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Immunology |
H. Lee Moffitt Cancer Center and the Department of Interdisciplinary Oncology, University of South Florida, Tampa, Florida
Requests for reprints: Dmitry I. Gabrilovich, H. Lee Moffitt Cancer Center, University of South Florida, MRC 2067, 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-903-6863; Fax 813-745-1328; E-mail: dgabril{at}moffitt.usf.edu.
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Introduction |
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Recent data from a number of groups have shown that ImC accumulated in tumor-bearing hosts play an important role in tumor nonresponsiveness by suppressing antigen-specific T-cell responses (2, 6–8). Earlier experiments from H. Schreiber's group (9) and from V. Bronte and N. Restifo (4) showed that depletion of murine Gr-1+ cells significantly improved CD8+ T-cell immune response and allowed for eradication of tumor. More recent work of J. Berzofsky's group showed that depleting Gr-1+ myeloid cells in vivo prevented tumor recurrence (10). ImC suppress immune response via number of different mechanisms, including loss or significant decrease of the expression of the T-cell receptor 
chain (CD3), which is the principal part of the T-cell receptor complex (11); inhibition of CD3/CD28–induced T-cell activation/proliferation by production of reactive nitrogen and oxygen intermediates (12); inhibition of IFN-
production by CD8+ T cells in response to the specific peptide presented by MHC class I molecules (5); prevention of the development of CTLs in vitro (13, 14); and induction of antigen-specific CD8+ T-cell tolerance in vivo (15). When cultured in vitro in the presence of appropriate growth factors, ImC could be differentiated into dendritic cell or macrophages (16–18). However, their differentiation is blocked when cells are transferred into tumor-bearing mice (17), indicating that tumor-derived factors play critical role in preventing differentiation of these cells. The role of these factors was further supported by the fact that surgical resection of the murine tumor decreased the number of ImC (19).
In cancer patients, ImC are defined as cells that express the common myeloid marker CD33 but lack expression of markers of mature myeloid and lymphoid cells and of the MHC class II molecule HLA-DR (7). An accumulation of phenotypic ImC was associated with the decreased number of dendritic cells in the peripheral blood of patients with head and neck, lung, or breast cancer (20). In functional testing, ImC isolated from peripheral blood of HLA-A2–positive cancer patients inhibited production of IFN- by CD8+ T cells restimulated with specific peptide-pulsed dendritic cells (7). Cells with similar phenotype were shown to suppress T-cell function in patients with advanced cancer patients (21). Thus, accumulation of ImC could be one of the mechanisms by which a growing tumor may induce an antigen-specific CD8+ T-cell unresponsiveness. It seems logical that elimination of those immune-suppressive cells may help to enhance the immune antitumor mechanisms in patients.
A promising, clinically relevant approach to reduce the proportion of ImC in tumor-bearing hosts may be use of agents that promote the differentiation of myeloid progenitors. Retinoic acids, ligands of the retinoic acid receptors [RAR; retinoid X receptor (RXR)], are among the compounds that may stimulate differentiation of myeloid progenitors into myeloid dendritic cells (5, 22). Mice with vitamin A deficiency (23) and mice treated with a pan-RAR antagonist (24) show accumulation of CD11b+Gr-1+ myeloid cells similar to the ImC that accumulate in cancer patients. Physiologic concentrations of all-trans retinoic acid (ATRA) induced in vitro differentiation of ImC from tumor-bearing mice into CD11c+MHC class II+ myeloid dendritic cell (5). In vivo, parenteral or oral administration of ATRA significantly reduced the presence of ImC in two different tested tumor models (25), independent of a direct antitumor effect of ATRA or decreased production of growth factors by tumor cells. Experiments with adoptive transfer showed that ATRA differentiated ImC in vivo into mature dendritic cell, macrophages, and granulocytes. The decreased presence of ImC in tumor-bearing mice noticeably improved CD4- and CD8-mediated tumor-specific immune responses. The combination of ATRA with two different types of cancer vaccines, in two different tumor models, significantly prolonged the antitumor effect of the treatment (25). These data suggest that elimination of ImC with ATRA could be a promising approach to improvement of immune response in cancer.
