The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hombach, A.
Right arrow Articles by Abken, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hombach, A.
Right arrow Articles by Abken, H.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Cancer
The Journal of Immunology, 2006, 177: 5668-5675.
Copyright © 2006 by The American Association of Immunologists, Inc.

Human CD4+ T Cells Lyse Target Cells via Granzyme/Perforin upon Circumvention of MHC Class II Restriction by an Antibody-Like Immunoreceptor1

Andreas Hombach, Heike Köhler, Gunter Rappl and Hinrich Abken2

Klinik I für Innere Medizin, Labor Tumorgenetik, and Zentrum für Molekulare Medizin Köln, Universität zu Köln, Köln, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune elimination of tumor cells requires the close cooperation between CD8+ CTL and CD4+ Th cells. We circumvent MHC class II-restriction of CD4+ T cells by expression of a recombinant immunoreceptor with an Ab-derived binding domain redirecting specificity. Human CD4+ T cells grafted with an immunoreceptor specific for carcinoembryonic Ag (CEA) are activated to proliferate and secrete cytokines upon binding to CEA+ target cells. Notably, redirected CD4+ T cells mediate cytolysis of CEA+ tumor cells with high efficiencies. Lysis by redirected CD4+ T cells is independent of death receptor signaling via TNF-{alpha} or Fas, but mediated by perforin and granzyme because cytolysis is inhibited by blocking the release of cytotoxic granules, but not by blocking of Fas ligand or TNF-{alpha}. CD4+ T cells redirected by Ab-derived immunoreceptors in a MHC class II-independent fashion substantially extend the power of an adoptive, Ag-triggered immunotherapy not only by CD4+ T cell help, but also by cytolytic effector functions. Because cytolysis is predominantly mediated via granzyme/perforin, target cells that are resistant to death receptor signaling become sensitive to a cytolytic attack by engineered CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A major hurdle in the immunotherapy of malignant diseases by adoptively transferred CD8+ CTL is their dependency on CD4+ Th cells, which require Ag-specific activation to provide help in the eradiation of tumor cells (1, 2). By release of cytokines and interaction with professional APCs, CD4+ T cells recruit innate immune and nonimmune effector cells to exhibit their antitumor activity (3, 4). However, the majority of tumor cells lack MHC class II (MHC-II)3 expression, thereby preventing direct activation of CD4+ T cells. For use in adoptive immunotherapy of malignant diseases, a MHC-II-independent, but Ag-specific, activation of CD4+ T cells is desirable.

T cells can be genetically equipped with predefined specificity by expression of a recombinant immunoreceptor molecule with dual properties, i.e., specific binding and triggering cellular activation. To combine the advantages of MHC-independent target recognition with the induction of the plethora of T cell effector functions, we make use of an immunoreceptor format that harbors both an Ab-like binding domain for Ag targeting and a TCR-like intracellular domain for T cell activation (5). The extracellular binding domain consists of a single-chain Ab fragment (scFv) with MHC-independent binding specificity. The signaling domain is derived from the cytoplasmic part of a membrane bound receptor molecule, e.g., the Fc{epsilon}RI receptor {gamma}-chain or the CD3 {zeta}-chain, capable to drive T cell activation. T cells equipped with such type of immunoreceptors induce an Ag-specific cellular immune response in vitro and in vivo, indicated by induction of cytokine secretion, T cell proliferation, and target cell lysis (6, 7, 8). By circumventing their restriction to MHC-I and MHC-II, respectively, both CD8+ and CD4+ T cells can be activated upon triggering with an Ab-type immunoreceptor. Because CD4+ T cells provide help to boost the efficiency of a CD8+ T cell-mediated cytolytic attack, these cells are of high interest for immunotherapeutic strategies using engineered T cells. Previous work of our and other groups indicated that receptor-grafted CD4+ T cells drive directly specific cytolysis of target cells (9, 10). However, the mechanism of CD4+ T cell-mediated cytolysis is still debated controversially.

T lymphocytes use predominantly two pathways in executing cytotoxicity, i.e., exocytosis of perforin and granzyme granules and death receptor signaling via Fas/Fas-ligand (Fas-L) or TNF/TNF-R. Investigations using Fas mutant lpr (11), Fas-L mutant gld (12), perforin-deficient (13), and perforin/Fas-L double knockout (14) mice suggest that MHC-II-restricted cytolysis by murine CD4+ T cells is predominantly mediated by the death receptor system (15, 16), whereas MHC-I-restricted cytolysis by CD8+ CTLs relies on perforin and granzymes. Accordingly, murine CD8+ T cells engrafted with a recombinant receptor lyse Fas-resistant target cells, whereas CD4+ T lymphocytes do not (17). However, human CD4+ T cells seem to execute cytolysis by different mechanisms than do murine CD4+ T cells. Alloantigen-specific, human CD4+ clones, as well as bulk CD4+ T cell cultures, lyse their target cells predominantly by granule exocytosis and not by the Fas/Fas-L system (18). In contrast, CD4+ cells that were engrafted with a recombinant, MHC-I-restricted TCR were reported to lyse exclusively those target cells that are susceptible for death receptor signaling (10).

