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Immunology

Peripheral Burst of Tumor-specific Cytotoxic T Lymphocytes and Infiltration of Metastatic Lesions by Memory CD8+ T Cells in Melanoma Patients Receiving Interleukin 12

Roberta Mortarini, Alessandra Borri, Gabrina Tragni, Ilaria Bersani, Claudia Vegetti, Emilio Bajetta, Silvana Pilotti, Vincenzo Cerundolo and Andrea Anichini
DOI:  Published July 2000

Abstract

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 μg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-127–35-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CD8+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

INTRODUCTION

IL 3 -12 is a heterodimeric cytokine acting as key regulator of cell-mediated immunity (see Refs. 1 and 2 for review) and as potent inhibitor of angiogenesis (3) . Immune regulation by IL-12 involves induction of T Helper 1 (TH1) differentiation, as well as activation of cytokine secretion, proliferation and cytolytic activity in NK and T cells (1, 2) . These functions of IL-12 have prompted its evaluation as a potential modulator of antitumor responses. Experimental studies of systemic administration of the cytokine (i.v., i.p., or s.c.) have indicated that IL-12 exerts antitumor activity against pulmonary and hepatic metastases (4, 5, 6) and can even prevent spontaneous tumor development in HER-2/neu transgenic mice (7) . In addition, models based on intratumor cytokine delivery, or in vivo transfer of cytokine-secreting tumors have indicated that IL-12 has significant and dose-dependent antitumor activity against a wide spectrum of murine tumors including melanoma, breast, ovarian, and bladder tumors (8, 9, 10, 11, 12) . All of the these studies have contributed to showing that IL-12 can inhibit tumor growth, improve the survival of tumor-bearing animals, and induce a long-lasting state of tumor-specific immunity.

The efficacy of IL-12 as antitumor cytokine, documented in experimental models, has opened the way to clinical studies based on systemic administration of rHuIL-12 to cancer patients (13, 14, 15, 16, 17, 18) . The clinical studies conducted thus far have been designed differently in terms of cytokine dosage (fixed versus escalating dose), route (i.v. versus s.c.), and schedule of administration, as well as clinical characteristics of enrolled patients. Despite these differences among the trials, immunological monitoring has indicated common effects of IL-12, such as transient lymphopenia (13 , 14 , 16 , 17) and induction of IFN-γ in serum (13, 14, 15, 16, 17) . In addition, increased levels of IL-10 in serum (14) and the enhancement of NK activity and T-cell proliferation in vitro after therapy have been described (17) in some studies. However, it is still unknown whether IL-12 administration in cancer patients induces systemic effects on T-cell-mediated antitumor immunity, affects expression of adhesion/homing receptors regulating T-cell homing/migration, and promotes T-cell infiltration of neoplastic tissue. To this end, we looked for evidence of modulation of CTL-mediated antitumor response in vivo in melanoma patients enrolled in a rHuIL-12 clinical study (14) . In that study, 10 metastatic melanoma patients received s.c. injections of low-dose (0.5 μg/kg) rHuIL-12 once a week in three weekly doses (no dose in week 4) in each of two 28-day cycles. That regimen was well tolerated by all of the patients, and antitumor activity was clinically documented, as shown by regression of s.c. nodules, superficial adenopathies, and hepatic metastases in 3 of 10 patients (14) .

As we show here for the first time, rHuIL-12 administration has a systemic impact on T-cell-mediated antitumor immunity, enhances expression of T-cell and endothelial cell adhesion/homing molecules, and promotes infiltration of neoplastic lesions by CD8+ T-cells with a memory phenotype. In addition, in comparison with pretherapy metastases, enhanced tumor necrosis and/or fibrosis was documented in some posttherapy lesions.

MATERIALS AND METHODS

Patients and Study Design.

Ten patients with advanced metastatic melanoma were enrolled in a pilot study of rHuIL-12 given s.c. Study design, patients characteristics, selection criteria, and clinical course have been reported in details elsewhere (14) . All but one of the patients (patient 6 was removed from the study after the first cycle) received two identical 28-day cycles, with s.c. injections of rHuIL-12 being given on days 1, 8, and 15 of each cycle. A fixed dose of 0.5 μg/kg of rHuIL-12 was used throughout the study. The treatment protocol was approved by the Ethical and Scientific Committees of our Institute, and a written informed consent was obtained from each patient. rHu IL-12 was supplied by Hoffmann-La Roche (Milan, Italy). The patients who showed clinically significant tumor regressions were patients 1, 5, and 10 (14) . Two of the enrolled patients (patients 1 and 3) expressed the HLA-A*0201 allele. An additional panel of metastatic melanoma patients, matched for disease stage but not enrolled in the rHuIL-12 clinical study, was selected as control group for LDA and ELISA assays.

Melanoma Cells.

Melanoma cells were isolated from surgical specimens from 3 of 10 patients enrolled in the rHuIL-12 clinical study and from additional patients not enrolled in the clinical study. The tumor cells were established as cell lines and kept in culture as described previously (19) . Tumor cells to be used as stimulators and targets for LDA were cultured in 10% pooled human serum (PHS)-RPMI 1640 (BioWhittaker, Verviers, Belgium). Absence of Mycoplasma contamination was checked by an ELISA kit (Boehringer Mannheim, Milan, Italy). Melanoma lines used in this study expressed HLA-class I, HLA-DR, and -DP antigens and major adhesion molecules (ICAM-1 and LFA-3).

T-Cell Phenotype.

Lymphocyte phenotype was evaluated after isolation on Ficoll gradients by means of immunofluorescence followed by flow cytometry analysis using a FACScan instrument (Becton Dickinson, Sunnyvale, CA). The following mAbs were used: Leu4 (anti-CD3), WT31 (anti-αβ TCR) and 11F2 (anti-γδ TCR; Becton Dickinson). Expression of adhesion/homing receptors on CD3+ T cells was evaluated by two-color immunofluorescence. To this end, cells were first stained with mAbs to CLA (HECA452 mAb, Becton Dickinson), or integrin α4 chain, or CD44, or CD11a, or CD18 (Immunotech, Marseille, FR) followed by incubation with FITC-conjugated goat antimouse or antirat IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) depending on the primary mAb, and finally by staining with PE-conjugated anti-CD3 mAbs (Becton Dickinson). Expression of signal transduction molecules (ζ chain and lck) in CD3+ T cells was evaluated by simultaneous membrane (for CD3 detection) and intracytoplasmic (for ζ chain and lck detection) immunofluorescence as recently described (20) . Statistical comparison of fluorescence histograms was performed by Kolmogorov-Smirnov statistics.

Cell Cycle Analysis.

Cell cycle analysis in patients’ CD3+ T cells was carried out by two-color immunofluorescence as described by Lakhanpal et al. (21) on cells stained first with FITC-labeled anti-CD3 mAbs (Becton Dickinson) and then, after treatment with paraformaldehyde and ethanol, with propidium iodide (Sigma Chemical Co. LTD., St. Louis, MO). One hundred thousand events were acquired, and an analysis of cell cycle on CD3+ cells was performed with the aid of the Modfit software (Becton Dickinson).