However, all these studies were done in vitro or in vivo in mice. The critical questions are if and how to use ATRA therapeutically to eliminate ImC in cancer patients. For the first time, in this study, we evaluate the effect of ATRA treatment on phenotype and function of dendritic cells, the presence of ImC, and antigen-specific immune response in metastatic, clear cell type, and kidney cancer patients.
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Materials and Methods |
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2, adequate renal and liver function, and no signs of anemia and leukopenia. Primary tumor was removed via nephrectomy or partial nephrectomy at least 90 days before start of treatment, other tumor debulking surgery at least 30 days before start, radiation therapy completed at least 7 days before start, and no required steroid treatment. Main exclusion criteria were the presence of not-resected primary tumor; brain metastasis with <90 days of stability after the end of active treatment; 
3 days of steroids at greater than physiologic replacement dose within the last 90 days; HIV; pregnancy (ATRA is a known teratogen); seizure medication that was contraindicated with ATRA; or other medical contraindication such as nonhealing wound, peripheral vascular disease, or congestive heart failure worse than New York Heart Association class I, or myocardial infarction or hypertension within 1 year. ATRA (Vesanoid) was obtained from commercial supply and provided to the subjects. A range of ATRA dose based on the experience in treatment of promyelocytic leukemia patients was selected as follows: 50, 100, and 150 mg/m2/d. The ATRA treatment dose was divided in three portions using 10 mg Vesanoid capsules, with instructions to take thrice a day, with at least 6 hours between each dose; missed doses were not made up for 21 doses. One week after the finish of ATRA, therapy with interleukin-2 (IL-2) started. The IL-2 (Proleukin) treatment included 30 planned doses, given as five doses per week with 2-day breaks (weekends), over 6 weeks. The first five doses were 250,000 IU/kg, and 125,000 IU/kg for the next 25 doses (26). Observed toxicity was graded using CTCAE3 criteria and tabulated. Management of toxicity was based on dose omission (ATRA, IL-2) or dose reduction. Following the completion of the IL-2, there was a 2- to 3-week break, with clinical and radiologic evaluation of the treatment response. Eight healthy donors comprised a control group.
Fifteen subjects received i.v., high-dose IL-2 for therapy of kidney cancer or melanoma among patients on a separate trial testing a novel schedule using courses of five doses of 600,000 units/kg/dose i.v. IL-2 at 8-hour intervals, repeated on 4 consecutive weeks (clinical results to be reported separately). The specimens collected at baseline and at the start of the 3rd week (after planned 10 doses of 600,000 units/kg IL-2) comprised the specimens used for an IL-2–only treatment group. These are at a 14-day interval, for the purpose of assessing IL-2 effect, to the day 14 and day 28 specimens from the ATRA-treated subjects.
Collection of blood samples and ATRA plasma level testing. For evaluation of immunologic variables, blood was collected before start of ATRA treatment, after the end of ATRA treatment (7 days after start), 1 week after the end of ATRA treatment before start of IL-2 therapy (14 days after start), and then every week for 4 weeks. Mononuclear cells (MNC) isolated at each of these time points were cryopreserved and stored for future analysis.
