Sentinel node lymphocytes: tumour reactive lymphocytes identified intraoperatively for the use in immunotherapy of colon cancer

Patients

Fifteen patients with colon cancer, with no signs of distant metastases or lymph node involvement prior to surgery, seven men and eight women, with an average age of 71 years were included in the study (Table 1). The study was approved by the local ethical committee and informed consent was given by the patients.

Identification of sentinel nodes

The colonic tumour site was mobilised through division of peritoneal adhesions to facilitate inspection of tumour and mesentery. One ml Patent blue dye (Guerbet, Paris) was injected superficially in the serosa around the tumour. Within 5 min, one to four blue-coloured mesenteric lymph nodes were identified macroscopically as sentinel nodes and they were marked with sutures.

Preparation of specimens

The sentinel and nonsentinel nodes were cut in half. Slices <1 mm thick were cut from the central and the peripheral part of the nodes for flow cytometry and proliferation analysis. The rest of the node underwent routine histopathological examination. Tumours were histopathologically classified as Duke's stages A–D (Duke, 1932) (Table 1). One piece of the primary tumour (including part of the invasive margin) was removed for flow cytometry analyses and as an antigen source.

Flow cytometry analyses

Peripheral blood leukocytes (PBL), lymph node cells and tumour cell suspensions at 1 × 10⁶ cells sample⁻¹ were subjected to investigation using flow cytometry (FACS). Cells were washed in PBS containing 2% FCS and 0.05% NaN₃ and stained with fluorophore conjugated antibodies against the cell surface molecules CD4 PE, CD8 PerCp (Becton Dickinson, San Jose, CA, USA) and the very early activation marker CD69 FITC (Pharmingen, San Jose, CA, USA). After staining, cells were investigated using a FACSCalibur (Becton Dickinson) and data were analysed using the Cellquest computer software (Becton Dickinson).

Immunological evaluation

Single cell suspensions from lymph nodes and tumours were obtained by gentle pressure using a loose fit glass homogeniser. Peripheral blood leukocytes were purified by ficoll-paque (Pharmacia, Amersham, Uppsala, Sweden). Cells were resuspended and washed twice in RPMI 1640 (Life technologies, Rockville, MD, USA) containing 2.5% fetal calf serum (FCS) (Life technologies). Finally, cells were resuspended in RPMI 1640 proliferation media containing 10% human AB serum (Sigma-Aldrich, St Louis, MO, USA), 1% penicillin–streptomycin (Sigma) and 1% glutamine (Sigma). Tumour samples were homogenised using a Ultra-turrax in 5 v (w v⁻¹) of 2 × PBS followed by 5 min denaturation at 97°C. Tumour homogenates were diluted to 1 : 10 and 1 : 100 in complete proliferation media. Purified PBL and lymph node cells were used at 3 × 10⁵ cells well⁻¹ in time course proliferation assays against diluted tumour homogenate, Con A 10 μg ml⁻¹ (Sigma) or carcinoembryonic antigen 100 μg ml⁻¹ (Sigma) in triplicate. Proliferation was measured by adding 1 μC_i of ³H-Thymidine/well (Amersham) 18 h prior to harvesting. Samples were subjected to scintillation counting.

Stimulations for the measurement of IFN-γ secretion were performed in 96-well plates with 3 × 10⁵ cells well⁻¹ in triplicate with tumour homogenate diluted 1/10 and 1/100, or Con A 10 μg ml⁻¹ (Sigma). The amount of secreted IFN-γ was measured with ELISA (Human IFN-γ Duoset, R&D Systems) on culture supernatants in pooled samples of the triplicates.

Intraoperative identification of sentinel node and pathological classification

One to four sentinel nodes (average 2.3) were detected intraoperatively using Patent blue injection in the circumference of the tumour. Patient characteristics and location of the tumours are presented in Table 1. Upon macroscopical dissection of the removed specimens, between five and 29 lymph nodes (average 19) were identified and embedded for histopathological evaluation (Figure 1A and B, left panels). Nine of the patients (Table 1) showed no signs of metastatic spread to sentinel node(s), nor to other lymph nodes despite that tumours grew through the bowel muscular wall (Figure 1A left panel) and they were classified as Duke's B. Patients no 10–14 (Table 1) had metastatic spread to the sentinel node and were histopathologically classified as Duke's C (Figure 1B left panel). Between one and six metastatic lymph nodes were identified by histopathology (Table 1). Patient no 15 in addition to one metastatic sentinel node had distant metastases to the liver, found at surgery, and was consequently classified as Duke's D (Table 1).