To resolve this issue, we redirected human CD4+ T cells from the peripheral blood in a MHC-II-independent fashion toward predefined Ag by expression of a recombinant, Ab-like immunoreceptor. Dissecting the mechanism of cytolysis by a panel of blocking experiments revealed that human CD4+ T cells drive their cytolytic activities predominantly via the perforin and granzyme pathway and not via death receptor signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

The anti-carcinoembryonic Ag (CEA) mAb BW431/26 and the anti-id mAb BW2064/36 with specificity for the anti-CEA mAb BW431/26 were described earlier (19). The hybridoma cell line OKT3, which produces the anti-CD3 mAb OKT3, was obtained from American Type Culture Collection (ATCC; CRL 8001). mAbs were affinity purified from murine ascites or hybridoma supernatants using an agarose-immobilized goat anti-mouse IgG1 (Sigma-Aldrich) or a Sepharose (Amersham Pharmacia) immobilized goat anti-mouse IgG2a Ab (Southern Biotechnology Associates). The PE-conjugated anti-CD4 mAb and the PE- and FITC-conjugated anti-CD8 mAbs, respectively, were purchased from DakoCytomation. The PE-conjugated F(ab')2 anti-human IgG1 Ab was purchased from Southern Biotechnology Associates. The anti-human Fas-L mAb NOK-1, which blocks signaling via Fas-L, the PE-conjugated anti-granzyme A mAb, and unlabeled and biotin-labeled matched-pair Abs for detection of human IFN-{gamma} and TNF-{alpha}, respectively, were purchased from BD Biosciences. The neutralizing anti-TN-{alpha} mAb (clone 6401) was purchased from R&D Systems. The PE-conjugated anti-granzyme B mAb was purchased from Serotec. The human Fas-L fused to the extracellular domain of the murine CD8 and the PE-conjugated anti-Perforin mAb were purchased from Ancell.

Tumor cell lines

293T cells are human embryonic kidney cells that express the SV40 large T Ag (20). SW948 (ATCC CLL 237) and LS174T (ATCC CCL 188) are CEA-expressing colon carcinoma cell lines, A375 (ATCC CRL 1619) and Colo320 (ATCC CCL 220.1) are CEA-negative cell lines, Jurkat (ATCC TIB 152) is a Fas, and L929 (ATCC CRL-2148) is a TNF-{alpha}-susceptible cell line. The cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) FCS (all obtained from Invitrogen Life Technologies).

MACS

Peripheral blood lymphocytes from healthy donors were isolated by density centrifugation, and monocytes were depleted by plastic adherence. Nonadherent lymphocytes were washed with cold PBS containing 0.5% (w/v) BSA, 1% (v/v) FCS, and 2 mM EDTA, and CD3+, CD4+, or CD8+ T cells were isolated by MACS using the CD3, CD4, and CD8 T cell isolation kit from Miltenyi Biotec, respectively, as was recommended by the manufacturer. The number of isolated CD3+ and CD4+ T cells, respectively, was determined by flow cytometry as described below. The number of contaminating non-T cells and CD8+ cells in the isolated populations was always <2%. MACS-isolated T cells were washed and cultured for 48 h in RPMI 1640 medium supplemented with 10% (v/v) FCS in the presence of IL-2 (400 U/ml) (Endogen) and OKT3 mAb (100 ng/ml). For longer culture periods, the cells were maintained in the presence of 400 U/ml IL-2

Generation of chimeric receptors and transduction of T cells

The generation of the retroviral expression cassettes for the CEA-specific immunoreceptors BW431/26-scFv-Fc-{gamma} and BW431/26-scFv-Fc-{zeta} were recently described in detail (20, 21, 22, 23). Retroviral vector DNA was cotransfected with the retroviral helper plasmid DNAs pHIT60 and pCOLT (20) (each 1 µg DNA/1 x 105 cells) into 293T cells using Polyfect reagent (Qiagen). pHIT60 encodes the MuLV gag and pol genes, and pCOLT encodes the GALV-envelope gene under control of the CMV promoter/enhancer. MACS-isolated T cells from the peripheral blood were activated by the addition of IL-2 and OKT3 mAb as described above, washed, resuspended in RPMI 1640 medium with IL-2 (400 U/ml), and cocultivated for 48 h with transiently transfected 293T cells. T cells were harvested, and receptor expression was monitored by flow cytometric analysis.

Immunofluorescence analysis

Receptor-grafted T cells were identified by two-color immunofluorescence using PE-conjugated anti-human IgG1 (0.1 µg/ml) and FITC-conjugated anti-CD3 (5 µg/ml) Abs. Labeled cells were analyzed on a FACS Canto cytofluorometer equipped with the FACS-Diva research software (BD Biosciences). To identify T cells with recombinant receptor expression, we set markers with >99% of nontransduced T cells beyond. Expression of cytolytic effector molecules was monitored as follows: CD3+ isolated T cells were stained with an anti-CD4-FITC mAb and subsequently fixed and permeabilized using the cytofix/cytoperm kit (BD Biosciences) according to the manufacturer’s recommendations. Permeabilized cells were incubated with PE-conjugated anti-perforin, granzyme A and granzyme B Abs, respectively, and washed and analyzed as described above. Expression of membrane-bound TNF-{alpha} on CD4+ and CD8+ T cells was determined using an anti-TNF-{alpha} mAb (R&D Systems) (10 µg/ml) and a FITC-conjugated F(ab')2 anti-mouse IgG1 Ab (Southern Biotechnology Associates) as follows: Receptor-grafted CD4+ and CD8+ T cells were cultivated for 72 h in microtiter plates that were coated with the anti-idiotypic mAb BW2064 or an isotype-matched control mAb (4 µg/ml each). The cells were removed and analyzed for TNF-{alpha} expression as described above.

Death receptor-mediated apoptosis of target cells

Sensitivity of target cells to Fas-mediated apoptosis was monitored using recombinant Fas-L fused to the extracellular part of murine CD8. Briefly, cells were incubated in 96-well microtiter plates with recombinant Fas-L (2 µg/ml) (Ancell) for 24 h. Specificity of Fas-mediated cell lysis was demonstrated by coincubation with the blocking anti-Fas mAb NOK-1 (10 µg/ml; BD Biosciences). Susceptibility of target cells to TNF-{alpha}-mediated cell death was tested using recombinant TNF-{alpha} (BD Biosciences) and actinomycin D-pretreated cells. Briefly, cells were incubated in 96-well microtiter plates in the presence of increasing amounts of TNF-{alpha} (0.1–10 ng/ml; Immunotools) with or without 1 µg/ml actinomycin D (Sigma-Aldrich). Viability of cells was monitored by a XTT (2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-5((phenyl-amino)carbonyl)-2H-tetrazolium hydroxide)-based colorimetric assay as described below.