Tetramer Staining.

T-cell staining was performed with PE-conjugated HLA-A*0201/Melan-A/Mart-126–35 tetramers and HLA-A*0201/influenza matrix58–66 tetramers. HLA-peptide tetrameric complexes were synthesized as described previously (22, 23) , and specificity of tetramer staining was reported previously (22, 23) . Staining of PBLs was performed by incubating cells for 30 min at 4°C with PE-conjugated tetramers. As control, two-color fluorescence analysis was performed in some instances by staining T cells with FITC-conjugated anti-CD8 mAbs (Becton Dickinson) and PE-conjugated tetramers. The modified Melan-A/Mart-126–35 peptide (ELAGIGILTV), used for refolding the HLA-peptide complex has been shown to increase the binding affinity for HLA-A*0201 without affecting CTL recognition (24) . Negative controls for tetramer staining included PBLs from HLA-A*0201-negative healthy donors, whereas a Melan-A/Mart-127–35-specific CTL clone A83 (25) and fresh TILs, previously shown to contain a high frequency of Melan-A/Mart-127–35-specific T cells (22) , were used as positive controls. An influenza-matrix58–66-specific T cell line (25) , selected from PBLs after culture for 3 weeks with peptide-loaded autologous monocyte-derived dendritic cells (25) , was used as positive control for staining with influenza-matrix58–66/HLA-A*0201 tetramers. One × 106 cells for each PBL sample were analyzed on a FACScalibur instrument (Becton Dickinson).

Determination of Tumor-specific and Peptide-specific CTLp Frequency.

Frequency of CTLp directed to autologous tumor or to a peptide from the melanoma antigen Melan-A/Mart-1 (Melan-A/Mart-127–35; Ref. 26 ) was evaluated by a high-efficiency LDA (22 , 25) that, as recently described (22) , provides frequency estimation in fresh and activated T-cell populations in the same range as by HLA-peptide tetramer staining. Split-well analysis for HLA-restricted precursors was performed at day +28 of culture by comparing lysis of autologous tumor in the presence or absence of mAbs with CD3 (OKT3, American Type Culture Collection, Manassas, VA) or to HLA-class I antigens (w6/32; Ref. 27 ). The final cytolytic assay was performed in the presence of 1.5 × 102 tumor targets/well, and a threshold of 10% lysis was used. Comparison of two populations of lysis values from the whole LDA set by ANOVA, followed by the Student-Newman-Keuls multiple-range test, indicated that a difference between tumor lysis in the presence or absence of mAbs to CD3 or to HLA-class I ≥20 was significant, with a P ≤ 0.01. By these criteria, comparison of lysis values in the two aliquots from the same well allowed the scoring of the original well as containing or not containing a tumor-specific CTLp (22 , 25) . The LDA for detection of peptide-specific CTLp directed to Melan-A/Mart-127–35 peptide in the context of HLA-A*0201 were set up in the presence of a constant number (2000/well) of the TAP-deficient T2 cell line loaded with Melan-A/Mart-127–35 (AAGIGILTV) peptide (PRIMM s.r.l., San Raffaele Biomedical Science Park, Milan, Italy) as described previously (22 , 25) . Split-well analysis for peptide-specific CTLp was conducted at day +28 of culture by comparing lysis of an EBV-transformed, HLA-A*0201+ B-lymphoblastoid cell line (9742 LCL, 3 × 102/well), empty or loaded with Melan-A/Mart-127–35, as described previously (22 , 25) . Thresholds of lysis and criteria to score a well as containing a Melan-A/Mart-1-specific CTLp were as described for the LDA sets tested on autologous tumor. A percentage of negative wells was then evaluated for each lymphocyte dilution. Linear regression analysis of number of responder cells/well against logarithmic percentage of negative wells was then performed by using the Excel software (Microsoft). On the basis of the Poisson distribution, the slope of the regression line was used to evaluate the antitumor and peptide-specific CTLp frequency (25) . To compare frequency values in distinct LDA sets, the upper and lower 95% confidence limits of the regression line were evaluated by the Fig.P software (Biosoft, Ferguson, MO). By this LDA assay, the variation in CTLp frequency value was less than 15% in independent LDA from the same blood sample. Furthermore, in control melanoma patients matched for disease stage but not enrolled in the rHuIL-12 clinical study, the variation in CTLp frequency value against autologous tumor was less than 30% in blood samples taken 7 days apart (data not shown).

Detection of Soluble Adhesion Molecules in Serum of Patients by ELISA.

Serum samples, obtained before and during rHuIL-12 therapy from patients enrolled in the rHuIL-12 clinical study and from control patients, were assayed by commercially available ELISA kits (Bender MedSystems, Vienna, Austria). Presence of the following soluble adhesion molecules was evaluated: ICAM-1, VCAM-1, and E-selectin. Results of ELISA experiments were statistically evaluated by ANOVA followed by the Tukey multiple comparison test.

Immunohistochemical Analysis of Melanoma Lesions.

Immunohistochemical analysis was performed as described recently (22) routinely on formalin or on Bouin’s fixed and paraffin-embedded specimens. To optimize immunodetection of Melan-A/Mart-1, gp100, CD8, CD45RO, and TIA-1, nonenzymatic antigen unmasking was performed as described previously (22) . Primary antibody incubation was performed overnight at 4°C with the following antibodies: M2–7C10 (anti Melan-A/Mart-1, Lab Vision Corp., Fremont, CA), HMB45 (anti gp-100, Dako Corporation, Carpinteria, CA), CD8 (Dako), TIA-1 (anticytolytic granule marker, Beckman Coulter, Fullerton, CA), and CD45RO (Dako). Staining with polyclonal antibody CD3 (Dako) was performed after 0.1% trypsin treatment for 5 min as described for antigen unmasking. Sections were subsequently rinsed three times in PBS + Triton X 100 and treated with biotinylated goat antimouse immunoglobulin (Dako) or with biotinylated goat antirabbit immunoglobulin (Dako) for CD3 staining. The slides were covered with streptavidin-horseradish peroxidase (Dako) for 30 min and finally visualized with the use of 3-amino-9-ethylcarbazole (Sigma) in 0.05 m acetate buffer containing 0.015% H2O2. Sections were then counterstained with hematoxylin and mounted with glycerin-gelatin 4. Tissue sections subjected to the same treatment but without incubation with primary antibody were used as negative controls. A reactive lymph node was used as positive controls for CD3, CD8, TIA-1, and CD45RO. The positive controls for tumor antigens were a Melan-A/Mart-1+, gp100+ lymph-node metastasis of melanoma and a Melan-A/Mart-1+ human melanoma cell line grown in nude mice.

RESULTS

rHuIL-12 Administration Induces a Burst of Tumor-specific, HLA-restricted CTLp and of Melan-A/Mart-127–35-specific CTLp in Peripheral Blood.