Specimens for ATRA pharmacokinetics were collected after the 1st (day 1), 10th (day 4), and 21st (day 7) doses, at time points "baseline," 1, 2, 3, and 4 hours after ATRA administration. These time points were selected based on previously published pharmacokinetics of ATRA (27, 28). The measurement of ATRA level in plasma was done at the Clinical Pharmacology Laboratory at the H. Lee Moffitt Cancer Center & Research Institute. Heparinized plasma samples that had been stored frozen were thawed and processed as described (29, 30). ATRA used to prepare solutions for standards was obtained from Sigma-Aldrich (St. Louis, MO). The internal standard chosen was retinol acetate (Sigma-Aldrich). The chromatography was done on an Agilent 1100 high-performance liquid chromatography system using a Zorbax 5 µm RX-C18 column (Agilent Technologies, Wilmington, DE) with a mobile phase consisting of a gradient of acetic acid (0.1%, v/v)/methanol/methyl tert-butyl ether (20:65.5:14.5, v/v/v) that increased in organic content to 10:74:16 (v/v/v) over 5 minutes, then held constant for 6 minutes, and returned to original conditions in 2 additional minutes. The run time was 16.5 minutes at a flow rate of 0.8 mL/min. The compounds were detected at a wavelength of 354 nm, with a linear range from 10 to 500 ng/mL. Quality control samples were checked at 30, 80, and 250 ng/mL and the limit of quantitation for the assay was 10 ng/mL. Calibration curves were calculated by linear regression with 1/y weighting to determine the slopes, intercepts, and correlation coefficients. Unknowns were plotted against the regression analysis of each assay to determine the concentration.
Evaluation of cell phenotype. All samples from one patient were analyzed simultaneously. Peripheral blood MNCs were thawed, cultured overnight in complete culture medium (RPMI 1640 and 10% FCS), and then used for the analysis. Cells were labeled with the antibodies for 40 minutes on ice and analyzed the same day by flow cytometry. The cocktail of lineage (Lin)–specific antibodies included phycoerythrin-conjugated antibodies against CD3, CD19, CD56, and CD14 (all from BD PharMingen, Franklin Lakes, NJ). In addition, we used PerCP-conjugated anti-HLA-DR antibody; antigen-presenting cell–conjugated anti-CD33 and CD11c antibodies; and FITC-conjugated anti-CD86, CD83, and CD40 antibodies (all from BD PharMingen). FITC-conjugated antibodies against CCR7 and CD123 were obtained from R&D Systems (Minneapolis, MN) and Miltenyi (Auburn, CA) respectively. The phenotype of the cells was evaluated by multicolor flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). One hundred thousand cells were collected from each variable to obtain reliable data. The following subsets of cells were evaluated: (a) immature myeloid cells: Lin–HLA–DR– CD33+; (b) dendritic cells: Lin–HLA–DR+; (c) myeloid dendritic cells: Lin–HLA–DR+ CD11c+ CD123–; (d) plasmacytoid dendritic cells: Lin–HLA–DR+ CD11c– CD123+; (e) mature dendritic cells: Lin+ HLA–DR+ and either CD86+, CD40+, CD83+, or CCR7+; (f) regulatory T cells: the presence of T regulatory cells was determined by staining the cells with antigen-presenting cell–conjugated anti-CD4, FITC conjugated anti-CD25 (both of which were from BD PharMingen), and phycoerythrin-conjugated anti-GITR (R&D Systems, Minneapolis, MN) antibodies.
Allogeneic mixed leukocyte reaction. T cells were purified from a leukocyte-enriched buffy coat from healthy donors using T-cell enrichment column (R&D Systems) according to the instructions from the manufacturer. MNCs obtained from patients were first irradiated at 25 Gy and then incubated with donor T cells in triplicates in round-bottomed, 96-well plates at T cells/MNC ratios 1:1, 1:2, 1:4, and 1:8. On day 4, 3[H]thymidine was added (1 µCi/well) and the cells were harvested 18 hours later. Thymidine uptake was measured using a liquid scintillation counter (Packard Instrument, Meriden, CT). T cells in culture medium alone were used as a background control.
Evaluation of T-cell function. MNCs were cultured in triplicates in round-bottomed, 96-well plates (105 MNC/well) for 4 days with either 0.1 µg/mL tetanus-toxoid (List Biological Labs, Campbell, CA) or 5 µg/mL phytohemagglutinin (PHA; Sigma). These concentrations of tetanus-toxoid and PHA were selected after initial testing. [3H]thymidine (1 µCi/well) was added 18 hours before cell harvest. Thymidine uptake was measured using a liquid scintillation counter (Packard Instrument). MNC in culture medium without any antigen were used as background controls. To measure T-cell response to stimulation with immobilized anti-CD3 antibody, round-bottomed, 96-wells plates were coated overnight at 4°C with 1 µg/mL anti-CD3 antibody (BD PharMingen) diluted in 1x Dulbecco's PBS. Excess of unbound anti-CD3 antibody was washed off with Dulbecco's PBS. Then, 1 x 105 MNC in 100 µL were added in triplicates and incubated for 4 days. T-cell proliferation was measured as described above.