Stimulation of receptor-grafted T cells and cytokine ELISAs

Receptor-grafted and nontransduced CD4+ T cells were cocultivated in serial dilutions with tumor cells (5 x 104 per well) for 18–48 h in 96-well round-bottom plates. To block receptor-mediated cellular activation and Fas-induced apoptosis, the assay was also performed in the presence of the anti-idiotypic mAb BW2064/36 and the anti-Fas-L mAb NOK-1 (each 10 µg/ml), respectively. Viability of target cells was recorded as described below, and culture supernatants were analyzed for secretion of IFN-{gamma} and TNF-{alpha} by ELISA. Briefly, IFN-{gamma} and TNF-{alpha} in the supernatant was bound to a solid phase anti-human IFN-{gamma} and anti-human TNF-{alpha} mAb (each 1 µg/ml), respectively, and detected by a biotinylated anti-human IFN-{gamma} and anti-human TNF-{alpha} mAb (each 0.5 µg/ml), respectively. The reaction product was visualized by a peroxidase-streptavidin-conjugate (1/10,000) and ABTS (both purchased from Roche Diagnostics) as substrate. The amount of cytokine was calculated using reference standard curves with known amounts of cytokines.

Cytotoxicity assays

Specific cytotoxicity of receptor-grafted CD4+ T cells against target cells was monitored by an XTT-based colorimetric assay according to Jost et al. (24). Briefly, receptor-grafted and nontransduced T cells (1 x 105 cells per well) were cocultivated with tumor cells in triplicates in round-bottom microtiter plates for 18–48 h. XTT reagent (1 mg/ml) (Cell Proliferation Kit II, Roche Diagnostics) was added to the cells and incubated for 30–90 min at 37°C. Reduction of XTT to formazan by viable tumor cells was monitored colorimetrically at an absorbance wavelength of 450 nm and a reference wavelength of 630 nm. Maximal reduction of XTT was determined as the mean of six wells containing tumor cells only, the background as the mean of six wells containing RPMI 1640 medium, 10% (v/v) FCS. The nonspecific formation of formazan due to the presence of effector cells was determined from triplicate wells containing effector cells in the same number as in the corresponding experimental wells. The viability of tumor cells (%) was calculated as follows: viability (%) = OD(experiment wells – corresponding number of effector cells)/OD(tumor cells without effectors – medium) x 100. To block exocytosis of cytotoxic granules, the assay was performed in the presence of 4 mM EGTA. The viability of target cells was not affected by EGTA during the incubation period of 18 h.

Short-term cytoxicity was measured according to Ref. 25 with modifications. Briefly, SW948 target cells (1 x 107) were labeled with CFSE (Invitrogen Life Technologies) by incubation with CFSE labeling solution (1 µM final concentration) for 5 min on ice as described (26) and washed five times with cold RPMI 1640 medium, 10% (v/v) FCS. Receptor-grafted and nontransduced T cells (1 x 105 cells per well) were cocultivated with CFSE-labeled tumor cells in triplicates in round-bottom microtiter plates for 5 h. Cells were removed, stained with 7-aminoactinomycin D, and the number of CFSE-labeled dead cells was determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We isolated human CD4+ T cells from the peripheral blood by MACS techniques and retrovirally grafted the cells with the recombinant immunoreceptors BW431/26-scFv-Fc-{gamma} and BW431/26-scFv-Fc-{zeta}, respectively. The immunoreceptors harbor the same Ab-derived, extracellular binding domain for MHC-II-independent binding to CEA and intracellularly the Fc{epsilon}RI{gamma} or CD3{zeta} domain for T cell activation. Recombinant immunoreceptors were expressed in CD4+ and CD8+ T cells with similar efficiency, resulting typically in 10–50% of receptor-expressing T cells (data not shown). Upon coincubation with CEA+ LS174T and SW948 tumor cells, receptor-grafted CD4+ T cells lysed the CEA+ tumor cells with high efficiencies, whereas CEA– Colo320 cells were not affected (Fig. 1). CD4+ T cells without CEA-specific immunoreceptor lysed neither CEA+ nor CEA– tumor cells, demonstrating the targeting specificity of receptor-triggered cytolysis by CD4+ T cells. Moreover, the activation of grafted CD4+ T cells resulted in secretion of high amounts of IFN-{gamma}, but no secretion of TNF-{alpha} could be monitored (Fig. 2). We obtained essentially the same results upon redirecting CD4+ T cells with the {gamma}- or {zeta}-chain signaling immunoreceptor.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 1. Specific cytolysis of CEA+ target cells by human CD4+ T cells grafted with a CEA-specific immunoreceptor. CD4+ T cells from the peripheral blood were equipped with the CEA-specific immunoreceptor BW431/26-scFv-Fc-{zeta} and grafted cells (2.5 x 103 – 2 x 104 receptor-expressing T cells per well) and nontransduced T cells in same numbers were cocultivated for 48 h with CEA+ LS174T (A) and SW948 (B) tumor cells and for control with CEA– Colo320 cells (C) (each 2.5 x 104 cells per well) in 96-well tissue culture plates. The viability of tumor cells was determined by the XTT assay as described in Materials and Methods. The assay was done in triplicates.

