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Immunology |
Departments of 1 Pathology, 2 Surgery, and 3 Internal Medicine and 4 Tumor Immunology and Immunotherapy Program of the Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan
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ABSTRACT |
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50% of the donor cells recovered from these sites after 5 days. Finally, donor T cells entering metastases both early (within 1–2 days) and late (after 2 days) suppressed tumor growth, but the early recruits accounted for most of the therapeutic response. Thus, cultured TDLN cells migrate directly into tumor-bearing organs and seed the recirculating pool of lymphocytes after infusion. Small fractions of the later differentiate in lymphoid organs and migrate into the lungs but appear less effective than effector cells in the initial bolus. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The essential first step after infusion of the cultured T cells is their accumulation within metastatic lesions. A variety of murine studies demonstrate a direct correlation between the suppression of tumor growth and the number of adoptively transferred cells within lesions (8, 9, 10, 11) . The initial entry of infused cells into tumors requires T-cell receptor-triggered differentiation ex vivo, but it is not tumor antigen specific. Kjaergaard and Shu (12) observed that T lymphoblasts cultured from normal spleen or lymph nodes draining MCA-205 (methyl cholanthrene-induced murine fibrosarcoma) and MCA-207 tumors trafficked equally well into 10-day-old MCA-205 pulmonary metastases. In contrast, naïve, resting splenic T cells could not enter the pulmonary metastases. The fate of recruited cells within the tumor microenvironment influences the clinical outcome as well. Shrikant and Mescher (8) found that early control of i.p. tumor correlated with a spike in the accumulation of antigen-specific effector cells. However, the number of effector T cells in the peritoneal cavity rapidly diminished, accompanied by an increase in tumor burden (8) . Furthermore, Hanson et al. (9) observed that extracellular signal-regulated kinase 2-restricted transgenic murine T cells infiltrated and suppressed the growth of 4-day-old s.c. fibrosarcomas expressing mutated extracellular signal-regulated kinase 2 (CMS5). In contrast, these cells failed to infiltrate or control the growth of 7-day-old s.c. CMS5 tumors (9) . Thus, both the nonspecific recruitment of effector T cells and subsequent events within the tumor microenvironment influence the magnitude and specificity of the antitumor response after adoptive immunotherapy.
The physiological mechanisms responsible for effector T-cell accumulation within tumors are incompletely understood. Plautz and colleagues found that the accumulation of infused, cultured TDLN cells in murine lung metastases is exquisitely sensitive to pertussis toxin (PTX), indicating that G-protein-dependent chemokine receptors are necessary (13) . However, PTX inhibits the entry of T cells into both immune reactions in tissues and secondary lymphoid organs (14 , 15) . Therefore, donor cells in lung metastases may enter from the initial bolus exclusively and/or differentiate within secondary lymphoid organs and then traffick into the lung at later time points. In this regard, Dummer et al. (16) reported that naïve, tumor-specific T cells proliferated within the lymphoid organs of sublethally irradiated recipients then trafficked to s.c. tumor nodules and suppressed their growth. Thus homeostatic proliferation of naïve T cells in depleted lymphoid organs promotes their differentiation and delivery to metastatic lesions. Whether the ex vivo-expanded T-lymphoblast populations used for adoptive immunotherapy will also proliferate in secondary lymphoid organs and subsequently traffick to metastases remains to be determined.
In summary, the existing literature on adoptive immunotherapy with cultured tumor-reactive T cells provides important insights into the recruitment process, but it does not fully explore the route and time course for donor T-cell entry into tumor-bearing tissues. In particular, the relative contributions of donor cells that enter tumors early and those that differentiate in secondary lymphoid organs then enter tumors late are unknown. Such studies require experimental techniques that can distinguish donor T cells entering tumor-bearing organs early after infusion from those that enter late. Without this ability, one cannot identify the sources for donor T-cell recruitment or reliably evaluate the fate of donor cells after entry into tissues. Consequently, we used delayed, PTX-mediated blockade of lymphocyte recruitment to investigate the trafficking and accumulation of cultured TDLN cells during adoptive immunotherapy. This report demonstrates that the sources and clinical impact of donor T cells entering the lung change markedly over time.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumors.
The MCA-205 cell line is a 3-methylcholanthrene-induced fibrosarcoma (provided by Dr. J. C. Yang; NIH, Bethesda, MD). The preparation and passage of these tumors were described previously (17)
. The tumors were maintained by serial s.c. transplantation in syngeneic mice, and they were used between the 5th and 8th s.c. passage. Furthermore, tumors from the same s.c. passage were used to generate TDLN cells and establish pulmonary metastases in a given experiment. Consequently, "antigenic drift" that occurred during s.c. passage was present in the neoplastic cells used for immunization and as therapeutic targets. Tumor cell suspensions were prepared from solid tumors by enzymatic digestion and washed in HBSS before i.v. and s.c. tumor inoculation.
TDLNs.
In each experiment, tumors were established in the flanks of 5–10 C57BL/6 CD45.1 mice by s.c. injection of 1 x 106 disaggregated tumor cells in 0.1 ml of HBSS. Nine days later, the regional lymph nodes (TDLNs) were harvested, mechanically disrupted using the blunt end of a 10-cc syringe in an etched Petri dish, and filtered through a 50 µm filter to yield a single cell suspension.
In Vitro Activation.
The TDLN cells were resuspended in complete media consisting of RPMI 1640 with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 µg/ml streptomycin, 100 units/ml penicillin G, 0.1 mM Non-Essential Amino Acids, 0.5 µg/ml fungizone (all from Life Technologies, Inc., Grand Island, NY), and 50 µM 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO). The TDLN cells were then cultured for 2 days at 0.5–2 x 106 cells/well in 24-well plates coated with an anti-CD3 monoclonal antibody (2C11 hybridoma from Dr. Jeffrey Bluestone; University of Chicago, Chicago, IL). The cells were then resuspended at 2 x 105 cells/ml in fresh complete media containing 60 IU/ml interleukin (IL)-2 (Chiron Therapeutics, Emeryville, CA) and cultured for 3 days at 37°C.
Cell Labeling.
Before adoptive transfer into tumor-bearing mice, the TDLN cells were labeled with 1–5 µM 5-(and 6-) carboxyfluorescein diacetate, succinimidyl ester [CFSE (C-1157; Molecular Probes, Inc., Eugene, OR)]. The cells were washed twice in PBS to remove residual serum and resuspended at a concentration of 10 x 106 cells/ml PBS. CFSE at 1 µM [for fluorescence-activated cell-sorting (FACS) analysis] or 5 µM (for immunohistochemistry) was added for every 10 x 106 cells, and cells were incubated in the dark at room temperature for 10 min. The cells were then centrifuged and washed two to three times in PBS to remove excess CFSE.
Adoptive Immunotherapy.
Pulmonary metastases were established in C57BL/6 CD45.2 background mice by i.v. infusion of 3 x 105 freshly disaggregated tumor cells. Mice received a single i.v. infusion of cultured TDLN cells labeled with 1 µM CFSE (or 5 µM for immunohistochemistry) 3 or 10 days after infusion of tumor cells. All mice received supplemental IL-2 (7000 IU) by twice daily i.p. injections for 5 days after infusion of TDLN cells. Some experiments involved pretreating TDLN cells with PTX at a concentration of 0.1 µg/10 x 106 cells for 90 min at 37°C. In experiments where PTX was used in vivo, 1 µg of PTX was injected i.v. 24 h (1 day) after adoptive transfer of TDLN cells. At multiple time points after the adoptive transfer, the mice were sacrificed, and their lungs, lymph nodes, and spleen were harvested. Individual experiments included a minimum of 3–5 mice/cohort, and no animals were excluded from the statistical evaluation. The figures are representative of at least two independent experiments.
Immunocytochemical Assessment of TDLN Cell Trafficking.
Mice with 10-day-old pulmonary MCA-205 metastases received an i.v. bolus containing 10 x 107 cultured TDLN cells labeled with CFSE (5 µM). At the indicated time points, lungs were harvested, perfused with 10% phosphate-buffered formalin (through the trachea), and immersed for a minimum of 48 h in the fixative. The fixed tissues were embedded in paraffin and sectioned (4 µm) for staining. The deparaffinized sections were incubated with anti-FITC, horseradish peroxidase-conjugated Fab' fragments (DakoUSA, Carpinteria, CA) diluted 1:20 from the stock solution, washed, and developed for 20 min using a diaminobenzidine reagent (Zymed Laboratories Inc., South San Francisco, CA) according to the manufacturer’s protocol. The sections were counterstained with hematoxylin and mounted in permount. The sections stained with the anti-FITC horseradish peroxidase conjugate and diaminobenzidine showed intense, diffuse cytoplasmic staining of multiple lymphocytes. Sections stained with an isotype matched anti-digoxin horseradish peroxidase conjugate (DakoUSA) showed no reactivity, indicating that the reaction was specific for the CFSE-labeled cells (data not shown).
Tissue Processing for FACS Analysis.
The mice were sacrificed, and their lungs were flushed with PBS by injecting a 27-gauge needle into the right cardiac ventricle and flushing with 5 ml of PBS. The lungs were then minced and placed into a 35-µm medicon (DakoUSA) with 0.5 ml of PBS and processed for 45 s in the Medimachine (DakoUSA). Samples were withdrawn from the medicon in a 1-ml syringe and flushed through a 50 µm filter to create a single cell suspension. The processing was repeated for another 45 s, and the medicon was rinsed as cells were collected, filtered, and enumerated before FACS staining. Lymph node and spleens were harvested, mechanically disrupted using the blunt end of a 10-cc syringe in an etched Petri dish, and filtered through a 50 µm filter to yield a single cell suspension.
FACS Analysis.
Processed lung, lymph node, and spleen cells were enumerated for cell counts and then stained for three-color FACS analysis using the phycoerythrin-conjugated anti-CD45.1 and CyChrome-conjugated anti-CD4 or -CD8 (all obtained from PharMingen, San Diego, CA). Apoptosis studies used phycoerythrin-conjugated annexin V and annexin V buffer solution (PharMingen). All samples were fixed in a 2% buffered formalin solution and read 24 h after labeling except for apoptosis studies, where the samples were read immediately without fixation. Before analysis on a Coulter XL (Beckman Coulter, Chaska, MN), a known quantity of 15 µm of polystyrene microbeads (Bangs Laboratories, Fishers, IN) was added to each sample to calculate the absolute number of cells of interest during analysis using the following formula : [number of cells analyzed in sample] x [number of beads added to sample]/[number of beads analyzed in sample]. For analysis of lymph node and spleen cells, 2.5 x 105 events were collected and analyzed using the WinList 5.0 program (Verity Software House, Inc., Topsham, ME). Analysis of lung tissue involved the collection of 1 x 106 cells/sample.
Statistical Analyses.
Student’s t test (2 cohorts) and one-way ANOVA followed by Tukey test for multiple comparisons (> 2 cohorts) were used to evaluate normally distributed data sets. The Mann-Whitney rank-sum test (2 cohorts) and Kruskal-Wallis one-way ANOVA on ranks followed by Dunnett’s pairwise multiple comparison procedure (> 2 cohorts) were used when one or more data sets in an experiment did not show a normal distribution. These statistical analyses were conducted using Sigmastat Version 2.0 for Windows.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, the infused cells are not seen within metastases or associated with venules. The numbers of stained cells within capillaries gradually fell over the first hour, but TDLN cells were still readily detected in these vessels at the 1 h time point (Fig. 1, D-F
). In some fields, labeled cells within capillaries were in close proximity to metastases, yet they were rarely found within them. Occasionally one finds labeled cells attached to the luminal surfaces of venules, but these interactions were infrequent at the 1 h time point. In contrast, 4 h after infusion, most labeled cells were bound to the luminal aspects of venules or in the perivascular space immediately adjacent to venules (Fig. 1, G-I
). Stained cells were clearly present within metastases as well, but the numbers were still low. TDLN cells were associated with venules at the 8 h and, to a lesser extent, 16 h time points as well. By 24 h after infusion, very few cells were either associated with venules or visible within capillaries away from metastatic lesions (Fig. 1, J-L)
. On the other hand, large numbers of stained cells were readily visible in both pleural and parenchymal metastases. Therefore, TDLN cell distribution within the lung vasculature changed dramatically during the first 24 h after infusion. At the end of this period, most infused cells visible in the lung were associated with metastases. Consequently, quantitative trafficking studies using TDLN cells extracted from whole lung were performed beginning at the 24 h time point because these data best reflect donor cell accumulation within tumor nodules. Quantitative trafficking studies were performed by infusing 14 x 106 CFSE-labeled, cultured CD45.1 TDLN cells into CD45.2 congenic animals with 3-day-old pulmonary metastases. The lymphoid cells were recovered from the lungs, spleen, and inguinal lymph nodes 1, 3, and 5 days after infusion of donor cells. Lung tissue maceration was performed with the Medimachine (DakoUSA) without enzymatic treatment after preliminary studies showed that this method increased processing speed and consistency without sacrificing lymphocyte recoveries or viabilities. Immediately before FACS analysis, polystyrene beads were added to the cell suspensions prepared from each organ so that the volume of sample analyzed and the absolute number of donor cells/organ could be precisely determined as outlined in "Materials and Methods" and described previously (19) .
The number of cultured TDLN cells in all organs was highest at the 1 day time point after infusion and then declined (Fig. 2)
. The peak level and the subsequent decline were greatest in the spleen, with 2 x 106 donor cells recovered at 1 day, falling 8-fold by 3 days and 20-fold at the 5 day time point. The levels of cultured TDLN cells in the inguinal lymph nodes peaked at 1–2 x 104 cells at 1 day, were unchanged at 3 days, and then declined by 4-fold at the 5 day time point. The levels in tumor-bearing lungs peaked at 4–8 x 104 cells at 1 day and then stabilized at 2–4 x 104 cells at both later time points. The CD8:CD4 ratio in the lung declined from 10 initially to 5 after 5 days. The ratio in the spleen fell from 10 to 1 between the 1 and 3 day time points. In lymph nodes, a CD8:CD4 ratio of 1 was maintained throughout.
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). Inguinal lymph nodes, which do not have a large intravascular pool, showed no decline in cell number between the 1 and 3 day time points. Therefore, donor cells recovered from the lung at the 1 day time point may include both cells that have migrated into the tissue and cells that are located within blood vessels. The later time points are less likely to include nonspecifically trapped cells; however, the potential impact of in situ proliferation and apoptosis grows as the in vivo incubation period increases.
Divided TDLN Cells Are Preferentially Recovered from the Tumor-Bearing Lungs.
The cycling activity of CFSE-labeled cultured TDLN cells was determined in tumor-bearing lungs and lymphoid organs by flow cytometric analysis of CD45.1-positive cells at intervals beginning 1 day after i.v. infusion (Fig. 3)
. The cultured TDLN cells showed a tight, unimodal CFSE distribution at the time of infusion (data not shown). TDLN cells fixed immediately after labeling and reanalyzed in parallel with the test samples maintained this staining pattern. The TDLN recovered from all organs showed uniform dilution of CFSE (relative to fixed, control cells) at the 1 day time point, suggesting that most infused cells are cycling at the time of infusion. This is consistent with the observation that TDLN cells maintained in culture beyond the infusion date undergo several cell divisions as well (data not shown). One cannot, however, formally exclude other causes for the initial, uniform decay in fluorescence intensity observed at the 24 h time point. Nonetheless, there is a clear divergence in proliferative activity within the donor pool as time progresses. At the later time points, the donor cells recovered from the lung showed a higher number of cell divisions (more cells with low levels of CFSE) than the donor cells recovered from lymphoid organs (Fig. 3)
. Subset analysis showed that the difference was most striking for the CD4 subset (Table 1)
. In contrast, a significant percentage of the CD8 TDLN cells recovered from all organs showed evidence of persistent cycling activity. Thus, cultured CD8 and CD4 TDLN cells proliferate after infusion into unirradiated, syngeneic recipients receiving low-dose IL-2. The dividing cells were detected at different frequencies in tumor-bearing lungs, spleen, and systemic lymph nodes.
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. In absolute number, 3–4 x 104 untreated TDLN cells and <2 x 103 PTX-treated TDLN cells were recovered from tumor-bearing lungs 3 and 5 days after adoptive transfer. The recoveries from the spleen were reduced 4-fold (Fig. 4B)
, and virtually no PTX-treated TDLN cells were detected in the inguinal lymph nodes at any point after infusion (Fig. 4C)
. As reported previously (13)
, PTX-treated cultured TDLN cells were completely inactive against pulmonary metastases (Fig. 5)
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. In addition, the frequency of apoptosis (annexin V staining) and necrosis (7-Amino-Actinomycin D staining) were identical in the control and PTX-pretreated cultures (Fig. 6B)
. The tumor-specific release of INF-
during coculture with irradiated tumors cells was also unaffected (data not shown). Thus, PTX showed no direct lymphocyte toxicity in vitro.
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. Donor T-cell levels in the inguinal lymph nodes were measured by FACS beginning the next day (Fig. 7B)
. The number of donor lymphocytes was reduced by 20-fold within 1 day of PTX infusion and remained at this level for the duration of the experiment (3 days). In studies not shown, the donor and endogenous T cells recovered from the spleen at each time point after PTX infusion did not respond to secondary lymphoid organ chemokine in Transwell chemotaxis assays. Therefore, 1 µg/animal PTX administered i.v. fully blocked chemokine-mediated signaling within 1 day. In fact, subsequent time course studies found that this dose of PTX blocked virtually all lymphocyte trafficking activity within 8 h of administration (data not shown).
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. The absolute number of donor TDLN cells recovered from the tumor-bearing lungs after PTX infusion declined gradually over the 3-day period of observation, falling to approximately 50% of the level observed in control mice (Fig. 7C)
. The decreased recovery did not result from enhanced apoptosis because PTX treatment did not increase annexin V expression on donor cells recovered from the lungs (Fig. 7D)
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The CFSE dilution profiles provided further insight into TDLN cell behavior after infusion (Fig. 8)
. The CFSE histograms showed that TDLN cells in the lung after PTX infusion continued to divide but suggested that the percentage of high cycle number cells was lower than that in control animals (Fig. 8B
, greater percentage of cells at low CFSE intensities in PTX- groups on day 3 and 4). Quantitative cell cycle analysis confirmed this impression and showed that the CD4 subset was impacted more than the CD8 subset (Fig. 8, C and D)
. Specifically, the percentages of high cycle number CD4 and CD8 cells in the lung continued to rise after PTX infusion; however, the rate of increase was reduced. The resulting fall in high cycle number cells was greatest for the CD4 subset, reaching 50% 3 days after PTX infusion. The reductions cannot be attributed to direct suppression of cell division because PTX did not inhibit T-cell proliferation in vitro (Fig. 6A)
, and T-cell proliferation in the spleens of the PTX-treated and untreated animals were the same (data not shown). Thus, TDLN cells that enter the lung continue to divide, and TDLN cells proliferating in the periphery traffick back to the lung at later time points.
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. After 10 days, the number of metastases was reduced by 90% and 80%, respectively, in mice that received vehicle alone or PTX compared with the IL-2-only control (Fig. 9B)
. Thus, PTX infusion produced a modest but statistically significant decrease in tumor suppression. Trafficking studies indicated that PTX infusion inhibited TDLN cell accumulation in the tumor-bearing lungs throughout the 10-day observation period (data not shown). Consequently, the small impact of PTX on clinical activity did not result from reentry of circulating TDLN cells into the lungs. Thus, both early and late TDLN cell trafficking into the lung contribute to tumor suppression, but the former accounts for most of the clinical activity against 3-day-old lung metastases.
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DISCUSSION |
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During the second phase (1 h to 8 h), robust adhesive interactions between the donor cells and venules developed along with transmigration into the perivenular space. These interactions were infrequent up to 1 h, but they were readily detected at the 4 and 8 h time points. The venules involved varied in caliber, but their internal diameters were all larger than the infused blasts. Consequently, direct adhesive interactions analogous to those documented in classic inflammatory models occur in tumor-bearing lungs as well.
The final phase (4 to 24 h) is characterized by progressive accumulation of labeled cells within pleural and parenchymal metastases. The donor cells were readily detected within metastases at the 4 h time point, and by 24 h, most labeled cells in the lung were associated with metastases. During early time points in particular (1 min to 1 h), donor cells within capillaries were frequently seen immediately adjacent to metastases. However, labeled cells within metastases were more common after large-scale adhesion to and transmigration across venules developed at the 4 h time point. In addition, some metastases appeared to grow within capillaries, suggesting that circulating TDLN cells can access tumor masses without crossing endothelial barriers. Thus, visual analysis revealed multiple potential entry points for circulating TDLN cells into tumor-bearing lungs and indicated that recruitment activity at these points changed over the first 24 h.
Direct visualization indicated that 1 day after infusion, most labeled TDLN cells in the lung were associated with metastases. Therefore, all quantitative trafficking studies began at this time point. CFSE-labeled TDLN cells accumulated in tumor-bearing lungs and secondary lymphoid organs after i.v. infusion into mice with 3-day-old MCA-205 pulmonary metastases. The TDLN cells isolated from tumor-bearing lungs showed asynchronous cell division, with most completing 4–8 cycles over 5 days. Cell division involved both the CD8 and the much smaller CD4 subset of TDLN cells; however, a significantly greater fraction of the former was proliferating at all time points and in all organs examined. The CD4:CD8 ratio in the lung rose over the 5-day period of observation, indicating that postinfusion cell division, trafficking, and/or enhanced survival may amplify the clinical activity of the CD4 subset over time. This change may, for example, explain how a subset that constitutes 5–10% of the infused population accounts for up to 50% of the antitumor activity against pulmonary micrometastases in the MCA-205 model (5 , 20) . On the other hand, a rise in the donor CD4:CD8 ratio may also reflect expansion, increased recruitment, or prolonged survival of regulatory CD4 T cells within the lung (21, 22, 23) . These cells suppress the antitumor activities of effector cells and may limit the overall therapeutic activity of the donor population. Thus, cultured TDLN cells are heterogeneous with respect to homing properties: some enter the tumor-bearing lung immediately; and others join the recirculating pool of lymphocytes in secondary lymphoid organs.
Many TDLN cells in secondary lymphoid organs ceased dividing within 24–48 h of infusion; however, a sizable subset in the spleen and a small subset in the inguinal lymph nodes showed robust proliferation with progressive CFSE dilution that overlapped the levels observed in the lung. In the current study, TDLN cells were infused after 5 days of culture in IL-2-containing media. Many of the cells were actively cycling at this time because continued in vitro culture resulted in an additional 5–6-fold expansion of TDLN over 3 days. It is well documented that CD8 cells undergo multiple rounds of antigen-presenting cell-independent cell division after T-cell receptor engagement in the proper costimulatory environment (24 , 25) . CD4 proliferation is more dependent on antigen presentation by antigen-presenting cells, but a prolonged antigen-independent phase of proliferation occurs as well (26) . Therefore, the continued in vivo division of TDLN cells may reflect, in part, residual nonspecific expansion triggered by the anti-CD3 treatment in vitro 3 days earlier. In addition, antigen-presenting cell and tumor antigen-driven proliferation of partially differentiated effector cells may occur as well because dissemination of tumor antigens to the spleen is a likely occurrence (27) .
Previous studies indicated that cultured TDLN cells are developmentally heterogeneous, with most tumor-suppressive activity concentrated in the subset of differentiated effector cells with low levels of CD62L at the time of infusion (12) . Only these CD62L-low cells entered tumors initially, suggesting that they acquired the adhesion/chemokine receptors necessary for trafficking into inflammatory sites during in vitro culture (28 , 29) . However, this study demonstrates that less differentiated TDLN cells contribute to the overall immunological response as well by joining the pool of endogenous lymphocytes recirculating through lymph nodes and spleen. These cells may resemble the "central-memory" cells described by Weninger et al. (28) in cultures supplemented with IL-15. The proliferative activity observed in the TDLN cells recovered from lymphoid organs suggested that these sites might provide donor cells for export to the lung at later time points.
CFSE dilution alone cannot determine whether the divided TDLN cells in tumor-bearing lungs are produced locally or proliferate at a distant site (e.g., lymphoid organs) and then traffick, via the bloodstream, to the lung. PTX inhibits signaling through a GTP-binding Gi-protein shared by most chemokine receptors (30 , 31) and blocks lymphocyte trafficking into multiple tissues including the lungs (19 , 32) . When administered in vivo, PTX inhibits the recirculation of endogenous lymphocytes through lymphoid organs (13 , 14) , resulting in a lymphocytosis that may persist for weeks (33) . Therefore, we blocked lymphocyte trafficking with i.v. PTX beginning 1 day after TDLN cell infusion to "isolate" the lungs from secondary lymphoid organs and thus clarify the origins of the divided TDLN cells that accumulated at later time points.
In our system, PTX infusion blocked the recirculation of CFSE-labeled donor TDLN cells through lymph nodes and the response to secondary lymphoid organ chemokine in Transwell assays, indicating that the relevant Gi-protein was inactivated within 8–24 h of treatment. In addition, pretreatment experiments confirmed that TDLN cell entry into tumor-bearing lungs was PTX sensitive (Gi-protein mediated). Therefore, it is reasonable to conclude that 8–24 h after PTX infusion, the lungs are no longer able to recruit circulating TDLN cells from any source. Consequently, the CFSE dilution observed at 2 and 3 days after PTX infusion provides strong evidence that TDLN cells continue proliferating after entry into the lung. Despite in situ cell division, the absolute number of TDLN cells recovered from tumor-bearing lungs fell to 50% of control levels 3 days after PTX infusion. This paralleled a statistically significant fall in the percentage of divided CD4 and, to a lesser extent, CD8 cells recovered from the lungs of PTX-treated animals. Because PTX does not directly affect T-cell growth or survival, these findings imply that donor cells cycling in secondary lymphoid organs are continuously trafficking into tumor-bearing lungs and account for a significant fraction of the donor cells in the lung at late time points.
The proliferation of cultured TDLN cells within tumor-bearing lungs differs from the behavior reported previously (34 , 35) for virus-specific CD8 effectors during pulmonary infections. Virus-specific CD8 effectors were unable to proliferate within the lung even when provided with exogenous antigen-pulsed dendritic cells. In contrast, they proliferated actively once they were removed from the lung, suggesting that unidentified factors within the pulmonary microenvironment actively inhibited further cell division. In addition, Reinhardt et al. (36) reported that CD4 T cells proliferated in lymph nodes draining cutaneous delayed-type hypersensitivity lesions then traffick to the lesions through the bloodstream; however, they did not detect further proliferation after entry into the lesion. These findings suggest that during normal immunological responses, T-cell proliferation and differentiation occur primarily in secondary lymphoid organs, with subsequent trafficking of nondividing cells to the active lesions in peripheral tissues. Therefore, therapy with cultured TDLN cells and systemic IL-2 may circumvent normal controls over T-cell proliferation in tissues, allowing expansion of both CD8 and CD4 donor T cells within tumor-bearing lungs. The contributions of the ex vivo activation conditions, the systemic IL-2 treatment, and the tumor microenvironment remain to be determined.
PTX treatment 1 day after TDLN infusion resulted in a small but statistically significant drop in antitumor activity. Specifically, the number of metastases increased 2-fold, and the overall level of suppression fell from 90% to 80% relative to the untreated control. The impact of PTX infusion on lymphocyte trafficking is long-lived. In previous studies, a single bolus of PTX inhibited recirculation through lymph nodes for several weeks (14
, 37)
. In this study, TDLN cells were excluded from lymph nodes beginning 1 day after PTX infusion and remained at reduced levels in both lymph nodes and lung until the latter were examined for metastases 10 days after PTX infusion. Therefore, a late "rebound" in the trafficking of donor T cells into the lung cannot account for the relatively modest suppression of antitumor activity after PTX infusion. Thus, TDLN cells that accumulate in the lung early and those that enter the lung late both influence the clinical outcome; however, the cells that accumulate early have the greatest impact.
The disproportionate contribution of early versus late T-cell recruitment suggests that the initial wave of cultured TDLN cells that enters the lung contains the most active effector cells. TDLN cultures contain a mixture of T1 pre-effectors/effectors capable of suppressing tumor growth (5) and T-regulatory cells that inhibit antitumor activity (21 , 22) . If donor proliferation within lymphoid organs increases the prevalence of regulatory cells, then late recruitment into the lung may diminish the overall therapeutic response. Alternatively, a late flux of donor effector cells from the periphery into the lung may augment tumor suppression. Consequently, the therapeutic response in a given case will depend on both the initial make-up of the infused population and whether subsequent in vivo proliferation/recruitment favors regulatory or effector cells.
In summary, donor T-cell accumulation in tumor-bearing lungs is a dynamic process involving the early recruitment of infused cells, their subsequent division and apoptosis within the lung, and ongoing late entry from proliferation centers in secondary lymphoid tissues. The proliferation of the donor cells in multiple sites, their continued influx from secondary lymphoid organs, and the diminished antitumor activity of donor cells entering the lung at late time points suggest that their functional characteristics are changing over time. The techniques described in this study distinguish donor cells that enter tumor-bearing organs early after infusion from those that differentiate in secondary lymphoid organs before trafficking to metastases. Consequently, one can now define the functional changes that occur in donor T cells after infusion, evaluate the impact of these changes on antitumor activities, and then optimize treatment protocols accordingly.
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FOOTNOTES |
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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.
Requests for reprints: Lloyd M. Stoolman, Department of Pathology, Room M4224 Medical Sciences Building 1, 1301 Catherine Road, Ann Arbor, Michigan 48103-0602. Phone: (734) 936-2459; Fax: (734) 763-6476; E-mail: stoolman{at}umich.edu
Received 9/ 4/03. Revised 1/ 9/04. Accepted 1/ 9/04.
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) or with (
) pertussis toxin (PTX). The bar charts show the mean cell counts (A) and percentages of annexin V + 7-Amino-Actinomycin D-positive cells (B) over the subsequent 3-day culture period.
