A critical role of T cell antigen receptor-transduced
MHC class I-restricted helper T cells
in tumor protection
Emma C. Morris*†, Aristotle Tsallios*, Gavin M. Bendle*, Shao-an Xue*, and Hans J. Stauss*
Department of Immunology, Imperial College, Du Cane Road, London W12 0NN, United Kingdom
Edited by N. Avrion Mitchison, University College London, London, United Kingdom, and approved April 20, 2005 (received for review January 14, 2005)
Adoptive transfer of antigen-specific CD4ⴙ and CD8ⴙ T cells is one
of the most efficient forms of cancer immunotherapy. However,
the isolation of antigen-specific CD4ⴙ T cells is limited because only
few tumor-associated helper epitopes are identified. Here, we used
T cell antigen receptor gene transfer to target CD4ⴙ T cells against
an MHC class I-presented epitope of a model tumor antigen.
IFN-␥-producing CD4ⴙ T cells were unable to expand in vivo and to
provide help for tumor rejection. In contrast, CD4ⴙ T cells produc-
ing high levels of IL-2 expanded in vivo, provided help for cytotoxic
T lymphocyte-mediated tumor rejection, and developed T cell
memory. The data demonstrate in vivo synergy between T cell
antigen receptor-transduced CD4ⴙ and CD8ⴙ T cells specific for the
same epitope resulting in long-term tumor protection.
T cell antigen receptor gene transfer 兩 tumor immunotherapy
T he infusion of allogeneic T cells into patients with leukemia can
provide long-term leukemia-free survival. Similarly, the adop-
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tive transfer of Epstein–Barr virus-specific donor-derived T cells
has been successfully used to treat Epstein–Barr virus-induced
malignancies after allogeneic stem cell transplantation (1, 2) and
solid organ transplants (3–5). More recently, it was demonstrated
that adoptive T cell transfer into patients conditioned by lymphoa-
blative chemotherapy resulted in strong antitumor effects in mel-
anoma patients (6, 7).
Indirect evidence suggests that the success of the adoptive
immunotherapy treatments described above may be related to the
use of antigen-specific CD4⫹ and CD8⫹ T lymphocytes. For
example, in melanoma the clinical response to infusion of CD8⫹ T
cell clones was much less impressive than the effect of adoptive
transfer of mixed CD4⫹ and CD8⫹ T cells, although a direct
comparison in patients after lymphoablative conditioning has not
yet been performed (6, 8). Administration of IL-2 can improve the
antimelanoma efficacy of transferred CD8⫹ T cells (9), suggesting
an important role of this cytokine that is typically produced by
CD4⫹ T helper cells. Superiority of mixed CD4⫹ and CD8⫹ T cells
was also suggested by adoptive cell transfer experiments in immu-
nocompromised patients at risk of CMV disease. The cell dose of
cloned CD8⫹ T cells used to control CMV was ⬇1,000-fold higher
than the dose of CMV-specific CD8⫹ T cells given together with
CMV-specific CD4⫹ T cells (10, 11).
The generation of tumor antigen-specific CD4⫹ helper T cells is
limited by the paucity of known MHC class II-binding tumor
epitopes and the lack of expression of MHC class II molecules on
most tumor cells. In contrast, a large number of CD8⫹ T cell-
recognized tumor epitopes presented by MHC class I molecules has
been identified, and in many cases cytotoxic T lymphocyte (CTL)
clones against such epitopes were isolated. In this study, we ex-
plored in a murine model system whether the T cell antigen
receptor (TCR) genes of tumor-specific CTL can be transferred to
CD4⫹ T cells to generate MHC class I-restricted, tumor-specific T
helper cells. We show that the TCR specific for an H2-Db-presented
epitope of influenza nucleoprotein (NP) can be used to produce
H2-Db-restricted, NP-specific CD4⫹ T cells. Adoptive transfer of
CD4⫹ T cells producing high levels of IL-2 and low levels of IFN-␥
7934 –7939 兩 PNAS 兩 May 31, 2005 兩 vol. 102 兩 no. 22
provides help for CTL-mediated rejection of NP-expressing EL4
tumors, whereas adoptive transfer of helper T cells producing high
levels of IFN-␥ was ineffective.
Materials and Methods
Mice. C57BL兾6 mice were purchased from Harlan (Harlan UK,
Loughborough, United Kingdom) and maintained in the animal
facility of Imperial College London. All procedures were carried
out according to United Kingdom Home Office Regulations.