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 2. CD4+ T cells secrete IFN-{gamma}, but not TNF-{alpha}, upon triggering via the anti-CEA immunoreceptor. CD4+ T cells grafted with the anti-CEA receptor BW431/26-scFv-Fc-{zeta} (2.5 x 103 – 2 x 104 receptor-expressing T cells per well) and nontransduced T cells in same numbers were cocultivated for 48 h with CEA+ SW948 and LS174T cells and for control with CEA– Colo320 tumor cells (each 2.5 x 104 cells per well) in a 96-well tissue culture plate. The supernatants were harvested and analyzed by ELISA for the presence of IFN-{gamma} (A, C, and E) and TNF-{alpha} (B, D, and F), respectively, as described in Materials and Methods. The assay was performed in triplicates.

 
MHC-I-restricted cytolytic activity by CD8+ T cells is executed predominantly via the perforin/granzyme pathway. However, the mechanism of cytolysis by MHC-II-independent CD4+ T cells is so far unresolved. To address this issue, we asked whether CEA+ SW948 and LS174T tumor cells, which are efficiently lysed by receptor-grafted CD4+ T cells, are susceptible for death receptor-induced lysis, particularly via TNF-{alpha} and Fas. In the first panel of assays, we incubated LS174T and SW948 tumor cells with serial dilutions of TNF-{alpha} in the presence or absence of actinomycin D (Fig. 3). Whereas LS174T and SW948 cells are susceptible to TNF-{alpha} induced cell death in the presence of actinomycin D, we could not record any cytolytic effects of TNF-{alpha} in these concentrations in absence of actinomycin D. TNF-{alpha}-susceptible L928 cells served as controls. Because TNF-{alpha} secretion by CD4+ T cells upon immunoreceptor triggering was below the detection limit of the ELISA system, i.e., 100 pg/ml, and because receptor-grafted CD4+ T cells lyse their target cells in the absence of actinomycin D, death signaling via soluble TNF-{alpha} is unlikely to account for CD4+ T cell mediated lysis of SW948 and LS174T cells.


Figure 3
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. Susceptibility of target cells for TNF-{alpha}-mediated cell death. SW948 and LS174T cells and for control L928 cells (each 5 x 104 per well) were incubated in 96-well microtiter plates for 48 h in the presence of increasing amounts of TNF-{alpha} (0.1–10 ng/ml) with (A) or without (B) actinomycin D (1 µg/ml). Viability of cells was monitored by a XTT-based colorimetric assay as described in Materials and Methods. The assay was done in triplicates.

 
Membrane-bound TNF-{alpha} can contribute to the cytolytic activity of cytotoxic CD8+ T cells (27). To monitor TNF-{alpha} expression on the cell surface of CD4+ T cells upon receptor-mediated activation, we incubated immunoreceptor BW431/26scFv-Fc-{zeta}-grafted CD4+ T cells, and for comparison, CD8+ T cells, in the presence of the solid-phase bound anti-idiotypic mAb BW2064, which binds to the BW431/26scFv domain of the immunoreceptor, or an isotype-matched control mAb. As summarized in Fig. 4, immunoreceptor-mediated activation moderately up-regulated TNF-{alpha} expression in 15% of CD4+ T cells, whereas 40% of CD8+ T cells increased TNF-{alpha} expression on the cell surface. To explore whether up-regulation of surface TNF-{alpha} contributes to immunoreceptor-triggered cytolytic activities, we incubated receptor-grafted CD4+ and CD8+ T cells, respectively, with CEA+ target cells while blocking TNF-{alpha} by a neutralizing Ab. Receptor-triggered cytolytic activities are slightly diminished in the presence of 1 µg/ml the blocking anti-TNF-{alpha} Ab, which are not further repressed at the concentration of 10 µg/ml (Fig. 5A). Obviously, membrane-bound TNF-{alpha} contributes to a minor extent but does not represent the predominant pathway to execute cytolysis by CD4+ T cells upon MHC-II-independent activation. We obtained essentially the same data for receptor-grafted CD8+ T cells, indicating that, for both T cell subsets, membrane-bound TNF-{alpha} has only minor impact on receptor-triggered cytotoxicity.


Figure 4
View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 4. Increased expression of membrane-bound TNF-{alpha} upon specific activation of receptor-grafted T cells. CD4+ and CD8+ T cells were equipped with the CEA-specific immunoreceptor BW431/26-scFv-Fc-{zeta} (anti-CEA-{zeta}), and grafted cells or nontransduced T cells (5 x 104 total cells per well) were cultivated in microtiter plates that were coated with the anti-idiotypic mAb BW2064 specific for the scFv domain of the recombinant immunoreceptor or an isotype-matched control mAb (each 4 µg/ml). After 72 h, TNF-{alpha} on the cell surface was determined by flow cytometry as described in Materials and Methods. The assay was done in triplicates, and cells were pooled before analysis. The percentage of receptor-grafted CD4+ and CD8+ T cells was 26 and 27%, respectively.

 

Figure 5
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 5. Blocking of TNF-{alpha} did not inhibit the cytolytic activity. Isolated CD4+ (upper) and CD8+ (lower) T cells were equipped with the CEA-specific immunoreceptor BW431/26-scFv-Fc-{zeta}. Grafted T cells (1 x 104 receptor-expressing T cells per well) or nontransduced T cells in same numbers were coincubated for 48 h with CEA+ SW948 tumor cells (2.5 x 104 per well) in the presence or absence of the blocking anti-TNF-{alpha} mAb or an isotype-matched control mAb (each 0.01–10 µg/ml). The viability of tumor cells was determined by the XTT assay as described in Materials and Methods. The assay was done in triplicates.