To evaluate possible systemic effects of rHuIL-12 administration on frequency of CTLp directed to tumor antigens, LDA was performed on PBLs from five of the enrolled patients (patients 1, 3, 5, 6, and 10). In three of these patients (patients 5, 6, and 10), the autologous tumor line was available, thus allowing us to evaluate the frequency of HLA-restricted CTLp directed to autologous melanoma. As shown in Fig. 1 ⇓ , in all of the three patients, a marked and significant increase in overall tumor-specific CTLp frequency (as evaluated by inhibition of tumor lysis with anti-CD3 mAbs), as well as in HLA-class-I-restricted antitumor CTLp frequency (as evaluated by inhibition with w6/32 mAbs) was observed between 4 and 7 days after the first rHuIL-12 administration. In patient 5, tumor-specific CTLp frequency values peaked at day +4, after the first rHuIL-12 administration, and dropped below threshold of detection at day +7 (Fig. 1) ⇓ . In blood samples taken 1 and 7 days after the last rHuIL-12 administration (patients 5 and 10, Fig. 1 ⇓ ), frequency of CTLp inhibited by anti-CD3 or by anti-HLA class-I mAbs was again higher than in pretherapy PBLs, although lower than the highest values seen between day +4 and +7 of the first cycle. CTLp determination in patient 6 could be performed in a blood sample taken even 1 day after the first rHuIL-12 administration, and at that time, frequency of CTLp inhibited by anti-CD3 mAbs showed a drop in comparison with pretherapy values (day −4) and to the subsequent burst (day +7).

Fig. 1.
Fig. 1.

Frequency of HLA-restricted, tumor-antigen-specific CTLp in PBLs of patients before and during rHuIL-12 therapy. Frequency of CTLp directed to autologous tumor and inhibited by anti-CD3 (left panels) or by anti-HLA-class I mAbs (right panels) was evaluated by LDA in PBLs from patients 5, 10, and 6. In patients 5 and 10, PBLs for LDA analysis were taken before therapy (day −4) and 4 and 7 days after the first IL-12 administration, as well as 1 and 7 days after the last IL-12 administration. In patient 6, PBLs were taken at day −4, and then 1 and 7 days after the first IL-12 administration. Patient 6 was removed from the trial after the first treatment cycle. Arrows, rHuIL-12 was given at day +1, +8, +15, +29, +36, and +43; close to each experimental point, observed values of CTLp frequency;∗ , CTLp frequencies are significantly different from pretherapy (day− 4 values) on the basis of the 95% confidence intervals of the regression lines.

The changes in CTLp frequency detected in patients 5, 6, and 10 were confirmed by repeated LDA assays on the same blood samples (data not shown). Furthermore, a frequency of precursors nonspecifically lysing the autologous tumor (i.e., lysis not blocked by either anti-CD3 or anti-HLA mAbs) was evaluable in all of the LDA sets from the three patients, including those sets showing tumor-specific CTLp frequency <5 CTLp/106 lymphocytes (data not shown).

In two patients (patients 1 and 3) expressing the restricting element (HLA-A*0201) for the tumor antigen Melan-A/Mart-127–35, the frequency of CTLp directed to this immunodominant epitope was evaluated in PBLs by LDA. In comparison with pretherapy values (day −4) in both patients (Fig. 2) ⇓ , a marked increase in peptide-specific CTLp in peripheral blood was observed between day +4 and day +7 after the first rHuIL-12 administration, as well as between 1 and 7 days after the last rHuIL-12 injection. To corroborate the LDA results, T cells from patient 1 were stained with HLA-A*0201/Melan-A/Mart-126–35 tetramers or, as control, with HLA-A*0201/influenzamatrix58–66 tetramers (Table 1) ⇓ . Analysis of day −4 and day +7 PBLs from patient 1 (Table 1) ⇓ did not detect T cells staining with HLA-A*0201/Melan-A/Mart-126–35 tetramers, in agreement with LDA (<5 CTLp/106 PBLs; see Fig. 2 ⇓ ). However, in day +1 PBLs, the T cells staining with HLA-A*0201/Melan-A/Mart-126–35 tetramers T cells were 252 CTLp/106 PBLs and at day +4 tetramer+ T cells were 239 CTLp/106 and 255 CTLp/106 lymphocytes (in two distinct experiments) in good agreement with LDA data in patient 1 (200 CTLp/106 PBL at day +4; see Fig. 2 ⇓ ). By contrast, staining of patient-1 PBLs with a control tetramer (HLA-A*0201 complexed with influenza matrix58–66 peptide) revealed a reduction in frequency of influenza-matrix-specific T cells at days +1 and +4 in comparison with day −4, followed by a rebound at day +7. Control experiments, in HLA-A*0201+ patients matched for disease stage but not enrolled in the rHuIL-12 study, indicated that the frequency of T cells staining with HLA-A*0201-Melan-A/Mart-1 tetramers was constant in independent blood samples taken weekly during 1 month (data not shown). Thus, the experiments with HLA-A*0201-Melan-A/Mart-1 tetramers confirmed the LDA data and indicated that rHuIL-12 administration transiently boosts the frequency of peptide-specific CTLp directed to a melanoma antigen peptide in peripheral blood.

Fig. 2.
Fig. 2.

Frequency of peptide-specific CTLp directed to Melan-A/Mart-127–35 peptide in two HLA-A*0201+ melanoma patients before and during rHuIL-12 therapy. PBLs from patients 1 and 3 were taken before therapy (day −4), 4 and 7 days after the first rHuIL-12 administration, as well as 1 and 7 days after the last IL-12 administration. ∗, CTLp frequencies are significantly different from pretherapy (day −4 values) on the basis of the 95% confidence intervals of the regression lines; arrows, timing of rHuIL-12 injections.

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Table 1

Staining of Melan-A/Mart-1-specific T cells by HLA-A*0201-Melan-A/Mart-126–35 tetrameric complexes

Staining with HLA-A*0201-Melan-A/Mart-126–35 PE-tetramers or HLA-A*0201-influenza-matrix58–66 PE-tetramers was performed on: PBLs of a HLA-A*0201− donor (negative control); a Melan-A/Mart-1-specific CTL clone A83 (25) , an influenza-matrix58–66-specific T cell line (25) , fresh TILs from a melanoma metastasis previously shown to contain a high frequency of Melan-A/Mart-1-specific T cells (22) (positive controls); and PBLs of a HLA-A*0201+ patient (Pt 1) enrolled in the rHuIL-12 study. Tetramer staining with positive and negative controls was used to set markers on FL-2 histograms to evaluate the numbers of tetramer+ T cells in the experimental samples from patient (Pt) 1. Frequency of tetramer+ T cells obtained by single staining with tetramers was confirmed by two-color analysis on cells stained with PE-labeled tetramers and FITC-labeled anti-CD8 mAbs (data not shown). PBLs of patient 1 were taken before (day −4) and after (day +1, +4, and +7) the first rHuIL-12 injection. One × 106 cells were analyzed for each sample.

