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Immunology |
1 Department of General Surgery, the University Hospital of Heidelberg; 2 Department of Cellular Immunology, Tumor Immunology Program, the German Cancer Research Center, Heidelberg, Germany; and 3 Berner Viszeralchirurgie Hirslanden, Klinik Beau-Site, Bern, Switzerland
Requests for reprints: Philipp Beckhove, Department of Cellular Immunology, Tumor Immunology Program, the German Cancer Research Center, INF 280, 69120 Heidelberg, Germany. Phone: 49-6221-423-745; Fax: 49-6221-423-702; E-mail: p.beckhove{at}dkfz.de.
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Abstract |
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rather than TH2 cytokines upon stimulation with tumor antigens. Although consistently induced during pancreatic cancer, T cells specific for pancreatic antigens were not detected during chronic pancreatitis, suggesting that their evaluation may be of diagnostic use in both diseases. Freshly isolated T cells from cancer patients recognized autologous tumor cells and rejected them in vitro and in a xenotransplant model in vivo, suggesting their therapeutic potential. Thus, tumor antigen–specific T cell responses occur regularly during pancreatic cancer disease and lead to enrichment of tumor cell–reactive memory T cells in the bone marrow. The bone marrow can therefore be considered an important organ for antitumor immune responses in pancreatic cancer. |
Introduction |
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Despite these observations, recent studies with peripheral blood of patients revealed that tumor-specific CTL responses could be generated in vitro upon repetitive T cell stimulation, suggesting the presence of tumor-specific CTL precursors in pancreatic cancer patients (8–12). However, a systematic characterization of spontaneous tumor-specific immune responses based on analyses of freshly isolated T cells has not been done to date. Although immune counteracting mechanisms might be especially efficient in the vicinity of solid tumor masses where the tumor microenvironment can limit the effectiveness of circulating antitumor lymphocytes (13) and may also affect immune responses in tumor draining lymph nodes, T cells resident in distant lymphatic organs might be less exposed to local suppressive influences.
Murine bone marrow has recently been shown to be an autonomous organ for T cell priming and generation of memory T cells with specificity for blood-derived and tumor-associated antigens (14) and to be a predominant site of homing, enrichment, and activation of memory T lymphocytes (15, 16). Similarly, in patients with breast cancer, multiple myeloma and malignant melanoma human bone marrow was found to contain large numbers of tumor-reactive memory T cells (17–20). Pancreatic tumor cells have been detected in the blood and bone marrow of 24% to 34% of investigated patients (21, 22) and might provide tumor antigens for induction or maintenance of local specific memory T cell responses.
In the present study, we addressed the question if and to what extent tumor-specific TH1 and cytotoxic effector T cells are generated in pancreatic cancer patients. To this end, we analyzed the presence, frequencies, and the functional properties of memory T cells specific for individual tumor antigens derived from autologous tumor cells or the defined tumor-associated antigen mucin-1 (MUC1), which is overexpressed in >90% of pancreatic cancers.
We compared specific memory responses of freshly isolated T cells from peripheral blood and bone marrow against cultured autologous tumor cells and dendritic cells presenting autologous tumor-associated antigens or synthesized polypeptides containing either the tandem-repeat sequence MUC1 (137-157)5 (ref. 23) or the signaling sequence MUC1 (1-100) of MUC1. We thereby show for the first time a regular spontaneous induction of tumor-reactive TH1 and cytotoxic T cells in pancreatic cancer patients capable of recognizing antigen-pulsed dendritic cells as well as autologous tumor cells with a predominant enrichment of such cells in the bone marrow.
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Materials and Methods |
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For transfer into NOD/SCID mice, 5 x 106 tumor cells were injected s.c. in 100 µL PBS per mouse into the flank region. Survival and general performance of mice was monitored daily. Tumor size was measured weekly (volume = length x width2 x 
/6; ref. 17).
Generation of bone marrow–derived T cells and dendritic cells. To obtain T lymphocytes, bone marrow mononuclear cells were incubated for 13 days in RPMI 1640 (Life Technologies, Eggenstein, Germany) with 10% human AB serum (PromoCell, Heidelberg, Germany), IL-2 (100 units/mL; Chiron, Ratingen, Germany) and IL-4 (60 units/mL; PromoCell) followed by overnight incubation in the same medium without interleukins (17). Depletion of contaminating B lymphocytes and myeloid precursors was done with magnetic beads conjugated with anti-CD19 and anti-CD15 monoclonal antibodies (mAb; both Dynal, Oslo, Norway).