Characterisation of immune cells in sentinel nodes

Single cell suspensions of lymphocytes collected from the tumour, sentinel and nonsentinel lymph node specimens, and peripheral blood (PBL), were prepared as described in Material and Methods. The number of sentinel nodes obtained and average cell yield from the sentinel node specimens for each patient are given in Table 1. Cell suspensions were triple stained with antibodies recognising the very early activation marker CD69 and the surface antigens CD4 (Figure 1A and B, middle panels) and CD8 (not shown) followed by flow cytometry analyses (FACS). The lymphocytic gate was established by gating on forward and side scatter characteristics. The percentage of CD4⁺ and CD8⁺ cells in the lymphocytic gate varied between lymph nodes (Table 1). No significant correlation was found between the ratio of CD4⁺ to CD8⁺ cells and tumour stage or proliferative response (data not shown). Similar numbers of activated CD4⁺CD69⁺ lymphocytes were found both in sentinel and nonsentinel nodes regardless of absence (Figure 1A middle panel) or presence of metastases (Figure 1B middle panel). All investigated tumours contained tumour-infiltrating lymphocytes (TILs) to a various extent. Tumour-infiltrating lymphocytes were both of the CD4 and CD8 subsets and the majority presented an activated CD69⁺ phenotype. In peripheral blood activated, CD69⁺ lymphocytes were absent (not shown).

Functional characterisation of lymphocytes

The proliferative responses of the cell populations were characterised by time course ³H-Thymidine incorporation assays using homogenised denatured autologous tumour cell extracts as antigen sources (Figure 1A and B right panels and Table 2). TILs did not proliferate in any of the investigated patients. By contrast, SN lymphocytes from eight patients classified as Duke's B proliferated in an antigen dependent manner against autologous tumour extract (Figure 1A, right panel and Table 2) with an average peak proliferation of 24567±6473 cpm (average±s.e.m.). Peak proliferation was most frequently observed on day 6. In patient no 5, thymidine incorporation was not performed. SN lymphocytes from five out of six metastatic nodes (patients no 10–12, 14 and 15) responded poorly, with an average proliferation of 2141±1002(average±s.e.m.) (Figure 1B, right panel). From one Duke's C patient (no 13) SN lymphocytes proliferated vigorously when stimulated with autologous tumour extract (30 469 cpm) and also exhibited a high spontaneous proliferation (Table 2). In addition, in two Duke's B patients the reactivity against a known colon cancer antigen, carcinoembryonic antigen (CEA) was tested. However, stimulation of SN lymphocytes with 100 μg ml⁻¹ of CEA did not result in proliferation (data not shown). In nonsentinel nodes proliferation upon stimulation with tumour extract was not detected, with the exception of three patients (no 6, 7 and 8) (Table 2). In PBLs the addition of tumour antigen did not result in vigorous proliferation in any case, but responses could be seen in patients no 7, 13 and 14. In the nonsentinel node in patient 11 and in SN lymphocytes from patient 12, high proliferation indexes were obtained (Table 2). They represent single-point measurements, lacking the characteristic dose–response and/or kinetics of antigen-driven proliferation.

To further investigate the functional state of the lymphocytes, T cells were stimulated with Con A which by-pass the antigen-specific activation through the T-cell receptor. TILs were unresponsive to Con A stimulation, as were SN lymphocytes from Duke's C patients no 11–12. SN lymphocytes from the remaining patients proliferated vigorously, as did PBLs and nonsentinel node lymphocytes from both C and D patients (Figure 2A and B).

The ability of the lymphocytes to produce IFN-γ when stimulated with antigen or with Con A was investigated in six patients, no 4, 5, 8, 13–15 (Table 3). Tumour-infiltrating lymphocytes from Duke's B patient no 4 secreted high amounts of IFN-γ when stimulated with Con A (717 pg ml⁻¹) and five times the background level upon addition of tumour extract, in spite of their inability to respond in the proliferation assay. Tumour-infiltrating lymphocytes from patient 5 (Duke's B) (Figure 3) and patient 15 (Duke's D) did not produce IFN-γ when stimulated with tumour homogenate, nor when they were stimulated with Con A (data not shown). In patients 8, 13 and 14 (Duke's C), the amount of TILs obtained, did not suffice for the IFN-γ ELISA.

SN lymphocytes from Duke's B patients no 4, 5 (Figure 3A) and 8, and from Duke's C patient no 13 (Figure 4B) secreted IFN-γ 4–38 times above background level when stimulated with autologous tumour homogenate. In patients no 14 and 15 SN lymphocytes did not produce IFN-γ above background, upon stimulation with tumour homogenate. Lymphocytes from nonsentinel nodes did not respond with any IFN-γ secretion above background levels (Figure 3A), with the exception of patient no 8, where the secretion was 30 times the background level. In patients no 4 and 8 secretory responses of four to five times background level were seen in PBLs. SN-, nonsentinel lymphocytes and PBLs displayed a vigorous secretory response when stimulated with Con A (data not shown).