Additional experiments were performed, before and during rHuIL-12 therapy, to characterize the peripheral T-cell population from 8 of 10 patients (patients 1 and 3–9). These experiments indicated that, at the same time points used for LDA and tetramer analysis, rHuIL-12 administration did not affect, in each patient, the proportion of circulating T lymphocytes nor the proportion of CD3+ cells expressing αβ or γδ TCRs (data not shown). Furthermore, the expression of two TCR signaling molecules (ζ chain and lck) that can be defective in cancer patients was normal in the treated patients and did not change after rHuIL-12 administration (data not shown). In addition, cell cycle analysis of patients’ T cells in PBLs did not show any difference in CD3+ T-cell proliferation in posttherapy (day +4 and +7) samples in comparison with pretherapy (day −4) T cells in any of the patients (see Fig. 3 ⇓ for representative data from one patient).

Fig. 3.
Fig. 3.

Cell cycle analysis of peripheral blood T cells before and during rHuIL-12 treatment. A, PBLs of a healthy donor were stimulated for 3 days with phytohemagglutinin followed by culture for 2 days in the presence 500 units/ml IL-2 (positive control for cell cycle analysis). Uncultured PBLs from a patient were taken before (day −4, B) and at day +4 (C), and at day +7 (D) after the first rHuIL-12 administration. Analysis of cells stained with anti-CD3 mAbs and propidium iodide was performed after gating on FL2-Area versus FL2-Width plots to exclude cell doublets and aggregates. The position of the vertical marker to discriminate cells in G1 from those in S and G2-M phases of the cell cycle was set with the aid of the Modfit software. In each dot plot, the upper left panel contains CD3+ T cells in G1 phase of the cell cycle, whereas the upper right panel contains CD3+ T cells in S and G2-M phases. A total of 100,000 cells were analyzed for each dot plot. Propidium iodide staining (FL2-Area) is reported on a linear scale, whereas staining for CD3 (FL1-height) is reported on a log scale.

Enhanced Expression of T-Cell Adhesion/Homing Molecules and Increased Serum Levels of Endothelial Cell Adhesion Molecules in Patients Receiving rHuIL-12.

Changes in expression of adhesion receptors regulating T-cell interaction with endothelial cells may have an impact on T-cell migratory patterns and, thus, promote T-cell infiltration of neoplastic tissues. To document evidence consistent with a systemic effect of rHuIL-12 on adhesion/homing molecules, analysis of expression of such receptors on circulating CD3+ T cells was performed. Within 24 h of the first rHuIL-12 administration, enhanced expression of the brightest fraction of T cells expressing the CD11a and CD18 subunits of LFA-1 and of the α4 subunit of VLA-4 (CD49d), as well as a clear increase in fluorescence intensity for CD44, was observed (Fig. 4) ⇓ . The increased expression was transient as, at day +7, fluorescence intensity for CD49d and CD44 dropped to values lower than those seen in pretherapy samples. In most patients a new transient increase in expression of LFA-1, VLA-4, and CD44 on circulating T cells took place between 1 and 4 days after the last rHuIL-12 administration of the second cycle (data not shown). In addition (see Table 2 ⇓ ), a clear increase in the proportion of T cells expressing CLA, the skin homing receptor, was documented, mainly after the first rHuIL-12 administration.

Fig. 4.
Fig. 4.

Expression of adhesion/homing molecules on peripheral blood T cells before and during rHuIL-12 treatment. Expression of CD11a, CD18, CD49d, and CD44 was evaluated in CD3+ T cells by two-color immunofluorescence and cytofluorometric analysis. PBLs were isolated at day −4 (before the first rHuIL-12 injection) and at day +1, +4, and +7. Expression of the four adhesion molecules was evaluated after gating for CD3+ T cells on FL2 versus forward-scatter plots. To ease the comparison of adhesion molecule expression in blood samples taken at different time points, an arbitrary dotted line, crossing each histogram, was drawn in correspondence to the main peak of fluorescence of day −4 histograms. Empty histograms, control staining with FITC-labeled secondary antimouse IgG. Expression of all of the adhesion molecules was significantly changed as a result of rHuIL-12 administration (P < 0.001, by Kolmogorov-Smirnov statistics for each comparison of pre- and post-IL-12 fluorescence histograms).

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Table 2

Expression of the skin-homing receptor CLA on CD3+ T cells in peripheral blood of patients receiving rHuIL-12

Expression of CLA antigen (on green fluorescence histograms) was evaluated by two-color immunofluorescence after gating on T cells stained by PE-conjugated anti-CD3 mAbs on red fluorescences versus forward-scatter plots. PBL samples were taken before (day −4) rHuIL-12 administration; 1, 4 and 7 days after the first cytokine injection; and 1 and 4 days after the last injection (days +43 and +47).

Within 24–48 h after the first rHuIL-12 administration, the phenotype changes for T-cell adhesion/homing receptors were matched by striking increases in the levels of soluble forms of three adhesion molecules (sICAM-1, sE-selectin, and sVCAM-1) that can be shed by activated endothelial cells (Fig. 5) ⇓ . In many instances, additional increases took place even at day +4 and+ 7 (Fig. 5) ⇓ . Soluble endothelial cell adhesion molecule levels declined after the first cycle of rHuIL-12 administration, but a new increase was detected in all of the patients at the second cycle (data not shown). Control experiments were performed by monitoring for 1 week the serum samples taken from 10 metastatic melanoma patients matched for disease stage with the patients receiving rHuIL-12. These experiments indicated that sICAM-1, sE-selectin, and sVCAM-1 levels were either constant or showed a maximum range of fluctuation within ±30% of the initial value (data not shown).

Fig. 5.
Fig. 5.

Soluble endothelial cell adhesion molecules in serum before and during rHuIL-12 therapy. Levels of soluble (s) forms of ICAM-1, E-selectin, and VCAM-1 were evaluated by ELISA in serum samples taken at days −4, +1, +2, +4, +7, and +11 of the first treatment cycle. Arrows, times of rHuIL-12 injection. ∗, serum levels of sICAM-1, sE-selectin, and sVCAM-1 were significantly different (P < 0.01; Tukey multiple comparison test) from pretherapy (day −4) values.

rHuIL-12 Administration Promotes Infiltration of Neoplastic Lesions by CD8+ T Cells with a Memory Phenotype.