Peptides, Cells, Cell Lines, and Transfectants. The influenza virus A
NP-derived peptide (ASNENDAM, pNP366), the control peptides
pMDM100 (YAMIYRNL) derived from the murine double
minute (MDM) 2 protein, and pSV9 (FAPGNYPAL) derived from
the Sendai virus (12), were synthesized by ProImmune (Oxford).
Murine splenocytes derived from C57BL兾6 mice were cultured in
RPMI medium 1640, 10% FCS兾1% penicillin兾1% streptomycin兾2
mM L-glutamine. Murine bone marrow-derived dendritic cells
(DCs) were isolated from C57BL兾6 mice and cultured for 6–7 days
in the presence of granulocyte兾macrophage colony-stimulating
factor. The murine lymphoma EL4 tumor cell line expresses H2-Db
MHC class I molecules but not MHC class II molecules (13). The
stably transfected EL4 NP tumor cells express the influenza virus
A NP and were a kind gift from B. Stockinger (National Institute
for Medical Research, London). Phoenix-Eco (PhEco) adherent
packaging cells (Nolan Laboratory, Stanford University, Stanford,
CA) were transiently transfected with retroviral vectors for the
generation of cell supernatant containing the recombinant retro-
virus required for infection of target cells.
Retroviral Transduction of Murine Splenocytes. The retroviral vector
pMX-TCR␣-IRES-TCR␤ (pMX-F5) was a kind gift from T.
Schumacher (Nederlands Kanker Instituut, Amsterdam). The ␣-
and ␤-chain TCR genes were cloned from the NP-specific CD8⫹
CTL clone F5 (14). The F5 TCR recognizes the influenza virus A
NP (NP366–379) peptide in the context of murine Db MHC class
I. The PhEco packaging cell line was cotransfected by calcium
phosphate precipitation with the pMX-F5 construct and the pCL-
Eco construct. Splenocytes were harvested from C57BL兾6 (H2-Db)
female mice and activated with conconavalin A (2 ␮g兾ml final
concentration) and IL-7 (1 ng兾ml final concentration) for 48–72 h.
After 48 h, the PhEco supernatant was harvested and used to
transduce activated splenocytes by coculture on fibronectin-treated
tissue culture plates in the presence of IL-2 (100 units兾ml). The
murine CD8␣ gene [a kind gift from R. Zamoyska (National
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TCR, T cell antigen receptor; TCR-td, TCR-transduced; mock-td, mock-
transduced; CTL, cytotoxic T lymphocyte; NP, nucleoprotein; pNP, peptide NP; PE, phyco-
erythrin; DC, dendritic cell; PhEco, Phoenix-Eco; MDM, murine double minute.
*Present address: Department of Immunology and Molecular Pathology, Royal Free Hos-
pital, University College London, London NW3 2QG, United Kingdom.
†To whom correspondence should be addressed. E-mail: emma.morris@medsch.ucl.ac.uk.
© 2005 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0500357102
 Institute for Medical Research, London)] was cloned into the
pMP71-pre retroviral vector [received from W. Uckert (Humboldt
University, Berlin) and originally made by C. Baum (Medical
School Hannover, Hannover, Germany)].
Purification of TCR-Transduced (TCR-td) CD4ⴙ Cells and TCR-td CD8ⴙ
Cells. Transduced splenocytes were stained with antimurine CD4,
CD8, NP tetramers, and V␤11 antibodies [antimurine CD4-FITC,
antimurine CD8-allophycocyanin, and antimurine V ␤ 11-
phycoerythrin (PE) antibodies were obtained from Pharmingen
and used as directed]. The PE-labeled NP tetramer (Db兾
ASNENMDAM) was synthesized by Proimmune and was used as
directed. Forty-eight hours after transduction cells were sorted into
CD4⫹ and CD8⫹ populations using PE-labeled anti-CD4 antibod-
ies and anti-PE beads (Miltenyi Biotec, Bergisch Gladbach, Ger-
many). Further enrichment for V␤11-expressing cells was per-
formed 72 h after transduction.
Functional Assays of TCR-td T Cells. For IFN-␥ release assays, 106
DCs were incubated for 30 min in 200 ␮l of assay medium (RPMI
medium 1640 with 5% heat-inactivated FCS) with synthetic pep-
tides. TCR-td CD4⫹ or TCR-td CD8⫹ T cells (5 ⫻ 104) were
incubated with 5 ⫻ 104 peptide loaded DCs (1:1 ratio) in duplicate
or triplicate. After 24 h, the supernatant was harvested and tested
in an IFN-␥ ELISA assay by using anti-IFN-␥ antibodies (Pharm-
ingen). Further IFN-␥ release assays used peptide-loaded EL4 cells
or EL4NP cells as targets.