 
In the second set of analyses, we asked whether CD4+ T cells perform cytolysis via the Fas pathway. We therefore monitored the susceptibility of the target cells SW948 and LS174T for Fas (CD95) induced apoptosis. As summarized in Fig. 6, recombinant Fas-L protein (sCD95L) did not induce apoptosis in SW948 cells, whereas LS174T cells are partly susceptible to Fas-mediated cell death. For control, incubation of Jurkat cells with Fas-L protein resulted in apoptosis of nearly all cells. Induction of cell death is due to Fas–Fas-L interaction, because preincubation of the cells with a neutralizing anti-Fas-L Ab resulted in abrogation of apoptotic cell death (Fig. 6). To analyze the contribution of Fas-signaling to receptor-mediated target cell lysis, we grafted CD4+ T cells with the CEA-specific receptor and recorded receptor-triggered lysis of CEA+ SW948 and LS174T cells in the presence of a neutralizing anti-Fas-L Ab. As summarized in Fig. 7, blocking of Fas-L has no effect on the efficiency of CD4+ T cell-mediated lysis of SW948 cells and a minor effect on LS174T cells, which are partly susceptible to Fas-induced cell death. In contrast, incubation with the anti-idiotypic mAb BW2064, which blocks the CEA-binding domain of the immunoreceptor, prevented lysis of both LS174T and SW948 target cells. As control, the anti-idiotypic mAb and the anti-Fas-L mAb had no influence on the viability of CEA– Jurkat cells. These data indicate that 1) target cell lysis is specifically mediated via the recombinant receptor, and 2) receptor-grafted CD4+ T cells lyse the Fas-resistant SW948 tumor cells exclusively and the Fas-sensitive LS174T cells predominantly in a Fas-independent manner.


Figure 6
View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 6. Susceptibility of target cells for Fas-L-mediated cell death. CEA+ LS174T (A) and SW948 (B) cells and, for control, Jurkat (C) cells (each 5 x 104 per well) were incubated in 96-well microtiter plates in the presence of recombinant Fas-L protein (2 µg/ml) for 24 h. Specificity of Fas-mediated cell lysis was demonstrated by coincubation with the blocking anti-Fas-L mAb NOK-1 or an isotype control mAb (each 10 µg/ml), respectively. Viability of cells was monitored by a XTT-based colorimetric assay. The assay was done in triplicates.

 

Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 7. Inhibition of cytolysis by blocking of the recombinant receptor and Fas-L. CD4+ T cells with and without the CEA-specific receptor BW431/26-scFv-Fc-{gamma} (1 x 105 total T cells per well) were cocultivated for 48 h with CEA+ SW948 and LS174T cells and, for control, with CEA– Jurkat tumor cells (each 5 x 104 cells per well) in the presence of the anti-Fas-L mAb NOK-1, the anti-idiotypic mAb BW2064/36 that blocks the CEA binding domain of the immunoreceptor, or an IgG1 isotype control mAb (each 10 µg/ml). The viability of cells was monitored by a XTT-based colorimetric assay. The assay was done in triplicates. The percentage of receptor-grafted CD4+ T cells was 32%.

 
We now asked whether CD4+ T cells mediate receptor-triggered cytolysis via the perforin/granzyme system as do CD8+ T and NK cells and tested by FACS analyses whether CD4+ T cells express the cytolytic effector molecules perforin and granzyme A and B. As summarized in Fig. 8, activated CD4+ T cells express perforin as well as granzyme A and B, respectively, as do CD8+ T cells. However, CD4+ T cells express substantially lower amounts of perforin and granzymes in both cell numbers and amount per cell, compared with CD8+ T cells.


Figure 8
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 8. Expression of perforin and granzymes in CD4+ T cells. CD3+ T cells were isolated by MACS techniques and activated as described in Materials and Methods. Cells were stained for CD4 expression by a FITC-conjugated anti-CD4 mAb, fixed, permeabilized, and stained intracellularly with PE-conjugated Abs specific for perforin, granzyme A, and granzyme B, and with a PE-conjugated isotype IgG control Ab, respectively, and analyzed by flow cytometry (A–D). Mean fluorescence intensities of the respective stainings are shown for CD8+ (E) and CD4+ (F) T cells.

 
To test whether expression of cytolytic effector molecules in CD4+ T cells is sufficient to mediate a cytotoxic T cell response by release of cytolytic granules, we incubated anti-CEA receptor-grafted CD4+ T cells together with CEA+ LS174T and SW948 cells and with CEA– Colo320 tumor cells, respectively, in the presence and absence of EGTA, which blocks the release of cytolytic granules. As summarized in Fig. 9, A–C, receptor-mediated lysis of CEA+ target cells is efficiently blocked in the presence of EGTA. Remarkably, lysis of the Fas-resistant SW948 cells was completely abrogated by EGTA, whereas lysis of the partially Fas-susceptible LS174T cells was not completely blocked. This indicates that CD4+ T cells engrafted by the recombinant immunoreceptor lyse their target cells predominantly via perforin/granzyme, but death receptor-mediated apoptosis may partially compensate for inefficient perforin/granzyme-mediated cytolysis.


Figure 9
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 9. Receptor-grafted CD4+ T cells lyse their target cells by release of cytotoxic granules. A–C, Inhibition of cytolysis by blocking of cytolytic granule release. CD4+ T cells equipped with the CEA-specific receptor BW431/26-scFv-Fc-{zeta} (2.5 x 104 receptor-expressing cells per well) and nontransduced T cells in same numbers were cocultivated for 18 h in 96-well tissue culture plates in the presence or absence of EGTA (4 mM) with CEA+ SW948 and LS174T cells and for control with CEA– Colo320 tumor cells (each 2.5 x 104 cells per well), respectively. Viability of cells was monitored by a XTT-based colorimetric assay. D, Receptor-grafted CD4+ T cells rapidly lyse their target cells in a short-term assay. CD4+ (2.5 x 104 receptor-expressing cells per well) and CD8+ (1 x 104 receptor-expressing cells per well) T cells equipped with the CEA-specific receptor BW431/26-scFv-Fc-{zeta} and T cells without immunoreceptor in same numbers, respectively, were coincubated for 5 h with CFSE-labeled CEA+ SW948 target cells (5 x 104 cells per well). Cells were removed, stained with 7-aminoactinomycin D, and the number of dead CFSE-labeled cells were determined by flow cytometry. The assays were done in triplicates.

