Full text of DOI:10.1078/0171-2985-00131

Immunobiol. (2002) 205, pp. 282–289
© 2002 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/immunobiol
Department of Hematology and Oncology, University Hospital of Regensburg, Germany
Analysis of the Immune Response against Tetanus Toxoid:
Enumeration of Specific T Helper Cells by the Elispot Assay
S
TEPHANIE
MAYER, MONIKA LAUMER, ANDREAS MACKENSEN, REINHARD
ANDREESEN, and STEFAN W. KRAUSE
Received August 7, 2001 · Accepted in revised form February 11, 2002
Abstract
Tetanus toxoid (TT) is an antigen known to induce strong T cell specific immune responses in
humans after vaccination. Here we have used the Elispot assay to assess the number of TT-specific
Interferon-g (IFN-g) secreting T cells present in individuals and monitored the number of TT specif-
ic T cells present in the donors for more than two years. In each of the 22 healthy volunteers tested,
TT-specific T cells could be detected. Six out of 7 repetitively tested donors showed a remarkably con-
stant number of TT-specific IFN-g secreting T cells over several months, whereas one donor demon-
strated a transient increase during a flu-like infection. Three healthy donors received TT booster-
immunizations and showed significant increases in the number of TT-specific IFN-g secreting T cells
which reached peak levels by 4 weeks after vaccination. Depletion of either CD4+ T cells, CD8+ T
cells or CD16+/CD56+ T cells by immunomagnetic separation demonstrated that TT-specific IFN-g
secretion is mediated exclusively by CD4+ T cells. In addition, HLA class-I and -II blocking studies
showed that IFN-g production is performed by HLA class-II restricted cells. Our data show that the
Elispot assay can be reliably used to assess the number of TT-specific CD4+ IFN-g producing cells (i.e.
probably T helper cells) and therefore maybe also useful for the assessment of reactions to other help-
er antigens.
Introduction
Tetanus toxoid (TT) is known to induce a strong and long lasting humoral immune res-
ponse in humans after vaccination (1). After TT vaccination, uptake of the antigen by
antigen-presenting cells (APC) leads to presentation via the MHC molecules (2, 3), fol-
lowed by induction of clonal T cell response (4–6). It has been demonstrated that TT-
specific T cells are mainly CD4+ cells secreting Th1 cytokines such as IFN-g (7).
Currently, antigen-specific proliferation of T cells is the standard method for
the assessment of CD4+ T cell activity (8). Secretion of cytokines is known to be
Abbreviations: TT = tetanus toxoid; IFN-g = interferon g; APC = antigen presenting cell; PBMC =
peripheral blood mononuclear cell.
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Elispot for TT-specific T cells · 283
another indicator of specific T cell responses (9) that can either be measured by
determination of soluble cytokines in cell culture supernatants or by intracellular cyto-
kine staining, a quantitative method to analyze T cell responses on an individual cell
basis (10).
The Elispot assay is another highly sensitive and reproducible method to determine
specific T cell responses by quantifying antigen-dependent cytokine secretion at a single
cell level. However, most of the studies using the Elispot assay were performed with
CD8+ cytotoxic T cells (11, 12). In the present study the Elispot assay was used to ana-
lyze TT-specific IFN-g secretion by peripheral blood mononuclear cells (PBMC) of
healthy donors. The Elispot protocol to monitor antigen-specific CD8+ T cells has been
established before (11, 13). Here, we analyzed the frequency of TT-specific T cells in
healthy donors before and after TT booster immunization. Our data show clear evidence
that the IFN-g-secreting T cell subpopulations specific for TT are CD4+ MHC class II
restricted T helper cells.
Materials and Methods
Blood donors and tetanus toxoid immunization
Heparinized blood samples were taken from healthy donors after obtaining informed consent. All
donors had been immunized previously against tetanus toxoid (TT) within one to ten years. Peripheral
blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll-
Hypaque 1.077 (Pharmacia, Freiburg, Germany). Cells were washed twice with phosphate-buffered
saline (PBS, Biochrom, Berlin, Germany). For booster-immunization some volunteers received a sin-
gle 0.5 ml (75 I. E.) intramuscular dose of aluminum-adsorbed TT (T-IMMUN, Immuno,
Heidelberg, Germany). Blood samples were collected before and at defined time points after vaccina-
tion as indicated in Figure 3.