For proliferation assays, 106 stimulator cells were incubated for
1 h in 200 ␮l of assay medium (as above) with 100 ␮M synthetic
peptide at 37°C. 5 ⫻ 104 TCR-td CD4⫹, or TCR-td CD8⫹ T cells
were incubated with 5 ⫻ 104 peptide-loaded DCs (1:1 ratio) or
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tumor cells in duplicate or triplicate. After 6, 24, or 48 h, 0.5 ␮Ci
(1 Ci ⫽ 37 GBq) [3H]thymidine was added to each well. The cells
were harvested by using a 96-well plate harvester (Amersham
Pharmacia), and thymidine incorporation was measured by using a
gamma counter (Wallace, Milton Keynes, United Kingdom).
IL-2 secretion of TCR-td T cells was measured by using IL-2-
dependent CTLL cells. The stimulation phase of the IL-2 assay was
performed exactly as the IFN-␥ and proliferation assays, and after
24, 48, or 72 h, the supernatant was harvested. CTLL cells (5 ⫻ 103)
Morris et al.
were added to 50 ␮l of harvested supernatant and incubated for 24 h
before [3H]thymidine pulsing and counting as above.
Adoptive Immunotherapy. C57BL兾6 mice (Thy1.2) were condi-
tioned with 600 rad on day ⫺2. Twelve hours later (day ⫺1), the
mice were injected s.c. with a tumorigenic dose of EL4 NP cells (3 ⫻
106 cells in 200 ␮l of PBS). After a further 24 h, mice were injected
i.v. with Thy1.1⫹ TCR-td T cells, Thy1.1⫹ Mock-transduced (mock-
td) T cells, or PBS alone (day 0). Mice were inspected daily and
killed when tumor burden exceeded 1 cm2 or if tumor ulceration
occurred regardless of tumor size. Representative surviving tumor-
free mice, were rechallenged with tumor cells on day ⫹90. These
mice were injected in the right lower leg with 1 ⫻ 106 irradiated EL4
NP cells. After 5 days, mice were killed, and right and left inguinal
and popliteal lymph nodes were stained with anti-Thy1.1 FITC,
anti-CD4-allophycocyanin, and anti-V␤11 PE antibodies before
analysis by flow cytometry.
Results
Generation of TCR-td CD4ⴙ, CD4ⴙ8ⴙ, and CD8ⴙ T Cell Populations.
Retroviral transduction of C57BL兾6 murine splenocytes with the
H2-Db-restricted, NP-specific F5 TCR resulted in successful intro-
duction and surface expression of the TCR into CD8⫹ T cells and
CD4⫹ T cells. Typically, after transduction, 6–12% of viable CD3⫹
IMMUNOLOGY
T cells were V␤11⫹ CD4⫹, and 10–15% were V␤11⫹ CD8⫹, as
determined by flow cytometry (Fig. 1 A and B). In each experiment,
mock-td T cells were used to determine the percentage of CD4⫹
and CD8⫹ T cells expressing endogenous V␤11 (Fig. 1 D and E).
Tetramer analysis revealed that the F5 TCR-td splenocytes bound
the NP (366–374) H2-Db tetramers, whereas the mock-td spleno-
cytes did not (Fig. 1 C and F). Typically, only 40–50% of transduced
T cells expressing the introduced V␤11 chain were able to bind NP
tetramers (Fig. 1, compare B, E, and C). TCR-td bulk T cells were
sorted 48 h after transduction into CD4⫹ and CD8⫹ cells by using
PE-labeled anti-CD4 antibody staining and anti-PE magnetic
beads. After 24 h, the sorted populations were further enriched for
V␤11⫹ cells, again by using magnetic bead selection. The TCR-td
CD4⫹ T cells used in all subsequent experiments contained ⬎99%
CD4⫹ T cells, always with ⬍1% contaminating CD8⫹ T cells (Fig.