Natural killer cells were removed using anti-CD56 mAbs (Beckman-Coulter, Krefeld, Germany) and magnetic beads conjugated with anti-mouse immunoglobulin mAbs (Dynal). After depletion, the suspension contained 85% to 95% CD3 T cells (
20% were CD8 T cells). To generate dendritic cells, bone marrow mononuclear cells were cultured for 14 days in serum-free X-VIVO 20 medium (Bio Whittaker, Walkersville, MD) with human granulocyte macrophage colony-stimulating factor (50 ng/mL; Behringwerke, Marburg, Germany) and IL-4 (1,000 units/mL; ref. 17). Afterwards, nonadherent dendritic cells were enriched by depletion of contaminating T and B lymphocytes using magnetic beads conjugated with anti-CD3 (Dynal), anti-CD56 (Beckman-Coulter), and anti-CD19 mAbs and pulsed for 20 hours with lysates (200 µg protein/106 cells/mL) from autologous tumor (cancer patients), from inflammatory pancreatic tissue (chronic pancreatitis patients), or normal peripheral blood mononuclear cells (PBMC) that were lysed by five freeze/thaw cycles (17) or with the same amount of tetanus toxoid.
IFN- ELISPOT assay. IFN- producing T lymphocytes were determined as previously described (17). Briefly, dendritic cells pulsed with different lysates or tetanus toxoid were coincubated with autologous T cells (dendritic cell/T cell ratio = 1:5) for 40 hours. The number of IFN- spot-forming cells was measured using a microscope Axioplan 2 and KS ELISPOT software (Carl Zeiss Vision, Hallbergmoos, Germany). Spots measured in the presence of dendritic cells pulsed with the autologous PBMC-lysate (as control for lysate-pulsed dendritic cells) or endobulin (as control for tetanus toxoid–pulsed dendritic cells) were considered as nonspecific background (negative control). In some cases, dendritic cells from healthy donors were pulsed with either allogeneic tumor cell lysate or PBMC-lysate from the same patient and analyzed in ELISPOT plates coated with mAbs against IFN-, IL-4, IL-10, and IL-12 (Mabtech, Nacka, Sweden) analyzed for respective cytokine secretion as indicators of different endogenous immunomodulating effects of the lysates.
Individuals were designated as responders if the numbers of spots in the presence of dendritic cells loaded with tumor antigens were significantly higher (P 
0.05 for tumor cell lysates and tetanus toxoid or P 0.1 for MUC1-derived peptides) than in negative control wells. The frequency of tumor-reactive bone marrow T lymphocyte was calculated as follows: (spot numbers in wells with tumor peptide–pulsed dendritic cells – spot numbers in negative control wells)/T cell numbers per well.
Mucin-1-derived peptides. Polypeptides containing the tandem-repeat sequence of the MUC1 antigen (p137-157)5 (ref. 23) and the signaling sequence of MUC1 (p1-100) were synthesized at the peptide facility of the German Cancer Research Center. As negative control antigen for MUC1-derived peptides, human immunoglobulin was used in the same protein concentration.
Cytotoxicity assays. Four-hour 51chromium release assays were done as described (17). Briefly, T cells were stimulated in a coculture with irradiated (100 Gy) autologous tumor cells pretreated with 100 units/mL IFN- in a ratio of 5:1 for 3 days. Chromium-labeled autologous tumor cells were used as targets. As control targets, the unrelated, allogeneic tumor cell lines U937 and K562 were used.
For detection of direct cytotoxicity, confluent monolayers of cultured tumor cells were generated in 48-well plates and cocultured with purified peripheral blood T cells in a ratio of 1:5 (test samples) or left untreated (control). For evaluation of antigen specificity, some tumor cultures were blocked with anti-HLA-I mAb prior to coculture with T cells. Dead tumor cells were quantified by trypan blue staining after 48 hours of coculture with autologous peripheral blood T cells.