To document possible evidence of enhanced T-cell infiltration of neoplastic lesions as a result of rHuIL-12 administration, metastatic lesions removed before and after rHuIL-12 therapy were compared by immunohistochemistry. All of the clinically regressing lesions (in patients 1, 5, and 10, as described previously in Ref. 14 ) could not be surgically excised. Nevertheless, another eight posttherapy metastases (clinically judged as nonregressing lesions) could be removed within 1–2 months after the last rHuIL-12 administration from three patients (patients 2, 3, and 10). From the same three patients, five pretherapy metastatic lesions could also be studied. All of these lesions were analyzed for patterns of infiltrating lymphocytes according to the“ brisk/non-brisk/absent” code proposed by Clark et al. (28) , for evidence of tumor necrosis and/or regression, and for expression of two melanoma antigens (Melan-A/Mart-1 and gp100). As shown in Table 3 ⇓ and exemplified in Fig. 6A ⇓ (lesion 1 of patient 10), none of the five pretherapy lesions from the three patients contained infiltrating CD3+ T cells and, thus, were classified as“ absent.” In contrast, from the same patients, 8 of 8 posttherapy metastatic lesions contained infiltrating T cells with either a“ brisk” or a “non-brisk” pattern (Table 3) ⇓ . None of the posttherapy s.c. lesions showed an enhanced infiltration of T cells in the areas of normal skin adjacent or overlaying the tumor tissue (data not shown). In addition, as shown in Table 3 ⇓ (and exemplified in Fig. 6B ⇓ ; lesion 2, patient 10), all of the T cells infiltrating the posttherapy lesions were CD8+ and expressed a memory phenotype (CD45RO+). Furthermore, a proportion between 10 and 100% of the T lymphocytes that infiltrated the lesions expressed the cytolytic granule-associated protein recognized by the TIA-1 antibody (Ref. 29 ; Table 3 ⇓ ). Histological evidence of increased tumor necrosis and/or regression was documented in posttherapy lesions from two patients (patients 10 and 2). In these instances, tumor necrosis areas were of the coagulative type, as defined by nuclear loss and marked cytoplasmic eosinophilia. In patient 2, histological evidence of tumor necrosis was observed only in posttherapy lesions (Table 3) ⇓ , whereas in patient 10, posttherapy samples showed larger areas of tumor necrosis in comparison with the pretherapy metastasis (Table 3 ⇓ ; Fig. 6B ⇓ ) and presence of fibrosis in two lesions (lesions 2 and 4 in Table 3 ⇓ ). In patient 3, no differences in the extent of tumor necrosis were observed between pre- and posttherapy lesions. Interestingly, none of the lesions in this HLA-A*0201+ patient expressed two tumor antigens (Melan-A/Mart-1 and gp100; Table 3 ⇓ ) that can be recognized by HLA-A2-restricted CTL, and the posttherapy lesion expressed HLA-A2 on only 30% of tumor cells (as evaluated by immunohistochemistry, data not shown). This suggested a possible mechanism of immune evasion, despite the non-brisk T-cell infiltrate found in the posttherapy lesion of this patient.

Fig. 6.
Fig. 6.

Immunohistochemical analysis of metastatic lesions isolated before and after rHuIL-12 therapy. A pretherapy nodal metastasis (A) and a posttherapy s.c. lesion (B) from patient 10 were characterized. Consecutive sections of paraffin embedded tumor fragments were conventionally stained with H&E (EE) or subjected to immunohistochemical staining with anti-CD3, anti-CD8, and anti-CD45RO mAbs. A, a nodal metastasis (lesion 1 in Table 3 ⇓ ) almost void of areas of necrosis or tumor regression (EE staining in A) lacked intratumoral lymphocytes CD3+, CD8+, and CD45RO+. B, a s.c. lesion (lesion 2 in Table 3 ⇓ ) showed intense intratumoral lymphocyte infiltrate characterized by CD3+, CD8+, and CD45RO+ cells. Original magnification for all of the panels, ×400.

View this table:
Table 3

Immunohistochemical analysis of neoplastic lesions from patients treated with rHuIL-12

Taken together, these data suggest that low-dose s.c. rHuIL-12 treatment in metastatic melanoma patients promotes infiltration of neoplastic tissue by CD8+ T cells with a cytolytic and memory phenotype.

DISCUSSION

The results of this study show that s.c. administration of low-dose rHuIL-12 in metastatic melanoma patients induces a peripheral burst of HLA-restricted, tumor-specific and peptide-specific CTLp, enhances expression of ligand receptor pairs contributing to the LFA-1/ICAM-1, VLA-4/VCAM-1 and CLA/E-Selectin adhesion pathways, and promotes infiltration of neoplastic tissue by CD8+ T cells with a memory and cytotoxic phenotype. Thus, in the clinical setting, rHuIL-12 can impact on three key immunological mechanisms that can regulate T-cell-mediated antitumor responses.

The rHuIL-12 effect on CTLp frequency was not attributable to a change in the proportion and phenotype of circulating CD3+ T cells. In fact, pretherapy values of circulating CD3+ T cells expressing either αβ or γδ TCRs indicated no significant differences in comparison with values detected at day +4 or +7, at times when LDA revealed a marked increase of tumor-specific CTLp. Furthermore, expression of two TCR signal transduction molecules (ζ chain and lck), which can be defective in cancer patients (30, 31) , was found to be normal in the enrolled patients, and rHuIL-12 treatment did not improve the expression of such molecules. Reversal of T-cell anergy by rHuIL-12 is another possible mechanism that may underlie the CTLp burst. IL-12 has indeed been shown to reverse antigen-specific T-cell anergy in vitro and in vivo (32, 33, 34) . However, this mechanism is not supported by the data obtained with tetramers. The tetramer experiments, with HLA-A*0201 complexed with Melan-A/Mart-126–35, indicated that an increase in the number of circulating peptide-specific CTLp did take place, as a result of rHuIL-12 administration, as early as 24 h after the first injection, which suggests rapid recruitment of tumor-specific CTLp into the blood as a possible mechanism of the observed burst.

Interestingly, rHuIL-12 administration did not similarly affect the peripheral frequency of any antigen-specific T-cell precursor. In fact, in patient 1, in contrast to the burst observed for Melan-A/Mart-1-specific precursors, the number of circulating influenza-matrix58–66-specific precursors dropped at day +1 and +4 after rHuIL-12 and showed a rebound only at day +7. In addition, evaluation of the frequency of Melan-A/Mart-1-specific T cells in peripheral blood by LDA and by tetramers provided similar values, in agreement with our recent results that indicated that the LDA technique, as modified by us, achieves the same efficiency in detecting antigen-specific T cells as the enzyme-linked immunospot or tetramer staining (22) .

Administration of rHuIL-12 may promote rapid clonal expansion of tumor-specific CTLp. This possibility is supported by several lines of evidence. For example, repeated s.c. injections of IL-12 can lead to selective expansion of a CD8+ T-cell subset characterized by high expression of CD18 and by non-MHC-restricted cytotoxicity after culture with IL-12 plus IL-2 (35) . In mice, the in vivo injection of tumor cells producing IL-12 has been shown to induce proliferation of CD8+ T cells with a memory phenotype (CD44hi) in both spleen and lymph nodes (36) . Furthermore, an increase in the frequency of antitumor CD8+ T cells, as inferred by TCR repertoire analysis, has been suggested in both blood and tumor site after local IL-12 gene therapy in mice (37) . More recently, IL-12 has been shown to act as a“ third signal” (in addition to TCR engagement and costimulation) promoting activation and clonal expansion of naive CD8+ T cells (38) . However, in our experiments of cell cycle evaluation, performed as early as 4 and 7 days after the first rHuIL-12 administration, no evidence was obtained of enhanced T-cell proliferation in vivo in peripheral blood. Nevertheless, we cannot rule out the possibility that rHuIL-12 did promote rapid CTLp proliferation in the tissues other than blood and before the peripheral burst of CTLp was induced and detected.