Cytokine bead array assay. Irradiated patient MNCs were cultured with donors' T cells at 1:1 ratio for 48 hours. The supernatants were collected and stored at –80°C, until the analysis. The profile of cytokines secreted by T cells after stimulation with patient's MNC was evaluated using the BD Cytometric Bead Array kit (BD Biosciences). Each sample tube was analyzed on the BD FACSCalibur flow cytometer using the BD FACSComp Software.
Statistical analysis. Patients were assigned to one of three ATRA dose levels, at a 1:1:1 ratio, using a randomly permuted list assignments, with the assignment generally being made on the initial day of treatment. The present report describes the experience in treatment of all 18 subjects; the original protocol allowed treatment of up to 36 subjects. The purpose of the randomization was to provide an unbiased assignment, not to compare the arms. Data were analyzed using two-sided Wilcoxon-Mann-Whitney test or Student's paired t test.
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Results |
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Among the side effects observed during the ATRA portion of the treatment were dry skin, myalgia, and headache, all mild to moderate grades; these typically remitted within a day or two of end of ATRA administration. Per-patient observed best clinical responses, using Response Evaluation Criteria in Solid Tumors, were evaluated after 11 to 12 weeks of treatment. Among 18 treated subjects, there were 1 complete response (lung, adrenal, and lymph nodes) ongoing at 9+ months, no partial responses, 11 stable diseases, and 3 progression. Three patients could not tolerate IL-2 treatment and it was discontinued before finishing planned treatment. Two of these three patients had progressive disease and one had stable disease.
Consistent with previous reports (31, 32), the peak of the plasma ATRA concentration was observed 2 to 3 hours after oral administration. Importantly, when assayed on day 4 or 7 of ATRA treatment, a smaller peak was observed (Fig. 1A ) in the same time frame. ATRA concentration in patients' plasma varied depending on dose of the drug (Fig. 1B). However, based on the day 1 peak ATRA level, the subjects were easily separated into two groups. Nine patients had peak of ATRA concentration <135 ng/mL and eight patients had ATRA concentration >150 ng/mL (one patient specimen not collected; Fig. 1C). These two groups were composed of patients who received different doses of ATRA (Fig. 1B and C). Subsequent analysis suggests a critical role of plasma ATRA concentration in the biological effects of the drug. In further discussion, we refer to the patients with lower ATRA concentrations (<135 ng/mL) as LC patients and patients with higher ATRA concentrations (>150 ng/mL) as HC patients.
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First, we evaluated the effect of ATRA on the number of WBC. Treatment with ATRA did not affect the total number of WBC, neutrophils, or lymphocytes but induced slight decrease in the number of monocytes, which was restored to pretreatment level 1 week after finish of the treatment (Table 1 ). IL-2 treatment resulted in significant increased in total number of WBC, neutrophils lymphocytes, but not monocytes (Table 1).