 
Perforin/granzyme-induced cytotoxicity is characterized by acute killing, whereas death receptor-mediated target cell lysis is more delayed. We therefore cocultivated anti-CEA-{zeta} receptor-grafted CD4+ and CD8+ T cells, respectively, with CEA+ SW948 tumor cells and determined cytotoxicity in a short-term, FACS-based killing assay as described in Materials and Methods. As summarized in Fig. 9D, receptor-grafted CD4+ T cells lysed SW948 target cells in a short-term killing assay within 5 h as do receptor-grafted CD8+ T cells, whereas nontransduced T cells did not. These data furthermore sustain our conclusion that immunoreceptor-triggered CD4+ T cells lyse their target cells preferentially via the perforin/granzyme pathway.

Because grafting with immunoreceptor converts CD4+ T cells to MHC-independent cytolytic effectors, we compared the efficiency of target cell lysis by receptor-grafted CD4+ and CD8+ T cells, respectively. As summarized in Fig. 10, A and B, both receptor-grafted CD4+ and CD8+ T cells lyse their target cells in an Ag-specific fashion. To achieve the same efficacy of target cell lysis, about twice the number of CD4+ effector T cells, compared with CD8+ T cells, are required. In contrast, receptor-grafted CD4+ T cells secreted higher amounts of IFN-{gamma} upon receptor cross-linking (Fig. 10, C and D).


Figure 10
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 10. Comparison of target cell lysis mediated by receptor-grafted CD4+ and CD8+ T cells. CD4+ and CD8+ T cells from the peripheral blood were equipped with the anti-CEA-immunoreceptor BW431/26-scFv-Fc-{gamma} and grafted cells (2 x 102 – 2.5 x 104 receptor-expressing T cells per well) and nontransduced T cells in same numbers were cocultivated for 48 h with CEA+ LS174T tumor cells and for control with CEA– A375 cells (each 2.5 x 104 cells per well) in 96-well tissue culture plates. A and B, The viability of tumor cells was determined by the XTT-based cytotoxicity assay. C and D, The culture supernatants were analyzed by ELISA for the presence of IFN-{gamma} as described in Materials and Methods. The assay was performed in triplicates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Whereas CD8+ T cells comprise most of the cytolytic effector lymphocytes in the peripheral blood, Ag-specific CD4+ T activity is pivotal to initiate complete T cell activation and execute effector functions. In contrast with the high frequency of CD4+ T cells in the peripheral blood, the number of potential targets for CD4+ effector cells is limited due to their MHC-II restriction. To bypass MHC restriction, we engrafted CD4+ T cells with an immunoreceptor molecule that binds CEA via an Ab-derived scFv domain (5, 8). Upon binding to target Ag, noteworthy, immunoreceptor-grafted CD4+ T cells exhibit highly efficient cytolytic effector functions toward CEA-positive target cells. CD4+ T cell directed cytolysis is Ag-specific and mediated via the recombinant immunoreceptor because 1) CEA-negative target cells are not lysed, 2) the cytotoxicity is inhibited by an anti-idiotypic mAb that blocks the binding domain of the immunoreceptor, and 3) nontransduced CD4+ T cells do not lyse the CEA-positive target cells. Whereas several reports describe the presence of cytolytic CD4+ T cells in humans (28, 29), the mechanism of target cell lysis by CD4+ T cells is still a matter of debate. Several mouse models imply that the cytolytic effector functions of murine CD4+ T cells depend predominantly on death receptor signaling, i.e., the Fas and/or TNF-R pathway (15, 16, 30), whereas CD8+ T cells mainly use the perforin/granzyme pathway to execute cytolysis (31).

In this report, we dissect the mechanism of CD4+ T cell-mediated target cell lysis using short-term cultured polyclonal human CD4+ T cells from the peripheral blood of healthy donors. Upon engraftment, immunoreceptor triggered cytolysis by CD4+ T cells is independent of death receptor signaling via the Fas pathway, because Fas-sensitive as well as Fas-resistant target cells were lysed with similar efficiencies and a blocking anti-Fas-L Ab did not inhibit receptor mediated cytolysis. Moreover, this indicates that CD4+ T cells lyse even Fas-sensitive target cells predominantly in a Fas-independent fashion. Additionally, death signaling via TNF-{alpha} seems to be unlikely because 1) the CEA+ target cells used in the assays are largely resistant to cell death mediated by soluble TNF-{alpha}, and 2) grafted CD4+ T cells up-regulate TNF-{alpha} on the cell membrane upon specific receptor cross-linking, but blocking of membrane-bound TNF-{alpha} did only slightly affect receptor-mediated target cell lysis. CD4+ T cell-triggered cytolysis, in contrast, is abrogated in the presence of EGTA, implying that cytolysis is mediated by release of cytolytic granules. Whereas death receptor signaling induces cell death of the delayed type, target cell lysis via perforin occurs more rapidly within some hours. Accordingly, CD4+ T cells lyse their targets rapidly (<5 h) in a short-term cytotoxicity assay, as do grafted CD8+ T cells. Taken together, our data indicate that redirected CD4+ T cells lyse their target cells predominantly via the perforin/granzyme pathway. The same mechanism has been described for CD8+ effector T cells (31). It was demonstrated recently that perforin is expressed differentially in human CD4+ and CD8+ T cells. Whereas the latter express perforin constitutively even in a resting state, synthesis of both perforin and granzymes is up-regulated in CD4+ T cells upon activation as it is in CD8+ T cells, however, at higher levels (cf Fig. 8) (32). However, the expression level of cytolytic effector molecules is sufficient to convert CD4+ T cells to highly efficient and MHC-independent cytolytic effector cells upon engraftment with recombinant immunoreceptors. In contrast, expression of cytolytic effector molecules correlates with cytolytic activity. Receptor-grafted CD4+ T cells that harbor about half amounts of granzyme B required about twice the number of effector cells to achieve the same cytolytic efficacy, compared with receptor-grafted CD8+ T cells (cf Figs. 8 and 10).