Interferon-g Elispot assay
The assay was adapted from an established protocol to monitor antigen-specific CD8+ cytotoxic T
cells (11;13). Concentrations of cells, antibodies and TT antigen as well as incubation times were opti-
mized in pilot experiments (data not shown). 96-well nitrocellulose plates (Millititer, Millipore,
Bredford, MA, USA) were coated overnight with 50 ml/well of anti-IFN-g monoclonal antibody
(MAb) (8 mg/ml, in PBS, code-no. 1598-00, Genzyme, Rüsselsheim, Germany) at 4°C. Wells were
washed with PBS and blocked with RPMI-1640 containing 10% human AB-blood group-serum for
two hours at 37°C. PBMC (1¥105 cells/well) in a total volume of 200 ml RPMI-1640 containing
10% AB-serum, 2 mM L-Glutamin, 100 U/ml penicillin, streptomycin and 1 mM MEM
Sodiumpyruvat (complete cell culture medium) were incubated with 3 mg/ml TT (Calbiochem-
Novabiochem, La Jolla, CA, USA; purity tested by SDS-PAGE according to the manufacturer). Each
sample was tested in triplicates. Unstimulated PBMC or PBMC stimulated with 1 mg/ml pokeweed
mitogen (PWM; Sigma, St. Louis, MO, USA) served as negative and positive controls, respectively.
After 20 hours of incubation at 37°C with 5% CO2, coated plates were washed six times with PBS
containing 0.05% Tween 20. The wells were then incubated for 24 hours at 4°C with 100 ml/well bio-
tinylated mouse anti-human IFN-g MAb (2.5 mg/ml in PBS; clone 4S.B3, Pharmingen, Hamburg;
Germany). After washing four times with PBS, 100 ml/well streptavidine-alkaline phosphatase
(BioRad, Munich,Germany) diluted 1:1000 in PBS was added to each well and incubated for an
additional 2 hours at room temperature. After two more washing steps with PBS, 100 ml/well
BCIP/NBT substrate (BioRad) was added for 20–30 min. Color development was stopped by wash-
ing the plates with tap water. Plates were then air-dried overnight. Spots were counted using a stereo-

284 · S. MAYER et al.
microscope. Mean numbers were calculated by counting spots from triplicate wells and the number of
spots from negative controls, wells without TT, were subtracted. In the vast majoritiy of tests negative
controls did not exceed two spots/105MNC.
Immunomagnetic cell separation
In some experiments CD4+ T cells, CD8+ T cells or CD16+/CD56+ T cells were depleted from PBMC
by two rounds of immunomagnetic separation using a MS+ selection column according to the manu-
facturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Thereafter, cells were washed
and resuspended in complete cell culture medium. Purity of the unseparated PBMC and the separat-
ed fractions were determined by flow cytometry. Phenotype analysis of unseparated PBMC revealed a
population of cells containing 30-38% CD4+ cells and 15–19% CD8+ cells. The CD8-depleted cell
fraction contained < 1% CD8+ T cells and 42–50% CD4+ T cells, while the CD4-depleted cell frac-
tion contained < 2% CD4+ T cells and 18–30% CD8+ T cells. The sample depleted of CD8+ and
CD16/CD56+ cells in an additional experiment contained <1% CD8+ T cells, <2% CD16+/CD56+
cells and 60% CD4+ cells. Monocytes (CD4 low, CD14+), required for antigen presentation, were still
present within the various T cell depleted cell subpopulations.
Blocking studies
Purified anti- HLA-DR (clone B8.12.2, Immunotec, Marseille, France) and mouse anti-human HLA-
ABC mAB (clone W6/32, Serotec, Kidlington, Oxford, GB) were used for the blocking studies.
Antibodies were used at final concentrations of 1 and 10 mg/ml and added to the cell cultures 30 min-
utes prior to the addition of TT. The antibody W6/32 was dialyzed before use to make it suitable for
cell culture.
Results
Frequency of TT-specific T lymphocytes in healthy immunized donors
The frequency of TT-specific, IFN-g secreting T cells present in 22 healthy blood
donors was established using the Elispot assay (see Fig. 1). All donors had previously
obtained active TT immunization within the last one to ten years. Results showed a
broad range in the total number of TT-specific T cells present in the different donors.