ELISPOT assays from tumor cell/peripheral blood mononuclear cell cocultures. Tumor cells were cocultured for 48 hours together with unseparated autologous PBMC in a ratio of 1:5 in ELISPOT plates coated with anti-IFN- mAb (Mabtech). Detection of spots was done according to the manufacturer's protocol (Mabtech). As controls, aliquots of tumor cells were incubated with anti-HLA I for 1 hour prior to coculture with T cells.
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Results |
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secretion, indicating an enrichment of tumor antigen–reactive memory T cells in that compartment (Fig. 1B and C; Supplementary Fig. S1).
Analysis of corresponding bone marrow and peripheral blood samples from four patients (Fig. 2; Supplementary Table S1) revealed an enrichment of IFN--secreting T cells in all bone marrow samples. In contrast, tumor-reactive T cells from only one patient secreted IL-10 and IL-4 in response to tumor antigens. This indicates a predominant TH1 profile of tumor-reactive bone marrow T cells. In contrast to bone marrow, we detected tumor antigen–specific memory T cells in corresponding peripheral blood samples of only two of the four patients.
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Tumor antigen–reactive memory T cells are regularly enriched in the bone marrow of pancreatic cancer patients. Short-term IFN- ELISPOT assays in the blood of 25 and in corresponding bone marrow from 15 patients revealed the presence of tumor-reactive memory T cells in all (100%) tested bone marrow samples but in only 52% (13 of 25) of blood samples (Fig. 3A). The overall numbers of tumor-reactive T cells were significantly higher in bone marrow compared with peripheral blood (Fig. 3A; Supplementary Fig. S1) and were comparable to frequencies of T cells reactive against the recall antigen tetanus toxoid (Fig. 3A) in the same patients or in healthy donors (data not shown).
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-secreting T cells reactive against autologous pancreatic tissue antigens is a unique feature of pancreatic cancer because the bone marrow and blood from a group of 11 patients undergoing pancreatic resection for chronic pancreatitis did not contain memory T cells capable of IFN- secretion after activation with autologous pancreatic tissue antigens, although chronic pancreatitis is characterized by phases of strong local and systemic inflammation. Interestingly, the only patient diagnosed for chronic pancreatitis that showed reactivity against autologous pancreas tissue in the peripheral blood contained a small invasive pancreatic carcinoma besides chronic pancreatitis (Fig. 3A; Table 2). Thus, the detection of pancreatic antigen-specific TH1 T cells might serve as a useful diagnostic tool to distinguish between pancreatic cancer and chronic pancreatitis in addition to pathologic evaluation. To evaluate if T cells reactive to a defined tumor-associated antigen are also induced and enriched during the course of pancreatic cancer, we synthesized two 100mer polypeptides from the well-defined tumor-associated antigen MUC1 (23). MUC1 was shown to be expressed in >90% of pancreatic cancers (27). The two polypeptides used contain the signal sequence and the tandem repeat region of MUC1, the latter being described previously to contain HLA-I and -II restricted epitopes that bind to a variety of HLA alleles allowing their application as antigen for functional T cell analyses without selection of distinct HLA types (23). As shown in Fig. 3B and C (and in Supplementary Fig. S2), we detected functional MUC1-specific T cells in the bone marrow of all five patients but in corresponding peripheral blood samples of only two out of five patients—a finding closely resembling that of the ELISPOT analyses with autologous tumor cell lysates. Although both peptides were recognized by T cells from pancreatic cancer patients, the peptide containing the signal sequence was recognized more frequently.
Direct recognition and rejection of autologous tumor cells by T cells from pancreatic cancer patients. Although we showed the presence and enrichment of functional tumor-specific T cells in bone marrow and peripheral blood of pancreatic cancer patients, their ability to directly recognize life autologous tumor cells remained unclear. We therefore stimulated freshly isolated PBMC from 10 patients with cultured autologous tumor cells for 48 hours in IFN- ELISPOT assays. As shown in Fig. 4, the stimulation with live tumor cells induced a significant induction of IFN- secretion. Such an effect was blocked partially by anti-HLAI mAb—indicating the presence of HLA-I-restricted T cells capable of recognizing autologous tumor cells. The remaining activity might be either due to replenishment of HLA complexes during the 48-hour coculture, or to natural killer cell activity.