Although our data imply that grafted CD4+ T cells may partly use death receptor signaling for the induction of cytolysis of sensitive target cells, the perforin/granzyme pathway is predominantly used for the cytolytic CD4+ T cell attack against both death receptor-resistant and -susceptible target cells. This conclusion is in striking contrast with a recent report (10) that implies activated human CD4+ T cells lyse target cells exclusively via Fas, but not via perforin and granzyme. Several technical and experimental differences exist between both studies. Those CD4+ T cells were grafted with a MHC-I-dependent immunoreceptor, whereas in this study, we used a MHC-independent receptor for activation. Moreover, the engrafted CD4+ T cells in our study showed significant cytolytic activities in low effector-to-target cell ratios (<1:10) whereas the MHC-dependent receptor-grafted T cells were reported to drive Fas-dependent cytolysis in much higher ratios, e.g., 30:1.

In summary, our results indicate that 1) human CD4+ T cells from the peripheral blood can be converted to highly efficient, MHC-II-independent cytolytic effector cells by engraftment with Ab-type immunoreceptors, and 2) the cytolytic response of CD4+ T cells is predominantly mediated via perforin/granzyme and, for the most part, is independent of death receptor signaling. These findings have significant consequences to expand the power of adoptive cellular immunotherapy in that CD4+ T cells from the peripheral blood can be efficiently recruited to eliminate tumor cells directly and independently from MHC-II restriction.


   Acknowledgments
 
We thank Birgit Hops, Frank Steiger, and Petra Hofmann for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Dr. Mildred Scheel Stiftung für Krebsforschung (Deutsche Krebshilfe), Bonn, Deutsche Forschungsgemeinschaft, Bonn, Fortune program of the Medical Faculty of the University of Cologne. Back

2 Address correspondence and reprint requests to Dr. Hinrich Abken, Klinik I für Innere Medizin, Labor für Tumorgenetik, Universität zu Köln, Josef Stelzmann Strasse 9, D-50924 Köln, Germany. E-mail address: hinrich.abken{at}uk-koeln.de Back

3 Abbreviations used in this paper: MHC-II, MHC class II; scFv, single-chain Ab fragment; Fas-L, Fas ligand; CEA, carcinoembryonic Ag; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-5((phenyl-amino)carbonyl)-2H-tetrazolium hydroxide. Back