However, we detected the presence of TT-specific IFN-g releasing cells in each of the 22
donors (Fig. 1). The number of TT-specific T cells present did not clearly correlate with
the time interval since the last TT immunization.
In 7 donors, the frequency of IFN-g releasing TT-specific T cells could be moni-
tored over several months. A reproducible and fairly constant number of TT-specific
T cells in the different donors (Fig. 2) could be observed. However, one donor sho-
wed a transient increase of TT-reactive IFN-g secreting T cells. This specific blood
sample was taken when the donor suffered an episode of flu-like illness with fever and
chills. After recovery, the number of TT-specific T cells present in this donor decrea-
sed to baseline.
Kinetics of TT-specific T cell responses in donors after TT vaccination
Three healthy volunteers received TT booster immunization i.m.. The frequency of TT-
specific T cells present was determined prior to and following immunization. A signifi-
cant increase in the number of TT-specific IFN-g secreting T cells was observed in all

Elispot for TT-specific T cells · 285
Figure 1. Baseline frequency of TT-specific cells. The number of cells secreting IFN-g in response to
TT in the Elispot assay is shown. PBMC from healthy donors, immunized with TT 1–10 years ago,
were cultured in the presence of 3 mg/ml TT for 20 hours. Results are shown as the mean number of
spots/1¥105 PMBC in 22 healthy donors. The numbers of spots from control wells without TT are
subtracted.
Figure 2. The number of TT-specific cells in seven donors repetitively determined over time is shown.
Blood was taken at different time points as indicated. One donor showed an increase in IFN-g secret-
ing cells during a flu-like infection (*).
three donors. The T cell population reached maximum at week 2 after vaccination in
two donors (no earlier time points were taken) and at week 4 in one donor (Fig. 3) and
demonstrated a slow decline over a period of several months.
TT-specific IFN-g secreting T cells are CD4+ helper cells
Two sets of experiments were performed to prove that TT-specific responses are induced
by HLA class II-restricted CD4+ T helper cells. Depletion of either CD4+- or CD8+
T cells demonstrated that IFN-g secreting TT-specific T cells reside within the CD4+
compartment (Fig. 4a). Almost no IFN-g secreting cells were found within the CD4+
depleted population whereas the number of cells present in CD8-depleted PBMC was
slightly higher than those present in the unseparated PBMC. In an additional experi-

286 · S. MAYER et al.
Figure 3. Increase in the frequency of TT-reactive cells post-immunization. TT-specific IFN-g secret-
ing cells of three volunteers were measured by the Elispot assay before (week 0) and several weeks after
booster-immunization with 75 I. U. TT injected i.m. Results are shown as the mean number of spots/
105 PBMC SD of triplicate wells.
ment, CD16+/CD56+ cells were depleted together with the CD8+ cells. Here we ob-
served 15 spots/well in unseparated PBMC, 3 spots/well in CD4+ depleted sample, 23
spots/well in the CD8+ depleted sample and 12 spots/well in the CD8+/CD16+/CD56+
depleted cells.
The specific release of IFN-g induced by TT-reactive T cells could also be inhibited
by increasing concentrations of an anti-HLA-DR mAb (B8.12.2). This antibody is
known to block 80% of the proliferative response in a mixed lymphocyte culture (15).
In control experiments, an anti-HLA-ABC mAb (W6/32) was used. This mAb had pre-
viously been shown to block the response of HLA class I restricted CD8+ influenza-spe-
cific T cells by 80% in a similar Elispot assay (11). However, W6/32 did not show any
inhibition of IFN-g secretion by TT-specific T cells (Fig. 4b).
Discussion
Using the Elispot assay we could quantify the T cell numbers against a typical T helper
cell antigen. TT-specific T cells could be detected in each of the 22 TT preimmunized
healthy blood donors. The frequency of TT-specific T cells measured by Elispot assay
was in the range of 1:3000–1:30000. Data obtained by others using limiting dilution
analysis of IL-2 secreting cells (up to 1/1500) are in accordance with these numbers (4).