Serum levels of soluble adhesion molecules and phenotype analysis of circulating CD3+ T cells provided evidence for an early and marked effect of rHuIL-12 on several receptor/ligand pairs involved in regulating interaction of T cells with endothelial cells and T-cell homing. The data suggested that s.c. rHuIL-12 treatment could activate a mechanism that may promote adhesion and then extravasation of activated/memory T cells by the LFA-1/ICAM-1, VLA-4/VCAM-1 and CLA/E-selectin pathways. This possibility is in agreement with experimental models that show that IL-12 therapy can induce T-cell infiltration of tumor tissue associated with enhanced VCAM-1 expression on tumor blood vessels (7) . Furthermore, in mice, IL-12-induced T-cell migration to tumor tissue can be inhibited by treating tumor-bearing animals with antibodies to either VLA-4 and LFA-1 (39) . Additional experimental evidence indicates that IL-12 can promote adhesion-dependent homing of T cells to both skin and liver (40, 41) , and in vivo treatment with antibodies to VCAM-1 can abrogate hepatic recruitment, in mice, of T and NK cells induced by systemic IL-12 (41) .

An increased proportion of CLA+ T cells was found in peripheral blood after rHuIL-12 administration. Interestingly, IL-12 has been shown to induce expression of this homing receptor (42) , and it is known that trans-endothelial migration of CLA+ T cells requires the VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways (43) . Inasmuch as circulating CLA+ T cells comprise memory/effector T cells (44) , then it is possible that rHuIL-12 treatment may promote adhesion-dependent extravasation of CLA+-activated/memory T cells. This possibility is in agreement with the memory phenotype (CD45RO+) expressed by T cells infiltrating posttherapy lesions in the present study. Furthermore, the increased expression of CLA antigens was more pronounced after the first cytokine injection than after the last one. This reduced biological effect of the last rHuIL-12 administration was observed also when looking at CTLp frequency and may be attributable to a reduced systemic availability of the cytokine, as previously described in these patients at the end of the therapy cycles (14) .

The immunohistochemical analysis of metastatic tissues that were removed from patients indicated the presence of a brisk, or non-brisk infiltrate of CD8+ T cells with a cytolytic (TIA-1+) and memory (CD45RO+) phenotype in posttherapy lesions. In addition, although all of the clinically regressing lesions could not be analyzed, other available posttherapy metastases showed increased histological evidence of tumor necrosis and/or regression in comparison with pretherapy tumor tissues. These data suggest that the antitumor activity of rHuIL-12, as evaluated at the histological level, may be greater than previously assessed at the clinical level in the same patients (14) . As suggested by some experimental models (45) and by clinical data obtained in cutaneous T-cell lymphoma patients treated with rHuIL-12 (18) , it is possible that the infiltrating CD8+, TIA-1+, CD45RO+ T cells may be involved in the observed tumor regressions. However, we cannot exclude the possibility that additional mechanisms, such as inhibition of angiogenesis and/or induction of endothelial wall injury, may contribute to the antitumor activity of rHuIL-12 in the clinical setting.

Finally, the increase in peripheral antitumor CTLp frequency was transient and subjected to attenuation after the last rHuIL-12 administration, in comparison with the peak value detected after the first injection. A similar “adaptive response,” was previously observed by us and others when looking at the serum levels of IL-12 and at induction of serum IFN-γ (13, 14) and IL-10 (14) after rHuIL-12 administration. In particular, in patients, a burst of IFN-γ was detected in serum within 1–2 days after the first rHuIL-12 injection, whereas lower levels were induced after the last rHuIL-12 administration (14) . The adaptive response could reduce the toxic effects associated with prolonged IL-12 therapy, but it might also contribute to reducing the antitumor efficacy of the cytokine. In support of this possibility, it has been shown (16) that a single IL-12 dose, given weeks before consecutive doses, can abrogate IL-12-associated toxicity but can also can block induction of IFN-γ, an important mediator of IL-12 biological effects (1) . At least two mechanisms have been described that could explain the adaptive response. On one hand, IL-12 administration to either cancer patients or mice, by fixed weekly doses, promotes enhanced IL-12 receptor expression in lymphoid cells, which may favor increased IL-12 clearance but leads also to reduced IFN-γ mRNA expression (46) . On the other hand, murine models of tumor immunotherapy have indicated the involvement of nitric oxide, produced by macrophages in response to IFN-γ, in the immune suppression induced by IL-12 given i.p. (47) . Additional studies are clearly needed to fully understand the mechanism of the adaptive response, thus improving the clinical efficacy of rHuIL-12 in anticancer therapy.

Acknowledgments

We gratefully thank Dr. G. Parmiani for advice and helpful discussions, Prof. E. Berti, (Institute of Dermatological Science, University of Milan, IRCCS Ospedale Maggiore, Milan, Italy) for the gift of mAbs, Dr. C. Lombardo (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan) for HLA tissue typing of patients. We gratefully acknowledge the skilful technical work of A. Molla and the excellent secretarial assistance of B. Canova.

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 in part by funds from the Italian Association for Cancer Research (AIRC, Milan, Italy) and the Italy-United States Program on Therapy of Tumors (Istituto Superiore di Sanità, Rome, Italy).

  • ↵2 To whom requests for reprints should be addressed, at Human Tumors Immunobiology Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390817; Fax: 39-02-2390630; E-mail: Anichini{at}istitutotumori.mi.it

  • ↵3 The abbreviations used are: IL, interleukin; CLA, cutaneous lymphocyte antigen; CTLp, CTL precursor(s); ICAM, intercellular adhesion molecule(s); LFA, lymphocyte function-associated antigen; LDA, limiting-dilution analysis; mAb, monoclonal antibody; Melan-A/Mart-1, melanoma antigen-A/melanoma antigen recognized by T cell; PBL, peripheral blood lymphocyte; rHu, recombinant human; TCR, T cell receptor; TIL, tumor-infiltrating lymphocyte; VCAM, vascular cell adhesion molecule(s); VLA, very late antigen; NK, natural killer; PE, phycoerythrin; FL, fluorescence.

  • Received December 28, 1999.
  • Accepted April 26, 2000.