1G). After enrichment, ⬎60% of the CD4⫹ T cells expressed the
Fig. 1. Retroviral gene transfer of TCR
chains, purification of TCR-expressing CD4⫹
and CD8⫹ T cells, and retroviral gene trans-
fer of CD8␣. (A–F) Flow cytometric analysis
of viable CD3⫹ murine splenocytes 2 days
after retroviral transfer with F5 TCR␣ and
TCR␤ genes (A–C) or mock transduction (D–
F). Cells were stained with PE-anti-V␤11,
PE-labeled NP tetramer, FITC-anti-CD4, or
allophycocyanin-anti-CD8. (G and H) Flow
cytometric analysis of purified CD4⫹ and
CD8⫹ T cells. Forty-eight hours after trans-
duction, TCR-td bulk T cells were sorted into
CD4⫹ and CD8⫹ T cells by using magnetic
beads, followed by enrichment for V␤11⫹
cells 1 day later. Cells were stained with
PE-anti-V␤11, FITC-anti-CD4, and兾or allo-
phycocyanin-anti-CD8, and quadrants
were set based on anti-CD4 and anti-CD8
stained mock-td cells. (I and J) Flow cyto-
metric analysis of purified TCR-td CD4⫹ T
cells (I) and TCR-td CD4⫹8⫹ T cells (J) after
retroviral cotransfer of F5 TCR␣, TCR␤, and
murine CD8␣ genes. After cotransfer of
CD8␣, ⬎95% of CD4⫹ T cells coexpressed
CD8␣ (TCR-td CD4⫹8⫹ T cells).
PNAS 兩 May 31, 2005 兩 vol. 102 兩 no. 22 兩 7935
 V␤11. The TCR-td CD8⫹ T cell population was typically ⱖ95%
pure (Fig. 1H). In some experiments, we introduced the CD8␣ gene
together with the F5 TCR genes and purified CD4⫹ T cells
expressing TCR and CD8. Most CD4⫹ T cells were found to
coexpress CD8, as determined by flow cytometry (Fig. 1J). This
expression pattern allowed us to assess the functional activity of the
NP-specific, class I-restricted F5 TCR in CD4⫹ T cells and in CD4⫹
cells that also expressed CD8, referred to as CD4⫹8⫹ T cells. Each
functional experiment was preceded by flow cytometric analysis to
confirm purity of the TCR-td T cell populations.

In Vitro Functional Activity of TCR-td CD4ⴙ and CD8ⴙ T Cells. TCR-td

bulk T cells secrete IFN-␥ in response to syngeneic bone marrow-
derived DCs coated with NP peptide (pNP) but not in response to
an irrelevant peptide (pMDM-100) or no peptide. Similarly, with
peptide-coated EL4 tumor cells as stimulators, the TCR-td bulk T
cells produced IFN-␥ in response to pNP but not pMDM-100 or no
peptide. Mock-td T cells did not secrete IFN-␥ in response to any
of the above targets. (Fig. 2A). Functional assays were repeated with
purified TCR-td CD4⫹ T cells and TCR-td CD8⫹ T cells. Using
peptide-coated syngeneic DCs as stimulator cells, 10 pM of pNP
peptide was required to induce IFN-␥ secretion by CD8⫹ T cells,
whereas a 10-fold higher peptide concentration (100 pM) was
required to trigger IFN-␥ secretion by the CD4⫹ T cells (Fig. 2B).
Once triggered, TCR-td CD4⫹ T cells secreted amounts of IFN-␥
equivalent to the TCR-Td CD8⫹ T cells when stimulated with DC.
However, the TCR-td CD4⫹ T cells produced little IFN-␥ in
response to peptide-coated EL4 tumor cells (Fig. 2C) or to EL4
cells transfected with NP (data not shown), whereas the purified
TCR-td CD8⫹ T cells mounted an efficient IFN-␥ response to
peptide-coated EL4 cells and EL4-NP cells. We explored whether
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transcostimulation and兾or the CD8 molecule could rescue the

antitumor IFN-␥ response of CD4⫹ T cells. The addition of
syngeneic DCs to the stimulation cultures restored the peptide-
specific IFN-␥ secretion of CD4⫹ T cells to EL4-NP tumor cells
(Fig. 2D). The same effect was observed with allogeneic DCs,
indicating that cross-presentation was not involved, and as few as
one DC per 1,000 tumor cells was sufficient (data not shown). The
introduction of CD8 into TCR-td CD4⫹ T cells abolished the DC
dependence of the IFN-␥ response. Purified TCR-td CD4 ⫹8⫹ T
cells produced IFN-␥ when stimulated with EL4-NP tumor cells in
the absence of DC. (Fig. 2E).
Surprisingly, we found that the ability of T cells to produce IFN-␥
did not correlate with their ability to proliferate after stimulation
with tumor cells. Despite the antitumor IFN-␥ response of TCR-td
CD4 ⫹ 8⫹ T cells, these cells proliferated poorly when stimulated
with peptide presenting tumor cells (Fig. 3A). The opposite pattern
was observed for TCR-td CD4⫹ T cells, which proliferated effi-
ciently after stimulation with tumor cells, although they produced
little IFN-␥ (Figs. 2C and 3A). The TCR-td CD8⫹ T cells efficiently
proliferated and produced IFN-␥ after stimulation with tumor cells.