Monitoring the frequency of TT-specific T cells for almost 2 years revealed that
their numbers remain remarkably constant. To our knowledge, such an analysis of the
frequency of TT-specific T cells has not been published before. Only in one donor
there was a strong, transient increase in the number of TT-specific IFN-g secreting
cells during a flu-like illness. This increase may be due to unspecific in vivo stimula-
tion and recruitment of TT-specific T cells for IFN-g secretion, mediated by macro-
phage cytokines such as TNF-a. Recruitment of NK cells after initial activation of T
helper cells is also possible. Crossreactivity with an infection-associated epitope and

Elispot for TT-specific T cells · 287
(A)
(B)
Figure 4. TT-reactive cells reside in the HLA-DR restricted CD4+ T helper cell compartment. (A) The
frequency of IFN-g secreting cells in response to TT was analyzed either in PBMC or in CD4- or CD8-
depleted cell fractions. Depletion of CD4+ or CD8+ cells was performed using magnetic beads as descri-
bed in “Materials and Methods”, with contaminating cells thereafter being < 2% of the population.
Mean results of three independent experiments are shown. (B) PBMC were cultured for 20 hours in
the presence of 3 mg/ml TT and 1 mg/ml or 10 mg/ml of either anti-HLA-DR mAB (B8.12.2), anti
HLA-ABC mAB (W6/32) or without antibody (control).
consecutive amplification of TT-specific T cells may be another explanation (14, 15).
The possibility that the obsereved IFN-g secreting cells are not truely TT-specific
rather are reacting to a contaminant (some bacterial product) present in the TT pep-
aration used for the Elispot assay is less likely, since the purity of the TT preparation
is high according to the information provided by the manufacturer. A nonspecific
increase of IFN-g production by T cells in vitro also does not seem likely since there
was no significant increase in background IFN-g secretion in the absence of antigen
(data not shown).
All three donors receiving TT booster-immunizations showed a significant increase in
the number of specific IFN-g secreting T cells. After several weeks, a slow decline in TT-
specific T cell frequencies could be observed, confirming the results of previous studies
which analyzed shorter time periods and used different tools of analysis (16).

288 · S. MAYER et al.
In previous reports, the Elispot assay has been established and been mainly used to
assess the number of HLA class I antigen-restricted CD8+ cytotoxic T cells (12, 17).
However, the human TT-specific T cell response is mostly confined to HLA class II
restricted CD4+ T helper cells (10, 18), which is in accordance with our data shown
above. In vitro data from KOZBOR et al. (3) suggest the additional existence of HLA-
DR4 restricted TT-specific CD8+ T cell clones. However, in our experiments there was
no evidence for a significant contribution of TT-specific CD8+ T cells to the total num-
ber of reactive cells since depletion of CD8+ T cells led rather to an increase in the num-
ber of IFN-g secreting T cells. There was a slight decrease in the number of IFN-g
secreting cells after the depletion of CD16+/CD56+ cells in addition to CD8+ cells. This
may either be due to positive feedback loops involving NK cells or by negative depletion
of preactivated CD56+ T cells. However, the strong residual activity within the
CD8+/CD16+/CD56+ depleted population and the almost complete abrogation of this
activity after CD4+ depletion corroborates the central role of CD4+ T lymphocytes for
IFN-g production in our system.
Although the unexplained “booster” of TT-reactivity during a common cold of one
donor leaves us with some questions about the specificity of the results in a situation of
general immune activation, the stable quantities of IFN-g secreting cells over time meas-
ured in the other donors and the specific increase after TT vaccination makes the results
plausible from a biological point of view. In conclusion, our data show that the Elispot
assay can be reliably used for the determination and monitoring of TT-specific T help-
er cells over time and may also be suitable for the quantification of T helper cells specif-
ic for other antigens. Furthermore, since most people are vaccinated against tetanus,
quantitation of TT specific T helper cells by Elispot can be used as a positive control for
the monitoring of specific T cell responses of patients in clinical protocols.
Acknowledgements
This work was supported by a grant of the University of Regensburg (ReForM). We would like to
thank all volunteers who kindly and frequently provided blood samples and K. MONDAL for critical-
ly reading the manuscript.
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Dr. S. MAYER, Department of Hematology and Oncology, University Hospital of Regensburg, F. J.
Strauss Allee 11, D-93042 Regensburg, Germany, Phone (49) 941 944 5501, Fax (49) 941 944 5502,
E-mail: stephanie.mayer@klinik.uni-regensburg.de