References

  1. ↵
    Trinchieri G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol., 70: 83-243, 1998.
    OpenUrlCrossRefPubMed
  2. ↵
    Gately M. K., Renzetti L. M., Magram J., Stern A. S., Adorini L., Gubler U., Presky D. H. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu. Rev. Immunol., 16: 495-521, 1998.
    OpenUrlCrossRefPubMed
  3. ↵
    Sgadari C., Angiolillo A. L., Tosato G. Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10. Blood, 87: 3877-3882, 1996.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Brunda M. J., Luistro L., Warrier R. R., Wright R. B., Hubbard B. R., Murphy M., Wolf S. F., Gately M. K. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J. Exp. Med., 178: 1223-1230, 1993.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Nastala C. L., Edington H. D., McKinney T. G., Tahara H., Nalesnik M. A., Brunda M. J., Gately M. K., Wolf S. F., Schreiber R. D., Storkus W. J., Lotze M. T. Recombinant IL-12 administration induces tumor regression in association with IFN-γ production. J. Immunol., 153: 1697-1706, 1994.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Mu J., Zou J. P., Yamamoto N., Tsutsui T., Tai X. G., Kobayashi M., Herrmann S., Fujiwara H., Hamaoka T. Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes. Cancer Res., 55: 4404-4408, 1995.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Boggio K., Nicoletti G., Di Carlo E., Cavallo F., Landuzzi L., Melani C., Giovarelli M., Rossi I., Nanni P., De Giovanni C., Bouchard P., Wolf S., Modesti A., Musiani P., Lollini P. L., Colombo M. P., Forni G. Interleukin-12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice. J. Exp. Med., 188: 589-596, 1998.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Tahara H., Zeh H. J., III, Storkus W. J., Pappo I., Watkins S. C., Gubler U., Wolf S. F., Robbins P. D., Lotze M. T. Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo. Cancer Res., 54: 182-189, 1994.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Tahara H., Zitvogel L., Storkus W. J., Zeh H. J., III, McKinney T. G., Schreiber R. D., Gubler U., Robbins P. D., Lotze M. T. Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector. J. Immunol., 154: 6466-6474, 1995.
    OpenUrlAbstract
  10. ↵
    Caruso M., Pham-Nguyen K., Kwong Y. L., Xu B., Kosai K. I., Finegold M., Woo S. L., Chen S. H. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc. Natl. Acad. Sci. USA, 93: 11302-11306, 1996.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Bramson J. L., Hitt M., Addison C. L., Muller W. J., Gauldie J., Graham F. L. Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum. Gene Ther., 7: 1995-2002, 1996.
    OpenUrlCrossRefPubMed
  12. ↵
    Chen L., Chen D., Block E., O’Donnell M., Kufe D. W., Clinton S. K. Eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer. J. Immunol., 159: 351-359, 1997.
    OpenUrlAbstract
  13. ↵
    Atkins M. B., Robertson M. J., Gordon M., Lotze M. T., DeCoste M., DuBois J. S., Ritz J., Sandler A. B., Edington H. D., Garzone P. D., Mier J. W., Canning C. M., Battiato L., Tahara H., Sherman M. L. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin. Cancer Res., 3: 409-417, 1997.
    OpenUrlAbstract
  14. ↵
    Bajetta E., Del Vecchio M., Mortarini R., Nadeau R., Rakhit A., Rimassa L., Fowst C., Borri A., Anichini A., Parmiani G. Pilot study of subcutaneous recombinant human interleukin 12 in metastatic melanoma. Clin. Cancer Res., 4: 75-85, 1998.
    OpenUrlAbstract
  15. ↵
    Motzer R. J., Rakhit A., Schwartz L. H., Olencki T., Malone T. M., Sandstrom K., Nadeau R., Parmar H., Bukowski R. Phase I trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma. Clin. Cancer Res., 4: 1183-1191, 1998.
    OpenUrlAbstract
  16. ↵
    Leonard J. P., Sherman M. L., Fisher G. L., Buchanan L. J., Larsen G., Atkins M. B., Sosman J. A., Dutcher J. P., Vogelzang N. J., Ryan J. L. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-γ production. Blood, 90: 2541-2548, 1997.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Robertson M. J., Cameron C., Atkins M. B., Gordon M. S., Lotze M. T., Sherman M. L., Ritz J. Immunological effects of interleukin 12 administered by bolus intravenous injection in patients with cancer. Clin. Cancer Res., 5: 9-16, 1999.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Rook A. H., Wood G. S., Yoo E. K., Elenitsas R., Kao D. M. F., Sherman M. L., Witmer W. K., Rockwell K. A., Shane R. B., Lessin S. R., Vonderheid E. C. Interleukin-12 therapy of cutaneous T-cell lymphoma induces lesion regression and cytotoxic T cell responses. Blood, 94: 902-908, 1999.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Anichini A., Mortarini R., Maccalli C., Squarcina P., Fleischhauer K., Mascheroni L., Parmiani G. Cytotoxic T cells directed to tumor antigens not expressed on normal melanocytes dominate HLA-A*0201-restricted immune repertoire to melanoma. J. Immunol., 156: 208-217, 1996.
    OpenUrlAbstract
  20. ↵
    Maccalli C., Pisarra P., Vegetti C., Sensi M., Parmiani G., Anichini A. Differential loss of T cell signaling molecules in metastatic melanoma patients’ T lymphocyte subsets expressing distinct TCR variable regions. J. Immunol., 163: 6912-6923, 1999.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Lakhanpal S., Gonchoroff N. J., Katzmann J. A., Handwerger B. S. A flow cytofluorometric double staining technique for simultaneous determination of human mononuclear cell surface phenotype and cell cycle phase. J. Immunol. Methods, 96: 35-40, 1987.
    OpenUrlCrossRefPubMed
  22. ↵
    Anichini A., Molla A., Mortarini R., Tragni G., Bersani I., Di Nicola M., Gianni A. M., Pilotti S., Dunbar R., Cerundolo V., Parmiani G. An expanded peripheral T cell population to a CTL-defined melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTL, but does not overcome tumor escape from immune surveillance in metastatic lesions. J. Exp. Med., 190: 651-668, 1999.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Ogg G. S., Dunbar P. R., Romero P., Chen J. L., Cerundolo V. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. J. Exp. Med., 188: 1203-1208, 1998.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Valmori D., Fontaneau J. F., Lizana C. M., Gervois N., Lienard D., Rimoldi D., Jongeneel V., Jotereau F., Cerottini J. C., Romero P. Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/Mart-1 immunodominant peptide analogues. J. Immunol., 160: 1750-1758, 1998.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Mortarini R., Anichini A., Di Nicola M., Siena S., Bregni M., Belli F., Molla A., Gianni A. M., Parmiani G. Autologous dendritic cells derived from CD34+ progenitors and from monocytes are not functionally equivalent APC in the induction of Melan-A/Mart-127–35-specific CTL from PBL of melanoma patients with low frequency of CTL precursors. Cancer Res., 57: 5534-5541, 1997.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Kawakami Y., Eliyahu S., Sakaguchi K., Robbins P. F., Yannelli J. R., Appella E., Rosenberg S. A. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2 restricted tumor infiltrating lymphocytes. J. Exp. Med., 180: 347-352, 1994.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Parham P., Barnstable C. J., Bodmer W. F. Use of monoclonal antibody (W6/32) in structural studies of HLA-A, -B, -C antigens. J. Immunol., 123: 342-349, 1979.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Clark W. H., Jr., Elder D. E., Guerry IV D., Braitman L. E., Trock B. J., Schultz D., Synnestvedt M., Halpern A. C. Model predicting survival in stage I melanoma based on tumor progression. J. Natl. Cancer Inst., 81: 1893-1904, 1989.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Anderson P., Nagler-Anderson C., O’Brien C., Levine H., Watkins S., Slayter H. S., Blue M. L., Schlossman S. F. A monoclonal antibody reactive with 15 kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes. J. Immunol., 144: 574-582, 1990.