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To evaluate the potential therapeutic capacity of reactivated memory T cells derived from pancreatic cancer patients in vivo, we transplanted tumor cells from three patients s.c. into NOD/SCID mice. All three patients had detectable tumor-reactive memory T cells in their bone marrow or peripheral blood. Tumor cells from patient P10, which did not express MHC molecules served as a control. All mice had developed progressively growing tumors before treatment. Thirty-five days after tumor injection, 5 x 106 purified T cells from peripheral blood (P8) or bone marrow (P12) of patients were cocultured overnight with autologous dendritic cells, which had been pulsed with lysate from autologous tumor cell lines and applied along with the dendritic cells i.v. to the tumor-bearing mice. As shown in Fig. 6, tumor growth was either not affected in the HLA-negative control (P10), was delayed (P8), or it was strongly inhibited (P12). The differences seen between P8 and P12 could have been due to the large difference in the size of the tumors at the time of the treatment, a tumor of 600 mm3 (P8) compared with a tumor of 100 mm3 (P12). The results suggest that reactivated tumor-reactive memory T cells from late-stage pancreatic cancer patients have the potential to exert antitumor effects in vivo.
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Discussion |
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upon stimulation by tumor antigen–presenting dendritic cells and are able to kill autologous tumor cells in vitro and to reject autologous tumor xenografts, thereby revealing their therapeutic potential. Previous studies on pancreatic cancer detected T cells from peripheral blood reactive to synthetic or allogeneic tumor-associated antigens upon repeated stimulations (8–11). Here, we analyzed a nonmodified repertoire of freshly isolated T cells and tested it directly for reactivity against autologous antigens upon one short-term restimulation. Because circulating T cells may not be representative for lymphatic organs (which are sites for generation and storage of T cells), we also analyzed T cells from the bone marrow of pancreatic cancer patients. Bone marrow is an intriguing organ for T cell immunity because: (a) it is an organ for homing of blood-derived T cells and contains resident dendritic cells and monocytes/macrophages which can function as antigen-presenting cells (14–16); (b) it has been recently shown to be a site of naïve T cell priming and memory T cell generation against blood-borne tumor-associated antigens (14); (c) in breast cancer patients, bone marrow has been shown to be enriched with therapeutically relevant tumor-reactive memory T cells (17, 18, 24); (d) in pancreatic cancer, bone marrow is known to be a homing site for disseminated tumor cells (21, 22); and (e) in comparison to tumor-draining lymph nodes, bone marrow is not so directly influenced by immunomodulatory factors from the tumor and may therefore allow polarization of TH1 responses.
Despite an observed predominance of TH2 responses in pancreatic cancer patients which was attributed to the secretion of IL-10 and TGF-ß by the tumor microenvironment (7), we found strong cytotoxic and TH1 responses characterized by induction of IFN- in the peripheral blood and bone marrow of tested patients. Such TH1 polarization requires release of IL-12 by antigen-presenting cells during the priming of naïve T cells (25, 26). IL-12 is induced in antigen-presenting cells during cognate interactions with TH1 cells (26).
However, its production is suppressed upon stimulation with IL-10 and TGF-ß. These cytokines are secreted in the microenvironment of pancreatic tumors. Thus, antigen-presenting cells that take up antigen in the vicinity of solid tumor masses may be influenced to induce tolerance or TH2 responses rather than TH1 responses in the draining lymph nodes. In contrast, the number of disseminated tumor cells found in bone marrow may not be sufficient to secrete enough of these immunomodulating cytokines to affect the generation and maintenance of TH1 cells in that microenvironment.
We hypothesized that the maintenance of a tumor-reactive memory T cell population in the bone marrow is a special property of this compartment. Such memory T cells could be hidden in the bone marrow and thus be out of reach of immunosuppressive influences from the primary tumor. Previous studies on murine models showed that tumor-specific cytotoxic memory T cells were induced in the bone marrow by disseminated tumor cells. These were kept under active control by CD8 memory T cells which prevented the outgrowth of distant metastases (28, 29). It cannot be excluded that pancreatic cancer induces a similar situation of continuous generation of immune competent tumor-reactive T cells controlling disseminated tumor cells in the bone marrow, whereas proportions of these cells upon release into the circulation are lost at the tumor site due to tumor-derived immunosuppressive factors.