There was a correlation between the proliferative potential of
helper T cells and their IL-2 production. Stimulation with tumor
cells triggered high levels of IL-2 production in CD4⫹ T cells and
reduced levels in CD4⫹8⫹ T cells (Fig. 3B). However, the CD4⫹8⫹
T cells were not defective in IL-2 production, suggesting that the
poor proliferation may be related to an antiproliferative effect of
IFN-␥ produced at high levels by these cells (15, 16).
TCR-Td CD4ⴙ T Cells Provide in Vivo Help for CD8-Mediated Tumor
Rejection. We performed adoptive immunotherapy experiments in
a syngeneic setting to determine whether CD4⫹ T cells with an
IFN-␥low兾proliferationhigh phenotype or CD4⫹8⫹ T cells with an
IFN-␥high兾proliferationlow phenotype were more efficient in pro-
viding help for tumor rejection. Two groups of 5 C57BL兾6 mice
received a tumorigenic dose of EL4-NP cells followed by adoptive
transfer of 106 purified TCR-td CD4⫹ or CD4⫹8⫹ helper T cells.
To assess the helper function in vivo, a further group of five mice
Fig. 2. In vitro functional analysis of TCR-td bulk T cells, CD8⫹ T cells, CD4⫹
T cells, and CD4⫹8⫹ T cells. (A) Peptide-specific IFN-␥ secretion of TCR-td bulk
T cells (black bars) in response to peptide-loaded DCs and MHC class-II negative
peptide-loaded EL4 tumor cells. Mock-td bulk T cells (gray bars) did not secrete
IFN-␥ in response to any of the targets. (B) Purified populations of TCR-td CD8⫹
T cells (black bars) and TCR-td CD4⫹ T cells (gray bars) secreted IFN-␥ in
response to peptide-loaded syngeneic DCs. TCR-td CD8⫹ T cells recognized 10
pM peptide, whereas a 10-fold higher concentration of peptide (100 pM) was
required to trigger IFN-␥ secretion by the TCR-td CD4⫹ T cells. (C) Using
peptide-loaded EL4 tumor cells as targets, TCR-td CD8⫹ T cells (black bars)
mounted an efficient IFN-␥ response, whereas the TCR-td CD4⫹ T cells (gray
bars) did not. (D) Transcostimulation by means of the addition of syngeneic
DCs rescued the peptide-specific IFN-␥ response of TCR-td CD4⫹ T cells (gray
bars) to MHC class II negative NP-expressing EL4 tumor cells (EL4 NP) compared
with that observed with TCR-td CD8⫹ T cells (black bars). (E) Coexpression
of murine CD8␣ abolished the DC dependence of the IFN-␥ response. TCR-td
CD4⫹8⫹ T cells (gray bars) and TCR-td CD8⫹ T cells (black bars) respond to
antigen presented by DCs and EL4 NP.
also received a small dose of 104 TCR-td CD8⫹ T cells, which was
insufficient to cause tumor rejection in the absence of help. Tumor
protection by large numbers of CTL (i.e., 106 TCR-td CD8⫹ T cells)
was T helper cell-independent (data not shown).
Fig. 4A shows poor survival of EL4-NP tumor-challenged mice
that were treated with PBS (n ⫽ 5) or mock-td CD4⫹ T cells (n ⫽
5). A small improvement was seen in the group of mice (n ⫽ 5) that
was treated with 106 TCR-td CD4⫹8⫹ helper T cells together with
104 TCR-td CD8⫹ T cell. In contrast, substantial protection was
seen in the five mice treated with 106 TCR-td CD4⫹ helper T cells
in combination with 104 TCR-td CD8⫹ T cells. To explore whether
the NP specificity of the CD4⫹ T cells was required, tumor-
challenged mice (n ⫽ 5) were treated in a separate experiment with
106 mock-td CD4⫹ T cells combined with 104 TCR-td CD8⫹ T cells.
This resulted in progressive tumor growth in all mice (Fig. 4B).