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We evaluated the presence of two major subtypes of dendritic cells: myeloid dendritic cells and plasmacytoid dendritic cells. The proportion of mature dendritic cells was evaluated within the population Lin–HLA–DR+ cells based on the expression of four markers typically associated with the phenotype of mature cells: CD86, CD40, CD83, and CCR7. The proportion of myeloid dendritic cells in renal cell carcinoma patients was significantly reduced (Fig. 3A ), whereas the proportion of plasmacytoid dendritic cells remained at the control level (Fig. 3B). This resulted in significant decrease in myeloid dendritic cell/plasmacytoid dendritic cell ratio (Fig. 3C). No changes in the levels of these dendritic cell populations were observed in patients with ATRA LC (Fig. 3A-C). Patients with ATRA HC showed slight increase in the proportion of myeloid dendritic cells and decrease in the proportion of plasmacytoid dendritic cells. Although these changes were not statistically significant, they resulted in a substantial improvement of myeloid dendritic cell/plasmacytoid dendritic cell ratio (Fig. 3A-C). Subsequent treatment with IL-2 equally reduced the proportion of both these populations and therefore did not change the proportion of myeloid dendritic cell/plasmacytoid dendritic cell (Fig. 3A-C). The proportion of mature dendritic cells was significantly reduced in patients with renal cell carcinoma. Treatment with ATRA resulted in slight increase in their presence. However, it did not reach statistical significance (Fig. 3D and data not shown).
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and IL-2 and decreased the production of Th2-type cytokine (IL-5). Another Th2-type cytokine IL-10 was below the level of detection. These changes resulted in increase of Th1/Th2–type cytokines ratios from 4.6 before ATRA treatment to 51.2, 7 days after ATRA treatment (P < 0.05) and 69.3 after 14 days of ATRA treatment (P < 0.05).
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ATRA treatment did not affect the proportion of CD4+CD25+GITR+ regulatory T cells (data not shown). IL-2 therapy initially increased that proportion albeit the change was not statistically significant (from 0.89 ± 1.09 before start of the treatment to 4.70 ± 7.42 on day 21; P = 0.06). It decreased slightly later during the course of IL-2 treatment (2.88 ± 4.24 on day 28 and 1.87 ± 1.72 on day 35, P > 0.06 in both cases).
The effect of IL-2 therapy alone on ImC, dendritic cells, and T-cell responses. The data presented above showed that effects of ATRA on ImC, dendritic cell subsets, and function were eliminated after start of IL-2 therapy. It was not clear whether those changes were indeed the effect of IL-2 therapy or the result of a time-dependent attenuation of the ATRA effect. The original design of the trial did not allow for long observation period between ATRA and IL-2 treatment. Therefore, to evaluate potential role of IL-2 on immunologic variables, we tested samples of blood obtained from patients enrolled in separate clinical trial.
Fifteen patients with metastatic cancer were treated as described in Materials and Methods. They have received several cycles of IL-2 therapy alone. Blood was collected before treatment and after the first 3-week cycle. Treatment with IL-2 alone produced significant decrease in the proportion of dendritic cells and myeloid dendritic cells but not plasmacytoid dendritic cells (Fig. 5 ). Slight increase in the proportion of ImC was observed. As a result, dendritic cell/ImC and myeloid dendritic cell/plasmacytoid dendritic cell ratios were reduced significantly (Fig. 5). IL-2 therapy also significantly decreased the ability of MNC to stimulate allogeneic T cells. The response of T cells to stimulation with tetanus-toxoid or anti-CD3 antibody was reduced. However, those differences did not reach statistical significance (Fig. 5). In addition, IL-2 significantly increased the proportion of regulatory T cells.