Efficient activation of T cells including their TH1 polarization, particularly against low-affinity antigens such as the majority of tumor-associated antigens, requires stimulation by activated, mature antigen-presenting cells. Such activation can be achieved via cognate interactions of antigen-presenting cells via CD40/CD40 L interactions with antigen-specific, activated T cells, by pattern recognition signals present on pathogens, such as bacteria and viruses, or through endogenous Toll-like receptor-activating agents such as heparin sulfate (25, 30). Heparin sulfate is released by a matrix-degrading enzyme, heparanase, which is involved in the metastatic potential of tumor cells and which is strongly overexpressed in many tumors including pancreatic cancer (31). Thus, promalignant tumor cell–derived factors could facilitate or even induce an immune response in the absence of exogeneous inflammatory stimuli.
Besides a potential induction within the bone marrow, circulating tumor-reactive memory T cells induced elsewhere might also accumulate in the bone marrow upon selective homing, where they may acquire an activated state due to an appropriate microenvironment (32, 33). This possibility is supported by the observation of a similar enrichment of tetanus toxoid–reactive T cells in that compartment, whereas long-term persistence of tetanus toxoid in the bone marrow of the patients seems unlikely.
Although tumor-immune memory T cells were found in the bone marrow of all tested pancreatic cancer patients, no such specific cells could be found in the bone marrow of patients with chronic pancreatitis. This difference is not only of theoretical interest but may be very useful as an additional diagnostic test parameter for distinction between chronic pancreatitis and pancreatic cancer, which is currently based solely on histopathologic assessment.
A comparison of tumor-associated antigen–specific T cell responses with those against recall antigens from the same patients revealed similar numbers of memory T cells reactive to tetanus toxoid and tumor-associated antigens in the peripheral blood of cancer patients. The levels of tetanus toxoid–reactive T cells from the blood of healthy donors were similar to those of healthy donors. Therefore, the prevalence of tumor antigen–specific memory T cells in patients with pancreatic cancer can be considered rather high, in the same order as T cells reactive to tetanus toxoid.
In conclusion, our data show for the first time the regular occurrence of tumor antigen–specific T cell responses during the course of pancreatic cancer leading to the generation and enrichment of tumor-immune memory T cells. These T cell responses are of TH1 rather than TH2 type and induce the generation of cytotoxic T cells capable of direct tumor cell recognition and destruction. Because such T cells could be isolated from the bone marrow of all tested patients but only from 50% of the tested peripheral blood samples, the bone marrow seems to play an important role in the generation and storage of immune-competent, tumor-reactive T lymphocytes.
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Acknowledgments |
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Footnotes |
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Received 3/31/05. Revised 7/12/05. Accepted 8/19/05.
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/black columns) and peripheral blood (PB or PBTC, 
/white columns) from four different pancreatic cancer patients (P1-3 and P26) in an autologous setting (using autologous dendritic cells pulsed with autologous tumor- or PBMC-lysates) or from five different healthy donors (ND 1-5) tested in an allogeneic settings (using autologous dendritic cells pulsed with allogeneic tumor- or PBMC-lysates). Values represent the difference between the number of spots observed with the tumor lysates and the PBMC lysates. T cell frequencies in samples containing significantly elevated numbers of cytokine-secreting cells (responders) compared with control wells are shown. Nonresponders are depicted at the bottom line. The lower panels depict mean frequencies of all four patients and all five healthy donors (thin horizontal lines, no specific reactivity detected).
) as antigen-presenting cells. Values from corresponding peripheral blood and bone marrow samples of the same patients were connected by interrupted lines.
) or NK-sensitive K562 cell line (erythromyeloid leukemia cell line, 
). One representative out of eight experiments is shown. B, HLA-I 25-dependent induction of tumor cell death during 48 hours of coculture with freshly isolated, purified autologous peripheral blood T cells from four different cancer patients. Cultured tumor cells were cocultured with purified peripheral blood T cells in a ratio of 1:5 (test samples, light gray columns) or left untreated (control, black columns). For evaluation of antigen specificity, some tumor cultures were blocked with anti-HLA-I mAb prior to coculture with T cells (dark gray columns). Dead tumor cells were quantified after 48 hours of coculture with autologous T cells by trypan blue staining.
) or were left untreated (
). As control, animals bearing HLA-I-negative tumors were treated similarly with autologous, tumor-associated antigen–activated T cells, and dendritic cells (P10). Three animals per group and time point were analyzed. Columns, mean; bars, ± SD of tumor diameters.