Similarly, treatment of five more mice with TCR-td CD4⫹ T cells
in the absence of CD8⫹ T cells was ineffective, indicating that
protection required the combination of antigen-specific CD4⫹ T

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Fig. 3. In vitro proliferative potential and IL-2 production of TCR-td CD4⫹ T
cells and TCR-td CD4⫹8⫹ T cells. (A) TCR-td CD4⫹8⫹ T cells proliferated poorly
in response to peptide-presenting tumor cells (white bars) compared with
TCR-td CD4⫹ T cells (gray bars) and TCR-td CD8⫹ T cells (black bars). The
proliferative potential of the TCR-td T cell populations correlated well with
IL-2 production. (B) TCR-td CD4⫹8⫹ T cells (white bars) secreted reduced levels
of IL-2 in response to EL4 NP tumor cells as compared with TCR-td CD4⫹ T cells
(gray bars). TCR-td CD8⫹ T cells (black bars) did not secrete IL-2 in response to
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any of the targets. Proliferation of the IL-2-dependent cell line CTLL was used
to measure the IL-2 content in the supernatant taken from the stimulated T
cells.
cells together with small numbers of TCR-td CD8⫹ T cells. In-
creasing the dose of TCR-td CD8⫹ T cells from 104 to 2 ⫻ 104
improved the level of tumor protection (Fig. 4, compare A with B).
TCR-td CD4ⴙ T Cells Develop Long-Term T Cell Memory. Spleens of all
mice used in the tumor challenge experiments in Fig. 4A were
analyzed by FACS to determine the number of adoptively trans-
ferred Thy1.1⫹兾V␤11⫹ TCR-td T cells at the time of antigen
exposure in tumor-bearing mice and at later time points to analyze
T cell persistence and recall memory responses in tumor-free mice.
No Thy1.1⫹兾V␤11⫹ T cells were identified in the control PBS-
treated mice (Fig. 5A). In tumor-bearing mice treated with mock-td
CD4⫹ T cells, between 0.5% and 1.7% of the gated V␤11⫹ cells
were the transferred T cells (Fig. 5B). A similar proportion of
transferred T cells, 0.5–3.3% of the gated V␤11⫹ cells, was seen in
three tumor-bearing mice treated with TCR-td CD4 ⫹ 8⫹ T cells,
suggesting that there was no antigen-driven expansion of these cells
compared with mock-td CD4⫹ T cells (Fig. 5C). Analysis of a
tumor-bearing mouse treated with TCR-td CD4⫹ T cells showed a
large expansion of transferred CD4⫹ cells (34% of gated V␤11⫹ T
cells) and an expansion of the cotransferred TCR-td CD8⫹ T cells
(13.6% of V␤11⫹ cells) (Fig. 5D).
Analysis of tumor-free mice 35 and 90 days after adoptive T cell
transfer showed persistence and memory development of TCR-td
CD4⫹ T cells. On day 35, the transferred CD4⫹ T cells were present
at a higher frequency than CD4⫹8⫹ cells (Fig. 6 A and B), and
CD4⫹ but not CD4⫹8⫹ T cells responded to tumor rechallenge at
day 90 (Fig. 6 C–H). The memory response of the TCR-td CD4⫹
T cells at day 90 was associated with a recall response of the TCR-td
CD8⫹ T cells (Fig. 6 F and H).
Discussion
Several groups have used retroviral TCR gene transfer to produce
antigen-specific human and murine T cells (17–27). This strategy
Morris et al.
IMMUNOLOGY
Fig. 4. TCR-td CD4⫹ T cells provide in vivo help for CD8 T cell-mediated tumor
rejection. C57BL兾6 mice received nonmyeloablative radiation (600 rad) and
then 12 h later were injected s.c. with 3 ⫻ 106 EL4 NP tumor cells. (A) A further
24 h later, mice received 106 TCR-td CD4⫹ T cells with 104 TCR-td CD8⫹ T cells
(open triangles), 106 TCR-td CD4⫹8⫹ T cells with 104 TCR-td CD8⫹ T cells
(crosses), 106 mock-td bulk T cells (open squares), or PBS (diamonds). (B) In a
separate tumor protection experiment, mice were conditioned and tumor-
challenged as above, followed by transfer of 106 mock-td CD4⫹ T cells with 104
TCR-td CD8⫹ T cells (open squares), 106 TCR-td CD4⫹ T cells with anti-CD8-
blocking antibody (filled diamonds), or 106 TCR-td CD4⫹ T cells with 2 ⫻ 104
TCR-td CD8⫹ T cells (open triangles).
