oxford, UK.
*The principle of the technique is that peptide/MHC complexes, as TCR ligands, can be used to identify T
cells with particular antigenic specificity.
*In vitro, a single, soluble peptide/MHC complex has a low affinity for its corresponding TCR, resulting
in a weakly bound complex with a rapid dissociation rate.
*MHC class I/peptide multimers, however, can greatly enhance the avidity of the binding. Such tetrameric
complexes can be tagged with fluorescent dyes, allowing individual T cells to be identified by flow cytometry
analysis.
* If the specific T cell is co-tagged with different fluorescent-conjugated antibodies that are specific for cell
surface markers (cytokine or chemokine) the phenotype of the cell can be elucidated simultaneously.
MHC class I multimers and the response to tumors
*Direct evidence for the role of CTLs in the immune response to tumours has been obtained
using MHC class I tetrameric complex technology.
*In patients with chronic myelogenous leukaemia treated with either IFN- or allogeneic bone
marrow transplantation, tumour-specific CTLs have been detected using specific MHC class I
tetramers complexed with PR1 (a peptide derived from proteinase 3).

*The presence of PR1-specific T cells correlates with disappearance of the tumour (by
cytogenic analysis) after treatment.

*Theoretically, CTLs with specificity against tumors could be expanded ex vivo and then
re infused as therapy for tumor patients. In practice, however, it has proved difficult to
isolate sufficient CTLs due to their low frequency.

*Single cell FACS of MHC class I tetrameric complex-stained CTLs has proved to be an
efficient method of obtaining significant numbers of melanoma-specific CD8 + CTLs, which kill
in vitro, and this procedure could be utilized for therapy .
Generation of MHC-Tetramer:
The strategy relies upon the use of BirA enzyme [bifunctional biotin-[acetylCoA carboxylase]
holoenzyme synthetase], which is capable of biotinylating a lysine contained within a particular
recognition sequence.

-Leu-His-His-Ile-Leu-Asp-Ala-Gln-Lys-Met-Val-
Trp-Asn-His-Arg-

*A BirA substrate peptide (usually the 15 mer, LHHILDAQKMVWNHR) is fused to the

extracellular portion of the MHC molecule,
*Both the heavy chain and 2-microglobulin are over expressed in E.coli as insoluble protein,
solubilized in denaturing buffer, then it is solubilized and allowed to folding by dilution in buffer
containing antigenic peptide.
*The folding reaction is concentrated using stirred cell with 10-kD cutoff membrane and the
trimolecular complex MHC/ 2m / peptide are purified by gel filtration.
Tetramer analysis to detect T lymphocytes (T cells) that have specific T-cell receptors
on their cell surface

(a) Soluble versions of the heavy chain of major

histocompatibility complex (MHC) class I
molecules are synthesised in E. coli bacteria.

(b) The molecules adopt an appropriate

conformation following the addition of β2
microglobulin (β2m) and a synthetic peptide that
represents the epitope that is recognised by the T-
cell receptor (TCR) of interest.

(c) Four MHC–biotin complexes are linked to a

single streptavidin molecule, using the specific
biotin–avidin interaction, to form a tetramer. The
streptavidin molecule is ‘tagged’ with a
fluorochrome (e.g. phycoerythrin; PE).

(d) Tetramers are mixed with the cell population that is to be analysed [e.g. total peripheral blood mononuclear cell
(PBMC) populations or CD8+ T lymphocytes (T cells)]. Only T cells that have TCRs that are capable of binding to the
particular MHC–peptide combination that is present in the tetramer are able to bind the tetramer; thus, such cells will
become labelled with the PE fluorochrome [shown in red on the graph in (e)].

(e) The cells are then analysed using flow cytometry; the proportion of the CD8+ T-cell population that stains positively
with the tetramer can be determined (top, right-hand quadrant).
Limitation of Tetramer Tech
*It requires knowledge of tumor antigen which is not often available.
* MHC-teteramer select T cell with single specificity, i.e, there is risk of
selection for tumor escapee variant, do not select for functional set and
may select T cell may be anergic.
*Individual MHC/peptide tetrameric complexes have to be produced
separately, and production is both technically difficult and time
consuming.
*These complexes have a limited storage stability. Flow cytometry
equipment is required for analysis of tetrameric staining.
Comparison of the sensitivity of detection of CD8+ T cells in currently
available assays

a
The assay used to measure human T-cell responses in vitro; this might follow an extended period
of restimulation and expansion in vitro.

b
The lowest frequency of specific T cells in the cell population at the start of the assay that can be
detected using the assay (if pre-culture and re-stimulation have been used, the cells might have been
expanded in relation to their frequency in the peripheral blood).

c
In tetramer analysis, a complex of four recombinant major histocompatibility complex (MHC)
class I molecules with β2 microglobulin and a peptide is labelled first with biotin, and then with
streptavidin to allow the complex to be further labelled with a fluorochrome. Binding of this
complex can be detected using a flow cytometer and is a measure of the number of specific T cells
in a cell population (such as peripheral blood) that can bind to the MHC–antigen-peptide complex.