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Discussion |
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, RARß, RAR and RXR, RXRß, RXR—activate transduction of target genes via complex genetic and epigenetic mechanisms (41). In therapeutic concentrations, ATRA does not directly inhibit the growth of most solid tumors. This could be partly explained by decreased expression of the receptors (42). In this study, we addressed, for the first time, the question whether ATRA can affect ImC and dendritic cells in cancer patients. This question is critical not only for understanding the biology of ATRA effects on human myeloid cells but also for the potential use of this treatment in association with cancer vaccines. The trial was done in patients with metastatic clear cell kidney cancer. The ATRA treatment was followed by an IL-2 schedule for which there is extensive experience. Although generally well tolerated, ATRA has pharmacokinetics that are complicated by heterogeneity of absorption and clearance between individuals, and the capacity to induce its own metabolism with chronic dosing. Therefore, it was important to establish an optimal dose schedule of the drug. We used a 3-fold range of dose levels, encompassing doses previously identified to be generally well tolerated and safe as well as having a biological effect (induced maturation and apoptosis of APL cells of promyelocytic leukemia; refs. 31, 43). As anticipated, ATRA pharmacokinetics exhibited wide interpatient variability. Median peak levels declined over the 7-day ATRA treatment period, consistent with previous observations that continuous administration of ATRA resulted in decreased plasma level of this drug (31). Analysis of ATRA levels in patients' blood revealed the existence of two groups with relatively low and high peak ATRA concentrations. It is important that low concentration group was composed of all six patients that received lowest dose of ATRA (50 mg/m2/d) but also two patients who received the 100 mg/m2/d dose and one patient with 150 mg/m2/d dose. To evaluate the relationship between ATRA concentration and its effect on ImC and dendritic cells, we compared ATRA HC and LC patients. This plasma level–based analysis is more logical than the dose-assigned one, because of the heterogeneity issue. Each dose group was relatively small (only six patients) and some patients in the groups with higher dose had low ATRA concentration in plasma. The mechanism of this heterogeneity is not entirely clear. ATRA does not require a specific transport mechanism; therefore, general conditions that influence absorption of the drug during oral administration like quantity and quality of recent food intake, the condition of gastrointestinal tract, etc., may affect the absorption and serum concentration of ATRA. The heterogeneity observed within dose cohorts seems comparable with that seen in previous studies. It is likely that in a larger cohort, these differences will be less prominent and patients can be separated based only on dose administered. Our data indicated that ATRA concentration below 135 ng/mL did not significantly affect either phenotype or function of dendritic cells and ImC, whereas higher concentration (>150 ng/mL) showed a significant effect. This points to the specific nature of the observed effects of ATRA and suggests that ATRA dose of 150 mg/m2/d could be the best for achieving a biological effect. At the highest dose level, ATRA concentrations in plasma on days 4 and 7 were similar to the ineffective level observed on day 1 of the lower ATRA dose. This provides a basis to conjecture that a shorter duration at the higher dose level could be sufficient to achieve optimal effect.
ATRA treatment did not change the number of WBC. Only slight decrease in monocyte count was found immediately after finish of the treatment. It returned to pretreatment level 7 days later. To reduce interexperimental variations, MNC collected at different time points from the same patient were thawed and analyzed at the same time. Although no additional cell purification was done, MNCs were cultured overnight before the analysis. This was important for two reasons. First, the phenotype and especially the function and of the cells analyzed immediately after the thawing can be significantly affected. Second, normal peripheral blood contains small proportion of immature myeloid cells and immature dendritic cells that quickly differentiate during overnight culture. This probably reflects the process that is taking place in tissues. We wanted to distinguish those cells from abnormal ImC accumulated in cancer patients. Overnight incubation would help to achieve this goal. As previously observed in other advanced cancer patients (7, 20), subjects with metastatic kidney cancer have almost 3-fold excess of ImC. ATRA significantly reduced that proportion. This was evident when samples from all patients on trial were analyzed together. However, the differences became stronger when we compared patients with low and high ATRA plasma concentration. Slight decrease in ImC was found in former group, whereas the latter one showed dramatic reduction of the proportion of these cells, restoring them to the control level. Thus, these data confirmed previous in vitro observations (7, 35). ATRA had little effect on the proportion of total dendritic cells. The ratio between myeloid and lymphoid dendritic cells may play an important role in tumor-associated immune defects in light of observations that myeloid dendritic cells primarily responsible for induction of immune response and plasmacytoid dendritic cells under certain circumstances can have immunosuppressive features (2, 44–46). In a number of studies, preferential loss of myeloid dendritic cells but not plasmacytoid dendritic cells in cancer patients was reported (20, 47, 48). A similar effect was observed in the patients with metastatic renal cell carcinoma. The proportion of myeloid dendritic cells was significantly reduced, whereas plasmacytoid dendritic cells were not affected. This resulted in dramatic drop in myeloid dendritic cell/plasmacytoid dendritic cell ratio. At "effective" concentrations, ATRA slightly increased the proportion of myeloid dendritic cells and decreased the proportion of plasmacytoid dendritic cells that restored myeloid dendritic cell/plasmacytoid dendritic cell ratio to the control level. Our previous study in vitro has shown that ImC isolated from cancer patients suppressed allogeneic MLR and antigen-specific T-cell responses (7). Elimination of ImC and increase in myeloid dendritic cell/plasmacytoid dendritic cell ratio after ATRA treatment apparently was responsible for the significant improvement of allogeneic MLR and antigen-specific T-cell response (tetanus-toxoid), which depends on adequate function of antigen-presenting cells. ATRA did not affect T-cell response to immobilized CD3 antibody and PHA-induced T-cell proliferation that requires little or no antigen-presenting cell participation. Consistent with preferential effect on myeloid rather than T-cell compartment, ATRA did not affect regulatory T cells.