provides for the production of tumor-specific T cell populations for
adoptive immunotherapy without the need for in vivo immunization
and in vitro T cell expansion. In a previous study (20) and in this
report T cells were transduced once on day 2–3 after polyclonal
activation and subsequently used on days 5–7 for adoptive transfer
without any further in vitro expansion. TCR gene transfer also
provides a strategy to generate antigen-specific helper T cells for
adoptive cancer immunotherapy. This strategy can be achieved by
transfer of MHC class II-restricted TCRs into CD4⫹ cells (19, 27)
or, as shown recently, by transfer of MHC class I-restricted TCRs
into CD4⫹ T cells (28). One recent publication showed that TCRs
isolated from CD8-independent CTL can be functionally active in
CD4⫹ T cells, and another study showed that the cotransfer of
CD8␣ and a TCR兾CD3␨ hybrid construct can be used to produce
HLA class I-restricted CD4⫹ T cells (29, 30). To date, a possible in
vivo role of CD4⫹ T cells transduced with a class I-restricted TCR
in tumor protection has not yet been explored.
Here, we have generated MHC class I-restricted helper T cells
that respond to antigen-presentation by MHC class II-negative
EL4-NP tumor cells. TCR-td CD4⫹ T cells proliferated and pro-
duced high levels of IL-2 but little IFN-␥, whereas CD4⫹ T cells
transduced with the same TCR and CD8␣ produced high levels of
IFN-␥ and reduced levels of IL-2 and proliferated poorly when
stimulated with tumor cells. The mechanism by which CD8␣
expression modifies the T cell response is currently unknown.
Altered cytokine production might be due to the ability of CD8 to
enhance the avidity of TCR兾MHC peptide interaction. For exam-
ple, a recent study demonstrated that high avidity TCR兾MHC
peptide interactions selectively impaired IL-2 production, which is
similar to our observation with CD8-expressing helper T cells (31).
Alternatively, the CD8 molecule may alter signal transduction in
PNAS 兩 May 31, 2005 兩 vol. 102 兩 no. 22 兩 7937

Fig. 5. Detection and in vivo persistence of TCR-td T cells in tumor-bearing
mice. Shown is the analysis of splenocytes prepared from mice in Fig. 4A at the
time they were killed because of tumor burden. Adoptively transferred T cells
were identified by triple staining with antibodies specific for Thy1.1, V␤11,
and CD4. Representative examples are shown for each group of mice, and all
FACS plots display viable lymphocytes gated on V␤11⫹ T cells. (A) No Thy1.1⫹
V␤11⫹ T cells were identified in four tumor-bearing PBS-treated control mice.
(B) In four mice treated with mock-td CD4⫹ T cells, between 0.5% and 1.7% of
the gated V␤11⫹ cells were the CD4⫹ Thy1.1⫹ transferred T cells. (C) Similar
numbers of TCR-td CD4⫹8⫹ cells were detected in three mice (0.5–3.3%)
suggesting a lack of antigen-driven expansion of the TCR-td CD4⫹8⫹ as
compared with the Mock-td CD4⫹ T cells. (D) Analysis of the spleen of a
tumor-bearing animal that received TCR-td CD4⫹ T cells showed a large
Downloaded from https://www.pnas.org by 143.208.36.172 on November 12, 2022 from IP address 143.208.36.172.
transduced CD4⫹ T cells reflect a feature of CD8␣兾␣ homodimers.
For example, the expression of CD8␣兾␣ homodimers in TCR-td
CD4⫹ cells may result in redirected homing to the gut, as is seen
with CD8␣兾␣ positive intraepithelial lyphocytes, which might result
in low numbers of T cells in lymphoid tissues. In fact, gene transfer
into CD4⫹ T cells will provide an interesting model to dissect
functional activities of CD8␣兾␣ homodimers and CD8␣兾␤ het-
erodimers in mature T cells.
We show that IL-2high兾proliferationhigh CD4⫹ T cells effectively
provide in vivo help for tumor rejection, whereas IFN-␥high兾
proliferationlow CD4⫹ T cells are unable to do so despite having the
same specificity. A similar in vivo protective effect has been
described for IL-2high兾proliferationhigh CD4⫹ T cells in HIV-
infected patients. Large numbers of these CD4⫹ T cells were
observed in patients controlling virus load, whereas the presence of
IFN-␥high兾proliferationlow CD4⫹ T cells was associated with un-
controlled virus replication (34–37).
In addition to the efficient help for tumor rejection, we also
showed that IL-2high兾proliferationhigh CD4⫹ T cells persist and
readily respond to tumor cell rechallenge 90 days after adoptive
transfer. This finding is in line with a recent report showing that
murine CD4⫹ T cells producing little IFN-␥ were able to persist in
vivo and develop into long-term memory cells, whereas CD4⫹ T
cells producing high levels of IFN-␥ survived poorly in vivo (38).