    OpenUrlAbstract
  30. ↵
    Finke J. H., Zea A. H., Stanley J., Longo D. L., Mizoguchi H., Tubbs R. R., Wiltrout R. H., O’Shea J. J., Kudoh S., Klein E., Bukowski R. M., Ochoa A. C. Loss of T-cell receptor ζ chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res., 53: 5613-5616, 1993.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Zea A. H., Cutri B. D., Longo D. L., Alvord W. G., Strobl S. L., Mizoguchi H., Creekmore S. P., O’Shea J. J., Powers G. C., Urba W. J., Ochoa A. C. Alterations in T cell receptor and signal transduction molecules in melanoma patients. Clin. Cancer Res., 1: 1327-1335, 1995.
    OpenUrlAbstract
  32. ↵
    Van Parijs L., Perez V. L., Biuckians A., Maki R. G., London C. A., Abbas A. K. Role of interleukin 12 and costimulators in T cell anergy in vivo. J. Exp. Med., 186: 1119-1128, 1997.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Ushio H. R., Tsuji F., Szczepanik M., Kawamoto K., Matsuda H., Askenase P. W. IL-12 reverses established antigen-specific tolerance of contact sensitivity by affecting costimulatory molecules B7–1 (CD80) and B7–2 (CD86). J. Immunol., 160: 2080-2088, 1998.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Ryan K. R., Evavold B. D. Persistence of peptide-induced CD4+ T cell anergy in vitro. J. Exp. Med., 187: 89-96, 1998.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Gollob J. A., Schnipper C. P., Orsini E., Murphy E., Daley J. F., Lazo S. B., Frank D. A., Neuberg D., Ritz J. Characterization of a novel subset of CD8(+) T cells that expands in patients receiving interleukin-12. J. Clin. Investig., 102: 561-575, 1998.
    OpenUrlCrossRefPubMed
  36. ↵
    Belardelli F., Ferrantini M., Santini S. M., Baccarini S., Proietti E., Colombo M. P., Sprent J., Tough D. F. The induction of in vivo proliferation of long lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-α1 into syngeneic mice. Cancer Res., 58: 5795-5802, 1998.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Fernandez N. C., Levraud J. P., Haddada H., Perricaudet M., Kourilsky P. Higher frequency of specific CD8+ T cells in the tumor and blood is associated with efficient local IL-12 gene therapy of cancer. J. Immunol., 162: 609-617, 1999.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Curtsinger J. M., Schmidt C. S., Mondino A., Lins D. C., Kedl R. M., Jenkins M. K., Mescher M. F. Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells. J. Immunol., 162: 3256-3262, 1999.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Ogawa M., Tsuitsui T., Zou J. P., Mu J., Wijesuriya R., Yu W. G., Hermann S., Kubo T., Fujiwara H., Hamaoka T. Enhanced induction of very late antigen 4/lymphocyte function-associated antigen 1-dependent T cell migration to tumor sites after administration of interleukin 12. Cancer Res., 57: 2216-2222, 1997.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Myers K. J., Eppihimer M. J., Hall L., Wolitzky B. Interleukin-12-induced adhesion molecule expression in murine liver. Am. J. Pathol., 152: 457-468, 1998.
    OpenUrlPubMed
  41. ↵
    Fogler W. E., Volker K., Watanabe M., Wigginton J. M., Roessler P., Brunda M. J., Ortaldo J. R., Wiltrout R. H. Recruitment of hepatic NK cells by IL-12 is dependent on IFN-γ and VCAM-1 and is rapidly down-regulated by a mechanism involving T cells and expression of Fas. J. Immunol., 161: 6014-6021, 1998.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Leung D. Y., Gately M., Trumble A., Ferguson-Darnell B., Schlievert P. M., Picker L. J. Bacterial superantigens induce T cell expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin-12 production. J. Exp. Med., 181: 747-753, 1995.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Santamaria Babi L. F., Moser R., Perez Soler M. T., Picker L. J., Blaser K., Hauser C. Migration of skin-homing T cells across cytokine-activated human endothelial cell layers involves interaction of the cutaneous lymphocyte-associated antigen (CLA), the very late antigen-4 (VLA-4), and the lymphocyte function-associated antigen-1 (LFA-1). J. Immunol., 154: 1543-1550, 1995.
    OpenUrlAbstract
  44. ↵
    Picker L. J., Treer J. R., Ferguson-Darnell B. B., Collins P. A., Bergstresser P. R., Terstappen L. W. Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyte-associated antigen, a tissue-selective homing receptor for skin-homing T cells. J. Immunol., 150: 1122-1136, 1993.
    OpenUrlAbstract
  45. ↵
    Cavallo F., Signorelli P., Giovarelli M., Musiani P., Modesti A., Brunda M. J., Colombo M. P., Forni G. Antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (IL-12) or other cytokines compared with exogenous IL-12. J. Natl. Cancer Inst., 89: 1049-1058, 1997.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Rakhit A., Yeon M. M., Ferrante J., Fettner S., Nadeau R., Motzer R., Bukowski R., Carvajal D. M., Wilkinson V. L., Presky D. H., Magram J., Gately M. K. Down-regulation of the pharmacokinetic-pharmacodynamic response to interleukin-12 during long-term administration to patients with renal cell carcinoma and evaluation of the mechanism of this “adaptive response” in mice. Clin. Pharmacol. Ther., 65: 615-629, 1999.
    OpenUrlCrossRefPubMed
  47. ↵
    Koblish H. K., Hunter C. A., Wysocka M., Trinchieri G., Lee W. M. Immune suppression by recombinant interleukin (rIL)-12 involves interferon γ induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med., 188: 1603-1610, 1998.
    OpenUrlAbstract/FREE Full Text
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July 2000
Volume 60, Issue 13

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Peripheral Burst of Tumor-specific Cytotoxic T Lymphocytes and Infiltration of Metastatic Lesions by Memory CD8+ T Cells in Melanoma Patients Receiving Interleukin 12
Roberta Mortarini, Alessandra Borri, Gabrina Tragni, Ilaria Bersani, Claudia Vegetti, Emilio Bajetta, Silvana Pilotti, Vincenzo Cerundolo and Andrea Anichini
Cancer Res July 1 2000 (60) (13) 3559-3568;
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