All patients on this trial were treated with s.c. IL-2 therapy following ATRA. Therefore, the duration of ATRA effect could not be clearly established in this study. IL-2 therapy substantially increased the total number of lymphocytes. This effect was reported in previous clinical trials and was attributed to accumulation of natural killer (NK) and lymphokine-activated killer (LAK) cells (reviewed in ref. 49). Seven days after start of IL-2 therapy, the proportion of ImC was significantly increased. It was especially dramatic when taking into account the significant increase in the total number of MNCs. Apparently, in addition to stimulation of lymphocyte production, IL-2 also stimulates myeloid lineage that resulted in increased production of immature cells and decreased proportion of dendritic cells. Increased number of neutrophils observed in these patients was consistent with this fact. Importantly, IL-2 also decreased antigen- and mitogen-driven T-cell proliferation. Although the precise mechanism of this effect is not clear, it could be attributed to a significant increase in the proportion of regulatory T cells observed in these patients. Investigation of the effect of IL-2 was not a goal of this study. However, it is consistent with a hypothesis that limited clinical success of IL-2 therapy could be attributable to the accumulation of immunosuppressive ImC, which are able to suppress the very same antigen-specific immune response IL-2 therapy is trying to enhance. It has been previously reported that some patients showed profound activation of NK and LAK without any evidence of tumor regression (50).
Observed IL-2 effects raised important question, whether elimination of the positive effect of ATRA on ImC and dendritic cells was due to IL-2 effect or due to the short duration of ATRA effect. Precise analysis would require new trial in a different group of patients where the effect of ATRA could be monitored for a longer period of time. However, in a framework on this study, we could partially address this question by evaluating the effect of IL-2 in patients who did not receive ATRA treatment. Our data showed that IL-2 alone exerted exactly the same effect as in combination with ATRA. This strongly suggests that abrogation of ATRA effect was probably due to the effect of IL-2. Our study suggests that for the purpose of improvement of host immune responses and to provide the support for cancer vaccines ATRA probably should not be combined with IL-2. However, IL-2 therapy can have a major therapeutic effect in a subset of patients with renal cell cancer. It is possible that accumulation of ImS and Treg in patients treated with IL-2 could limit the clinical efficacy of this therapy. If this is the case, then ATRA could be considered as potentially useful addition to IL-2. Specifically designed clinical trials are needed to address this question.
Thus, at an effective dose, ATRA treatment substantially reduced the presence of ImC and improved myeloid/lymphoid dendritic cell ratio. This resulted in improved dendritic cell function and antigen-specific immune response. This indicates that impaired dendritic cell phenotype and immune function in patients with advanced cancer can be effectively modulated pharmacologically and provides strong rationale for combining ATRA treatment with different cancer vaccines.
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Acknowledgments |
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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.
We thank John Seigne, M.B., B.Ch., for help in the development of the clinical trial.
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Footnotes |
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Received 5/10/06. Revised 6/29/06. Accepted 7/10/06.
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References |
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chain of T-cell receptor complex and antigen-specific T-cell responses. Proc Natl Acad Sci U S A 1996;93:13119–24.This article has been cited by other articles:
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