Our experiments suggest that an important mechanism by which
proliferation competent helper T cells contribute to tumor immu-
nity is by triggering the in vivo expansion of antigen-specific CD8⫹
T cells. Given separately, neither antigen-specific helper T cells nor
CD8⫹ T cells were able to mediate protection in tumor-challenged
mice. It is likely that antigen-specific IL-2 production by helper T
cells at the site of tumor growth may enhance local proliferation and

expansion of transferred Thy1.1⫹ CD4⫹ T cells (34.0% of gated V␤11⫹), to-
gether with an expansion of the cotransferred Thy1.1⫹ CD8⫹ T cells (13.6% of
gated V␤11⫹). Staining of lymph node cells of mice in the four treatment
groups showed similar levels of transferred Thy1.1⫹ V␤11⫹ CD4⫹ T cells (data
not shown).
helper T cells because of its ability to recruit LAT, an adaptor
molecule that preferentially associates with CD8 compared with
CD4 (32, 33). It is possible that the observed functional changes of
retention of CD8⫹ T cells, as described previously in a murine
tumor model (39).
In our experiments, TCR-td CD4⫹ and TCR-td CD8⫹ T cells
expanded and persisted at similar frequencies, even when 100-fold
more CD4⫹ T cells were adoptively transferred into the mice.
Although this result suggested a competitive advantage for CD8⫹
T cells, it is possible that some cross-regulation may determine the
relative expansion rate of CD4⫹ and CD8⫹ cells. For example, the
adoptive transfer of 106 TCR-td CD4⫹ cells along with 104 or 106
TCR-td CD8⫹ cells (i.e., 100:1 and 1:1 ratios) resulted in similar size

Fig. 6. TCR-td CD4⫹ T cells persist and

develop long-term memory in vivo.
Shown is the analysis of splenocytes and
lymph node cells prepared from tumor-
free mice in Fig. 4 A on day 35 or day 95
after T cell transfer. Adoptively trans-
ferred T cells were identified by triple
staining with antibodies specific for
Thy1.1, V␤11, and CD4, and all FACS
plots display viable lymphocytes gated
on V␤11⫹ T cells. (A and B) A day-35
comparison of splenocytes of mice
treated with TCR-td CD4 ⫹ (A) and
CD4⫹8⫹ (B) cells. Similar frequencies of
transferred Thy1.1⫹ CD4⫹ T cells were
detected in lymph node samples (data
not shown). (C–H) At day 90, three tu-
mor-free mice were challenged s.c. with
1 ⫻ 106 irradiated EL4 NP tumor cells in
the right lower leg. After 5 days, ex vivo
FACS analysis was used to analyze T
cell responses in the tumor-antigen-
exposed right inguinal and popliteal
lymph nodes, by using the nonexposed
left inguinal and popliteal lymph nodes as a baseline control. The TCR-td CD4⫹8⫹ T cells did not respond to tumor rechallenge at day 90 (C and D), whereas
both mice that received TCR-td CD4⫹ cells demonstrated an expansion of TCR-td CD4⫹ and CD8⫹ T cells in the lymph nodes of the tumor antigen exposed
right side (E–H).

7938 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0500357102 Morris et al.

 CD8⫹ T cell populations 5 weeks after T cell transfer. A simple
feedback loop may involve IL-2 that is provided by TCR-td CD4⫹
T cells and that controls the extent of expansion of TCR-td CD8⫹
T cells with the same specificity.
The observation that IFN-␥high helper T cells were unable to
contribute to tumor protection was surprising, considering the
important role of this cytokine in preventing tumor growth by
inhibition of tumor stroma (40). In addition, IFN-␥ can lead to FAS
ligand (CD178) up-regulation in T cells and FAS (CD95) up-
regulation in target cells, thus enhancing CTL killing of target cells
by means of the FAS pathway (41, 42). However, our data do not
rule out a role of IFN-␥ in tumor immunity. Rather, the data
demonstrate that CD4⫹ T cells, which are unable to expand and
persist in vivo, cannot exert antitumor effects even when they
produce high levels of IFN-␥. It is possible that the tumor control,
which is mediated by CD8⫹ T cells in our experiments, involves
CD8⫹ T cell-derived IFN-␥.
The ability to produce CD4⫹ and CD8⫹ T cells specific for the
same tumor antigen provides a powerful model to dissect the
mechanisms of in vivo interaction between these T cell populations
and provides an opportunity to design strategies to optimally exploit
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PNAS 兩 May 31, 2005 兩 vol. 102 兩 no. 22 兩 7939