Received for publication December 19, 2005. Accepted for publication August 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421: 852-856. [Medline]
  2. Melief, C. J., R. E. Toes, J. P. Medema, S. H. van der Burg, F. Ossendorp, R. Offringa. 2000. Strategies for immunotherapy of cancer. Adv. Immunol. 75: 235-282. [Medline]
  3. Mumberg, D., P. A. Monach, S. Wanderling, M. Philip, A. Y. Toledano, R. D. Schreiber, H. Schreiber. 1999. CD4+ T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-{gamma}. Proc. Natl. Acad. Sci. USA 96: 8633-8638. [Abstract/Free Full Text]
  4. Qin, Z., T. Blankenstein. 2000. CD4+ T cell–mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN{gamma} receptor expression by nonhematopoietic cells. Immunity 12: 677-686. [Medline]
  5. Eshhar, Z., T. Waks, G. Gross, D. G. Schindler. 1993. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the {gamma} or {zeta} subunits of the immunoglobulin and T-cell receptors. Proc. Natl. Acad. Sci. USA 90: 720-724. [Abstract/Free Full Text]
  6. Moritz, D., W. Wels, J. Mattern, B. Groner. 1994. Cytotoxic T lymphocytes with a grafted recognition specificity for ERBB2-expressing tumor cells. Proc. Natl. Acad. Sci. USA 91: 4318-4322. [Abstract/Free Full Text]
  7. Hwu, P., J. C. Yang, R. Cowherd, J. Treisman, G. E. Shafer, Z. Eshhar, S. A. Rosenberg. 1995. In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes. Cancer Res. 55: 3369-3373. [Abstract/Free Full Text]
  8. Abken, H., A. Hombach, U. Reinhold, S. Ferrone. 1998. Can combined T-cell- and antibody-based immunotherapy outsmart tumor cells?. Immunol. Today 19: 2-5. [Medline]
  9. Hombach, A., C. Heuser, T. Marquardt, A. Wieczarkowiecz, V. Groneck, C. Pohl, H. Abken. 2001. CD4+ T cells engrafted with a recombinant immunoreceptor efficiently lyse target cells in a MHC antigen- and Fas-independent fashion. J. Immunol. 167: 1090-1096. [Abstract/Free Full Text]
  10. Kuball, J., F. W. Schmitz, R. H. Voss, E. A. Ferreira, R. Engel, P. Guillaume, S. Strand, P. Romero, C. Huber, L. A. Sherman, M. Theobald. 2005. Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR. Immunity 22: 117-129. [Medline]
  11. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356: 314-317. [Medline]
  12. Takahashi, T., M. Tanaka, C. I. Brannan, N. A. Jenkins, N. G. Copeland, T. Suda, S. Nagata. 1994. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76: 969-976. [Medline]
  13. Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. Huang, J. D. Young, R. Ahmed, W. R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91: 10854-10858. [Abstract/Free Full Text]
  14. Braun, M. Y., B. Lowin, L. French, H. Acha-Orbea, J. Tschopp. 1996. Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease. J. Exp. Med. 183: 657-661. [Abstract/Free Full Text]
  15. Shresta, S., C. T. Pham, D. A. Thomas, T. A. Graubert, T. J. Ley. 1998. How do cytotoxic lymphocytes kill their targets?. Curr. Opin. Immunol. 10: 581-587. [Medline]
  16. Graubert, T. A., J. F. DiPersio, J. H. Russell, T. J. Ley. 1997. Perforin/granzyme-dependent and independent mechanisms are both important for the development of graft-versus-host disease after murine bone marrow transplantation. J. Clin. Invest. 100: 904-911. [Medline]
  17. Darcy, P. K., N. M. Haynes, M. B. Snook, J. A. Trapani, L. Cerruti, S. M. Jane, M. J. Smyth. 2000. Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL. J. Immunol. 164: 3705-3712. [Abstract/Free Full Text]
  18. Yasukawa, M., H. Ohminami, J. Arai, Y. Kasahara, Y. Ishida, S. Fujita. 2000. Granule exocytosis, and not the fas/fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans. Blood 95: 2352-2355. [Abstract/Free Full Text]
  19. Kaulen, H., G. Seemann, K. Bosslet, W. Schwaeble, W. Dippold. 1993. Humanized anti-carcinoembryonic antigen antibody: strategies to enhance human tumor cell killing. Year Immunol. 7: 106-109. [Medline]
  20. Weijtens, M. E., R. A. Willemsen, E. H. Hart, R. L. Bolhuis. 1998. A retroviral vector system "STITCH" in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes. Gene Ther. 5: 1195-1203. [Medline]
  21. Hombach, A., D. Koch, R. Sircar, C. Heuser, V. Diehl, W. Kruis, C. Pohl, H. Abken. 1999. A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene Ther. 6: 300-304. [Medline]
  22. Hombach, A., C. Schneider, D. Sent, D. Koch, R. A. Willemsen, V. Diehl, W. Kruis, R. L. Bolhuis, C. Pohl, H. Abken. 2000. An entirely humanized CD3 {zeta}-chain signaling receptor that directs peripheral blood t cells to specific lysis of carcinoembryonic antigen-positive tumor cells. Int. J. Cancer 88: 115-120. [Medline]
  23. Hombach, A., C. Heuser, M. Gerken, B. Fischer, K. Lewalter, V. Diehl, C. Pohl, H. Abken. 2000. T cell activation by recombinant Fc{epsilon}RI {gamma}-chain immune receptors: an extracellular r domain impairs antigen-dependent T cell activation but not antigen recognition. Gene Ther. 7: 1067-1075. [Medline]
  24. Jost, L. M., J. M. Kirkwood, T. L. Whiteside. 1992. Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J. Immunol. Methods 147: 153-165. [Medline]
  25. Provinciali, M., G. Di Stefano, N. Fabris. 1992. Optimization of cytotoxic assay by target cell retention of the fluorescent dye carboxyfluorescein diacetate (CFDA) and comparison with conventional 51CR release assay. J. Immunol. Methods 155: 19-24. [Medline]
  26. Angulo, R., D. A. Fulcher. 1998. Measurement of Candida-specific blastogenesis: comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression. Cytometry 34: 143-151. [Medline]
  27. Ratner, A., W. R. Clark. 1993. Role of TNF-{alpha} in CD8+ cytotoxic T lymphocyte-mediated lysis. J. Immunol. 150: 4303-4314. [Abstract]
  28. Gagnon, S. J., F. A. Ennis, A. L. Rothman. 1999. Bystander target cell lysis and cytokine production by dengue virus-specific human CD4+ cytotoxic T-lymphocyte clones. J. Virol. 73: 3623-3629. [Abstract/Free Full Text]
  29. Guo, Y., H. Niiya, T. Azuma, N. Uchida, Y. Yakushijin, I. Sakai, T. Hato, M. Takahashi, S. Senju, Y. Nishimura, M. Yasukawa. 2005. Direct recognition and lysis of leukemia cells by WT1-specific CD4+ T lymphocytes in an HLA class II-restricted manner. Blood 106: 1415-1418. [Abstract/Free Full Text]
  30. Hsieh, M. H., R. Korngold. 2000. Differential use of FasL- and perforin-mediated cytolytic mechanisms by T-cell subsets involved in graft-versus-myeloid leukemia responses. Blood 96: 1047-1055. [Abstract/Free Full Text]
  31. Kreuwel, H. T., D. J. Morgan, T. Krahl, A. Ko, N. Sarvetnick, L. A. Sherman. 1999. Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways in CD8+ T cell-mediated insulin-dependent diabetes mellitus. J. Immunol. 163: 4335-4341. [Abstract/Free Full Text]
  32. Niiya, H., I. Sakai, J. Lei, T. Azuma, N. Uchida, Y. Yakushijin, T. Hato, S. Fujita, M. Yasukawa. 2005. Differential regulation of perforin expression in human CD4+ and CD8+ cytotoxic T lymphocytes. Exp. Hematol. 33: 811-818. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Azizi, D. E. Anderson, J. V. Torres, A. Ogrel, M. Ghorbani, C. Soare, P. Sandstrom, J. Fournier, and F. Diaz-Mitoma
Induction of Broad Cross-Subtype-Specific HIV-1 Immune Responses by a Novel Multivalent HIV-1 Peptide Vaccine in Cynomolgus Macaques
J. Immunol., February 15, 2008; 180(4): 2174 - 2186.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Koehler, D. Kofler, A. Hombach, and H. Abken
CD28 Costimulation Overcomes Transforming Growth Factor-{beta}-Mediated Repression of Proliferation of Redirected Human CD4+ and CD8+ T Cells in an Antitumor Cell Attack
Cancer Res., March 1, 2007; 67(5): 2265 - 2273.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hombach, A.
Right arrow Articles by Abken, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hombach, A.
Right arrow Articles by Abken, H.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Cancer

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS