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The Journal of Immunology, 2001, 167: 3089-3098.
Copyright © 2001 by The American Association of Immunologists

Tumor-Specific Responses in Lymph Nodes Draining Murine Sarcomas Are Concentrated in Cells Expressing P-Selectin Binding Sites1

Keishi Tanigawa2,*,{dagger}, Nobuhiro Takeshita2,*,{dagger}, Ronald A. Craig2,*, Katie Phillips*, Randall N. Knibbs*, Alfred E. Chang{dagger} and Lloyd M. Stoolman3,*

Departments of * Pathology and {dagger} Surgery, Division of Surgical Oncology, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized {alpha}CD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plighigh T cells) produced T lymphoblasts with the most tumor-specific IFN-{gamma} synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plighigh T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plighigh TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plighigh fraction before expansion (Pliglow cells). Tumor-specific IFN-{gamma} synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plighigh fraction accounted for the marked increase in antitumor activities after culture. The cultured Plighigh fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of {alpha}{beta} T cells accounted for 95–97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adoptive cellular immunotherapy for the treatment of metastatic cancer shows promise in animal models (1). One approach uses tumor-draining lymph nodes (TDLN)4 as a source for tumor-responsive pre-effector cells (2, 3). Activation of murine TDLN on immobilized {alpha}CD3 and growth in low-dose IL-2 produced T lymphoblasts showing strain-specific antitumor activities in vitro and in vivo. These lymphoblasts secreted IFN-{gamma} when cocultured with syngeneic tumor, and suppressed the growth of established pulmonary micrometastases following i.v. infusion (4). In a small phase I clinical trial, T cells cultured from lymph nodes primed with autologous tumor-derived vaccines showed tumor-specific IFN-{gamma} secretion in vitro and produced measurable tumor regression following adoptive transfer in 33% of patients with disseminated renal cell carcinomas (2 complete and 2 partial responses among 12 participants) (5).

One factor limiting the effectiveness of adoptive immunotherapy in both murine models and human clinical trials was the low frequency of tumor-reactive T cells in the seed populations that differentiate into Tc1 and Th1 (T1) effector cells during culture. The nonspecific expansion of all T lymphocytes in the seed population further diminished the frequency of tumor-reactive cells in the adoptively transferred lymphoblasts. Consequently, the isolation of the tumor-reactive T1 pre-effector/effector cells from draining lymph nodes before nonspecific expansion is likely to increase the therapeutic impact of adoptive cellular immunotherapy.

Multiple studies suggest that T1 effector cells developed distinctive adhesion receptor profiles in the course of differentiation. In particular, the synthesis of binding sites (ligands) for the P- and E-selectins, two vascular adhesion receptors involved in leukocyte trafficking into tissues, increased dramatically under culture conditions that generated T1 effector cells in vitro (6, 7). Selectin-ligand synthesis occurred during T cell proliferation and differentiation in vivo as well (8, 9); however, whether these binding sites appeared primarily on T1 effector cells in vivo remains unclear. The current study examined the hypothesis that selectin-ligand synthesis provides a surrogate marker for T1 pre-effector/effector cells in TDLN that can be used to enhance the effectiveness of adoptive immunotherapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free environment. Animals were used at age 8 wk or older. Recognized principles of humane laboratory animal care were followed, and the University of Michigan Department of Laboratory of Animal Medicine approved all experimental protocols.

Tumors

The MCA 205 and 207 lines are 3-methylcholanthrene-induced fibrosarcomas (provided by J. C. Yang, National Institutes of Health, Bethesda, MD). The preparation and passage of these tumors were described previously (4, 10). The tumors were maintained by serial s.c. transplantation in syngeneic mice, and they were used before the seventh generation. 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 20–50 C57BL/6 mice by s.c. injection of 106 disaggregated tumor cells in 0.05 ml HBSS. Nine days later, the regional lymph nodes (TDLN) were harvested and teased into suspension using sterile 20-gauge needles. Tissue fragments were disaggregated and then passed through nylon mesh to remove clumps.

Fractionation procedure

Nylon wool fractionation. TDLN cells (3–8 x 108 cells) were suspended in mouse lymphocyte culture medium (MLM) consisting of RPMI 1640 with 10% heat-inactivated FCS (Summit Biotechnology, Ft. Collins, CO), 1 mM sodium pyruvate, 50 µM 2-ME, 2 mM fresh L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin-G (all unattributed reagents from Life Technologies, Grand Island, NY). A nylon wool column was prepared by stuffing a 30-ml syringe barrel (BD Biosciences, San Jose, CA) with 2-g nylon wool fibers (Associated Biomedic Systems, Buffalo, NY). Up to 3 x 108 cells in 4 ml MLM were applied to a single column, followed by a 2-ml MLM wash. After 45 min at 37°C, unattached cells were washed out of the column with 50 ml MLM.

P-selectin fractionation. All steps were performed at 4–7°C in DPBS+ buffer consisting of Dulbecco’s PBS (Life Technologies) with 0.5% human serum albumin (Michigan Department of Public Health, Lansing, MI). The nylon wool-purified cells were washed in DPBS+, centrifuged, and resuspended in solution containing the human IgM:P-selectin chimera (IgM-CD62P). Initial experiments used high titer culture supernatants from COS cells transfected with the IgM-CD62P construct (11, 12). More complete recovery/depletion of the target population occurred with affinity-purified IgM-CD62P used at ~10 µg/ml. After 30–60 min, the cells were centrifuged, resuspended in 1 ml DPBS+ (up to 108 cells), and combined with 40 µl biotinylated goat anti-human IgM (Zymed Laboratories, South San Francisco, CA). After 20 min, the cells were centrifuged, resuspended in 90 µl DPBS+, and combined with 10–20 µl streptavidin-conjugated paramagnetic beads (Miltenyi Biotec, Auburn, CA). After 20 min, the cells were centrifuged, resuspended in 2 ml DPBS+, and applied to a magnetized Midi MACS column (Miltenyi Biotec). The fraction that eluted from the magnetized column with 10 ml DPBS+ was enriched for cells expressing no or low numbers of P-selectin binding sites (ligands); thus, it was designated the Pliglow fraction. The fraction that eluted with 10 ml DPBS+, after washing and removal of the column from the magnet, was enriched for cells expressing high numbers of P-selectin ligands; thus, it was designated the Plighigh fraction.

B cell depletions

B cells were depleted before P-selectin fractionation using MACS paramagnetic beads conjugated with anti-murine CD19 mAb (Miltenyi Biotec). The {alpha}CD19 MACS beads (10 µl) were combined with 1 x 107 nylon wool-processed TDLN cells (90 µl) in DPBS2+ (DPBS+ supplemented with 60 IU/ml IL-2) and incubated for 20 min at 4°C. After washing (10 ml DBPS2+ x 1), the cells were applied to a magnetized LS column as described for P-selectin sorting. The nonadherent cells were eluted and then subjected to P-selectin fractionation, as described above. The technique removed 97–99% of the CD19-positive B cells remaining after processing over nylon wool.

In vitro activation

The TDLN fractions were resuspended in MLM and cultured for 2 days at 0.5–2 x 106 cells/well in 24-well plates coated with an anti-CD3{epsilon} mAb (2C11 hybridoma from J. Bluestone, University of Chicago, Chicago, IL). The cells were then resuspended at 2 x 105 cells/ml in fresh MLM containing 60 IU/ml IL-2 (Chiron Therapeutics, Emeryville, CA) and cultured for 3 days at 37°C.

Measurement of in vitro cytokine release

Tumor-specific cytokine production in vitro was measured by coculture of irradiated tumor cells with cultured TDLN fractions, as described (4, 10). In brief, 1 x 106 freshly disaggregated and irradiated MCA 205 or 207 tumor cells were combined with equal numbers of cultured TDLN cells in 2 ml MLM containing 60 IU human rIL-2. After 48 h, the culture supernatants were collected for cytokine measurements using commercially available ELISA kits.

Adoptive immunotherapy

Pulmonary metastases were established in C57BL/6 mice by i.v. infusion of 3 x 105 freshly disaggregated tumor cells. Mice received a single i.v. infusion of cultured TDLN cells 3 days after infusion of tumor cells. Eleven to eighteen days later, the mice were sacrificed and examined for pulmonary metastases, as described (4, 10). ANOVA with multiple comparisons (Tukey test) was used to determine whether the mean numbers of pulmonary metastases under different treatment conditions were significantly different. Individual experiments included a minimum of three to five mice per cohort, and no animals were excluded from the statistical evaluation. The figures are either representative of at least two independent experiments or they contained data pooled from multiple independent experiments.

Subset depletion studies

In some experiments, PE-conjugated mAbs and anti-PE-coated MACS paramagnetic beads (Miltenyi Biotec) were used to deplete cultured Plighigh cells expressing CD4, CD8, or NK epitopes (NK1.1 and DX-5). Cells (1 x 107) and dialyzed, PE-conjugated mAb(s) (20 µg) were combined in 1 ml DPBS2+ and incubated for 20 min at 4°C. After washing (15 ml x 2 in DPBS2+), the cells were resuspended in 80 µl DPBS2+, combined with 20 µl anti-PE-conjugated beads, and incubated for 20 min at 4°C. After a final wash (10 ml x 1 in DPBS2+), the cells were applied to either an LS (CD4 or CD8) or LD (NK 1.1 and DX-5) Midi MACS column as described for P-selectin sorting. The nonadherent cells were eluted and used for adoptive transfer studies, as described in the text. The control populations were cultured Plighigh cells processed as above, except that an irrelevant isotype-matched mAb was substituted for the lineage-specific mAb. The control and subset-depleted populations were infused as quickly as possible after processing to minimize loss of tumor-suppressive activity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E- and P-selectin ligands on TDLN cells

Inguinal lymph nodes draining s.c. MCA 205 sarcoma implants were collected 9 days after inoculation of 106 viable tumor cells. Selectin ligands were expressed by minor populations of both the CD4 and CD8 T cell subsets, with the former significantly larger than the latter (Fig. 1GoA). The percentage of T cells with binding sites for P-selectin equaled or exceeded the number with binding sites for E-selectin in both subsets. The absolute percentages of these fractions varied from one experiment to the next, but the former was consistently higher than the latter. Similar observations were reported for T cells responding to nontumor Ags in vivo (8), and P-selectin ligand synthesis was strongly associated with Ag-specific T cell proliferation in an OVA transgenic model (9).



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  		FIGURE 1. A, P- and E-selectin binding sites on T cells in TDLN. The assay used FITC-conjugated CD4 or CD8 and a three-step indirect reaction for selectin binding sites (human IgM murine selectin chimera, a biotinylated anti-human IgM secondary, and streptavidin-conjugated CyChrome). The blackened areas denote cells with fluorescent intensities (FI) above the EDTA control (EDTA blocks nonspecific chimera attachment). The numerical values are the mean and SE of 10 independent measurements on TDLN cells pooled from 15 animals in a representative experiment. B, Activation epitopes on T cells with and without binding sites for P-selectin. The assay used FITC-conjugated CD4 or CD8; PE-conjugated CD25, CD44, CD62L, or CD69; and the three-step indirect CyChrome reaction for P-selectin binding sites (see above). The filled histograms show epitope distributions on cells with P-selectin binding sites. The open histograms show epitope distributions on cells without P-selectin binding sites.

 
Activation epitopes on T cells with and without P-selectin binding sites

A high percentage of the TDLN T cells (~40%) expressed activation epitopes (moderate-high levels of CD25, CD69, or CD44) and/or low levels of CD62L, implying that a robust proliferative response occurred in the lymph nodes draining MCA 205 sarcomas (Fig. 1GoB). The TDLN T cells with P-selectin binding sites expressed the highest levels of the CD44 and CD69 epitopes in both T cell subsets. In addition, these cells expressed variable levels of the CD25 and CD62L epitopes with discrete high and negative-low subpopulations detected. However, most CD4 and CD8 cells with high levels of CD44 or CD69 did not coexpress P-selectin ligands (compare areas under histograms in Fig. 1GoB). Thus, TDLN T cells expressing P-selectin binding sites constituted a subset of recently or previously activated T cells.

TDLN characteristics, fractionation, and culture

The fractionation procedure for Plighigh T cells used a nylon wool column to partially deplete costimulatory cells from TDLN, then a human IgM-murine P-selectin chimera (IgM-CD62P) and paramagnetic beads to collect/deplete the target population. The TDLN suspension consisted primarily of {alpha}{beta} TCR-positive T cells (~46%), CD19-positive B cells (~46%), and lesser numbers of cells expressing NK (NK1.1 and DX-5) and {gamma}{delta} epitopes (Table IGo). The nylon wool-processed fraction (referred to as T lymphocyte enriched or TLE) was substantially depleted of CD19-positive B cells (~9%) and enriched for {alpha}{beta} T cells (~84%). The CD4:CD8 ratio was greatest in the Plighigh fraction, reflecting the distribution of P-selectin ligands on these subsets (Table IIGo). The Plighigh fraction also contained variable numbers of B cells (15–70%), NK cells (~1–11%), and {gamma}{delta} (1–3%) cells. Murine macrophages and dendritic cells do not express high levels of lineage-specific Ags, and thus one cannot accurately count them by flow cytometry. Low levels of cells expressing CD14 or Mac-3 (1–3%) were detected in all populations, indicating that macrophages and dendritic cells, in addition to B cells, may provide costimulatory activity during culture.


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  		Table I. Expression of lineage restricted epitopes in fresh TDLN fractions1

 

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  		Table II. Expression of binding sites for P-selectin on freshly fractionated CD4 and CD8 subsets of TDLN1

 
P-selectin binding sites (ligands) were expressed by 4–20% of CD4 and 2–8% CD8 T cells in the TLE fraction across multiple experiments (Table IIGo). After sorting, P-selectin binding sites were detected on 30–85% of the CD4 or CD8 cells in the Plighigh fractions, as opposed to 0–3.5% of the CD4 and 0–0.6% of the CD8-positive cells in the Pliglow fractions. The Pliglow fractions were completely devoid of T cells expressing E-selectin ligands (not shown). Therefore, all T cells in TDLN that synthesized E-selectin ligands synthesized high levels of P-selectin ligands as well. However, the converse was not true because the percentage of T cells with P-selectin binding sites frequently exceeded the percentage with E-selectin binding sites (Fig. 1GoA).

The apparent variability in the percentage of cells with P-selectin binding sites in freshly sorted Plighigh T cells most likely reflected interference from IgM-CD62P/bead complexes. High levels of the chimera were used to ensure optimal recovery of the target population; consequently, most ligands were occupied when fresh chimera was added to assess enrichment after sorting. Furthermore, Table IIGo included measurements made with both monoclonal and polyclonal anti-human IgM secondary reagents. When directly conjugated polyclonal reagents were used, 75–85% of the Plighigh T cells showed high levels of reactivity. The polyclonal reagent binds to multiple epitopes in the IgM domain of IgM-CD62P; thus, it should detect both chimera complexed to beads and chimera added after sorting. It should be emphasized that, despite the variability in staining, the estimates of purity in Table IIGo demonstrated significant enrichment of the target T cell population when compared with unfractionated (7- to 17-fold) and Pliglow populations (40- to 50-fold).

Tumor-specific responses in cultured fractions

The cultured T lymphoblasts were infused into syngeneic animals bearing 3-day established MCA 205 sarcoma lung micrometastases to assess the activity of tumor-reactive cells in vivo. Cultured unfractionated TDLN and TLE showed similar activities when run in parallel. The animals receiving cultured Plighigh cells developed significantly fewer visible metastases on the pleural surface than animals receiving equal numbers of either the unfractionated populations or the cultured Pliglow cells (Fig. 2Go). The dose-response study indicated that 50% inhibition of metastases required ~0.1–0.3 x 106 cultured Plighigh cells, ~2–4 x 106 cultured TDLN cells, and >9 x 106 Pliglow cells per animal. Therefore, cultured Plighigh cells were 10- to 20-fold and >30-fold more potent than the cultured unfractionated and cultured Pliglow cells, respectively.



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  		FIGURE 2. Dose-response curves for suppression of lung metastases by cultured unfractionated TDLN, cultured Plighigh TDLN, and cultured Pliglow TDLN. Cells were administered i.v. 3 days after infusion of fresh disaggregated MCA 205 tumor cells. The numbers of visible pleural metastases were scored 11–18 days later. The pleural metastases in each group are reported as a percentage of the mean ± SE (n = 4–5) normalized to animals receiving IL-2 only (mean number of control metastases equaled 250 in experiment A, 600 in experiment B). When compared at 0.5–2 x 106 cells/animal, the difference between cultured Plighigh cells and either the cultured unfractionated or the cultured Pliglow cells is highly significant (p < 0.005 by ANOVA and Tukey multiple comparisons test). These findings are representative of 10 independent experiments.

 
These findings indicated that the IgM-CD62P chimera bound to tumor-reactive cells in TLE. However, additional experiments were performed to verify that the selectin domain of the chimera was responsible for the increased antitumor activity. Inclusion of EDTA with the IgM-CD62P chimera, which inactivates the selectin domain, or substitution of an IgM-CD45 chimera reduced the subsequent recovery of cells from the paramagnetic column by >90% (not shown). Therefore, the P-selectin domain of the chimera was necessary for the reversible attachment of cells to the paramagnetic column. A depletion experiment verified that neither the IgM-CD45 chimera nor the paramagnetic beads bound significantly to the tumor-reactive T cells in the TLE (Fig. 3Go). The tumor-reactive pre-effector cells in TLE remained after sorting was performed with the IgM-CD45 chimera. Subsequent sorting of the IgM-CD45-depleted TLE with the IgM-CD62P chimera generated the expected level of antitumor activity in vivo. Consequently, the depletion and concentration of tumor-reactive cells involved a specific adhesive interaction between the P-selectin domain of the chimera and target cells in TLE.



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  		FIGURE 3. Tumor-responsive cells in TDLN do not react with IgM-CD45 chimeras. A standard fractionation was conducted with an IgM-CD45 chimera. The IgM-CD45 chimera-treated cells that flowed through the paramagnetic column should be depleted of cells that bound significant levels of either the IgM-CD45 chimera or the paramagnetic beads (IgM-CD45 DP). These cells were then subjected to standard sorting with the IgM-CD62P chimera (IgM-CD45 DP Plighigh). For comparison, TLE cells were passed through the paramagnetic column, without prior incubation with chimera/beads (No chimera DP), and then subjected to standard sorting with the IgM-CD62P chimera (No chimera DP Plighigh). The TLE, No chimera DP, IgM-CD45 DP, No chimera DP Plighigh, and IgM-CD45 DP Plighigh fractions were cultured and evaluated for tumor-suppressive activity in vivo. The differences between the groups indicated on the figure are not significant (ANOVA and Tukey multiple comparisons test).

 
As noted above, the fresh Plighigh fractions contained significant numbers of potential Ag-presenting and costimulatory (AP/CS) cells, particularly B cells. Therefore, the contribution of AP/CS activity was evaluated directly. P-selectin fractionation was performed on TLE cells before and after depletion of CD19-positive B cells (Fig. 4GoA). The standard Plighigh fraction, the CD19-depleted Plighigh/low fractions, and the CD19-depleted fractions supplemented with irradiated splenocytes (2500 rad, 1:1 ratio) were cultured and tested for tumor-suppressive activities in vivo. The CD19-depleted Plighigh fraction slightly exceeded the tumor-suppressive activity of the standard Plighigh fraction. Furthermore, the tumor-suppressive activity of the CD19-depleted Pliglow fraction did not improve when cocultured with the irradiated splenocytes. Finally, coculture of irradiated Plighigh cells with Pliglow cells (1:1) did not promote the development of antitumor activity (Fig. 4GoB). Therefore, a disproportionate concentration of AP/CS activity in the Plighigh fraction cannot account for their increased antitumor activity after culture.



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  		FIGURE 4. The impact of costimulatory activity in the development of effector T cells. A, P-selectin sorting was performed on TLE and TLE depleted of CD19-positive B cells, as described in Materials and Methods. The resulting Plighigh, Plighigh-CD19, and Pliglow-CD19 were then cultured as usual. The latter two fractions were also cultured with irradiated (2500 rad) syngeneic splenocytes (1:1) prepared from tumor-free animals (Plighigh or low-CD19 + IS). All animals received 2 x 106 cultured cells 3 days after infusion of MCA 205 tumor cells. The mean ± SE (n = 5) is reported for the number of pleural metastases detected ~2 wk after treatment. B, Tumor suppression by Pliglow cells cultured with and without irradiated fresh Plighigh cells. Pliglow cells were combined with {gamma}-irradiated (2500 rad) fresh Plighigh cells at a ratio of 1:1 and cultured as usual. All animals received 1 x 106 cultured cells 3 days after infusion of MCA 205 tumor cells. The mean ± SE (n = 5) is reported for the number of pleural metastases detected ~2 wk after treatment. The differences between the columns designated with * and ** are significant (p > 0.05 by ANOVA and Tukey multiple comparisons test).

 
The magnitude and specificity of IFN-{gamma} secretion during coculture with the immunizing tumor were compared in the cultured TDLN fractions. As reported, bulk culture of unfractionated TDLN resulted in tumor-specific secretion of IFN-{gamma} (Fig. 5Go). However, T lymphoblasts cultured from the Plighigh fraction of TDLN secreted substantially higher levels of IFN-{gamma} than unfractionated populations, whereas T lymphoblasts cultured from the Pliglow fraction secreted substantially lower levels than the unfractionated cells. The strain-specific IFN-{gamma} secretion was significantly higher than secretion in response to the antigenically distinct MCA 207 sarcoma in all populations except those cultured from the Pliglow fraction.



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  		FIGURE 5. Secretion of IFN-{gamma} by cultured TDLN fractions during coculture with irradiated MCA sarcomas. Fresh irradiated tumor was cocultured with either the immunizing tumor (MCA 205) or a distinct strain (MCA 207). Capture ELISA were performed on the culture supernatants. The means of three replicates for each group are shown for two independent experiments. Similar findings occurred in five additional experiments.

 
Active T cell subsets in the cultured Plighigh population

The cultured Plighigh population was more heterogeneous than the other cultured populations (Table IIIGo). Consequently, targeted paramagnetic depletions were performed to identify the T cell fractions responsible for suppression of MCA 205 growth in vivo. The control population of cultured Plighigh cells received treatment identical with the depleted fractions, except an isotype-matched control mAb was substituted for the lineage-restricted mAb. This control is essential, because processing blast populations over the paramagnetic depletion column alone reduced antitumor activity (relative to unprocessed cells) ~2-fold. The depletions reduced the targeted population(s) by 95–100% (Fig. 6Go). When compared with the processed control population, selective depletion of the CD8 or CD4 fraction reduced tumor suppression by ~10 (8, 9, 10, 11, 12, 13, 14, 15)- and ~3-fold, respectively. Concurrent removal of both the CD8 and CD4 fractions reduced tumor suppression by 19- to 35-fold (95–97%), depending on whether one compares the doses required for a 40% or a 20% reduction in metastases. In contrast, the fractions depleted of cells expressing the NK epitopes NK 1.1 and DX-5 showed no loss of tumor-suppressive activity. Consequently, the CD8 and CD4 subsets of {alpha}{beta} T cells in the cultured Plighigh population contained the effector cells that suppressed MCA 205 sarcomas following adoptive transfer.


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  		Table III. Expression of lineage-restricted epitopes in cultured fractions of TDLN1

 


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  		FIGURE 6. Lymphocyte subsets with tumor-suppressive activity in the cultured Plighigh population. Plighigh cells were cultured as described, then depleted of cells expressing either CD8 only, CD4 only, CD8+4, or NK (NK 1.1 and DX-5) epitopes using paramagnetic techniques. The histograms in the upper panel show the distribution of each epitope in the population before (open histogram) and after (solid histogram) depletion. The tumor-suppressive activities of undepleted and depleted populations were then compared in animals with 3-day established pulmonary metastases. The outcomes for the depleted fractions are plotted at the Plighigh cell number before depletion. For example, the Plighigh - (CD8) cohort plotted at 1 x 106 normalized cells received the cells remaining after 1 x 106 Plighigh cells were depleted of all cells expressing the CD8 epitope. The means (±SE, n = 4) from three independent experiments are shown with best-fit regression lines (second order polynomial). (+), Plighigh - (NK); ({diamondsuit}), Plighigh; ({blacksquare}), Plighigh - (CD4); ({blacktriangleup}), Plighigh - (CD8); (x), Plighigh - (CD4+8). When compared at 0.5 - 2 x 106 cells/animal, the difference between cultured Plighigh cells and either the cultured Plighigh - (CD4), the cultured Plighigh - (CD8), or the cultured Plighigh - (CD4+8) cells is highly significant (p < 0.05 by ANOVA and Tukey multiple comparisons test).

 
Inhibition of MCA 205 metastases in mice with targeted deletion of P-/E-selectin

At the time of adoptive transfer into tumor-bearing animals, all cultured populations expressed binding sites for E-selectin on 56–69% of the CD4 and 73–80% of the CD8 T lymphoblasts (Table IVGo). However, the expression of P-selectin binding sites varied significantly among the cultured populations. The highest mean levels were measured in the cultured Plighigh cells (68% in the CD4, 77% in the CD8), and the lowest levels in the cultured Pliglow cells (33% in the CD4, 54% in the CD8). The direct role of P-selectin ligands in tumor suppression was evaluated by adoptively transferring cultured Plighigh TDLN into tumor-bearing wild-type animals and animals with targeted deletions in the P- and E-selectin genes (P-E- animals). The P-E- animals were backcrossed into the C57BL/6 strain five generations before use. Previous studies with these animals showed marked reductions in selectin-mediated leukocyte recruitment into cutaneous delayed hypersensitivity lesions (13). However, the cultured Plighigh TDLN from wild-type animals suppressed the growth of pulmonary MCA 205 micrometastases equally well in wild-type and P-E- animals (Fig. 7Go).


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  		Table IV. Expression of binding sites for P- and E-selectin on T lymphoblasts in cultured TDLN fractions at the time of adoptive transfer1

 


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  		FIGURE 7. Adoptive transfer of wild-type cultured Plighigh TDLN into wild-type (WT) and E-/P-selectin deletion mutants (E-P-) bearing 3-day established MCA 205 metastases. The TDLN were raised in wild-type animals, fractionated, cultured, and administered i.v., as described for other adoptive transfer experiments. Each animal received 0.9 x 106 cultured cells. The figure shows the mean number of pleural metastases in WT and E-P- animals treated with IL-2 alone or cultured Plighigh TDLN (n = 5). The results are representative of two independent experiments. The differences between the columns designated with * and ** are significant (p > 0.05 by ANOVA, WT and E-P- groups compared separately).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results showed that the Plighigh T cells in lymph nodes draining s.c. MCA sarcomas generated the most potent antitumor effector cells after Ag-independent culture. Tumor strain-specific IFN-{gamma} secretion was highest in the cultured Plighigh cells, intermediate in unfractionated TDLN cells, and barely detectable in the cultured Pliglow cells. After adoptive transfer, the cultured Plighigh cells were 10- to 20-fold and >30-fold more potent than cultured unfractionated TDLN and Pliglow cells, respectively.

The fresh Plighigh fractions consisted of {alpha}{beta} T cells, NK cells, {gamma}{delta} T cells, and variable numbers of potential AP/CS cells. Such heterogeneity is consistent with the reported distribution of P-selectin ligands in mononuclear leukocytes. Robust synthesis of P- and E-selectin binding sites during {alpha}{beta} T cell activation is well known (12, 14). In particular, T1 polarization is associated with selectin ligand expression both in vitro (6, 7, 15) and in vivo (8, 9). Selectin binding sites were also reported on resting and IL-12-stimulated human NK cells (16) and {gamma}{delta} T cells in ruminants (17). In addition, selectin ligands were detected on a variety of AP/CS cells, including activated B cells (18, 19), monocytes (20, 21), and some dendritic cells (22, 23).

The high percentage of AP/CS cells in the fresh Plighigh fraction suggested that these cells might account for the activity of cultured Plighigh cells in vivo. This explanation is unlikely for several reasons. The activation protocol used immobilized anti-CD3 to directly cross-link CD3 epitopes on T cells. As a result, endogenous APC were bypassed during ex vivo expansion of the TDLN fractions. Furthermore, {alpha}{beta} T cells expanded well in all fractions. The TDLN and TLE cultures showed the greatest expansion (CD8, 100- to 120-fold; CD4, ~20-fold). The Plighigh cultures were slightly less active (CD8, ~94-fold; CD4, ~9-fold). The Pliglow cultures showed lower, but still substantial increases in cell number (CD8, ~40-fold; CD4, ~9-fold). Thus, all fractions contained sufficient costimulatory activity for at least five to seven CD8 and three to four CD4 doublings. Furthermore, neither B cell depletion nor addition of exogenous irradiated splenocytes altered the difference between the cultured Plighigh and Pliglow populations. Finally, coculture of the Pliglow population with irradiated Plighigh cells failed to increase antitumor activity. Consequently, the increased antitumor activity in cultured Plighigh TDLN does not result from the endogenous AP/CS cells in the population. We conclude that the Plighigh T cells contained the most potent tumor-reactive preeffector/effector cells. P-selectin sorting concentrated these cells, thereby increasing the antitumor activity that developed after Ag-independent expansion in vitro.

The P-selectin ligands were not simply surrogate markers for T cell activation in TDLN because they were detected on <25% of the T cells showing phenotypic evidence of activation (low levels of CD62L; elevated levels of CD69 and CD44). Furthermore, the phenotype of fresh Plighigh T cells resembled the effector memory T cells characterized by Sallusto and colleagues in humans (24, 25). These investigators identified three populations of resting (CD69- and CD25-negative) T cells based on adhesion receptors, cytokine receptors, and functional characteristics. Naive T cells expressed uniformly high levels of CD62L, high levels of the CCR7 receptor, low levels of CD44, and no detectable selectin ligands (assessed by HECA-452 expression). The central memory subset expressed CD62L, CCR7, and intermediate levels of CD44. The effector memory subset expressed bimodal CD62L, minimal CCR7, and the highest levels of CD44 and HECA-452. The effector memory cells also expressed multiple chemokine receptors associated with recruitment into inflammatory sites, contained perforin, and rapidly synthesized IFN-{gamma} upon stimulation. These and other findings in humans were most consistent with a linear model of T cell differentiation that proceeded from naive, through central memory, and culminated in effector/effector memory cells.

The Plighigh T cells in TDLN expressed CD69 and some CD25, and thus differed from resting human effector memory cells in the peripheral blood. However, the adhesion receptor profile was similar, suggesting that the subset consisted of at least partially differentiated effector cells. The increased T1 effector activity in cultured Plighigh cells and the diminished levels in cultured Pliglow cells provided additional evidence for this assertion. Whether the tumor-responsive Plighigh T cells were irreversibly committed to T1 differentiation in vivo or represented partially differentiated cells that committed fully during the subsequent expansion in vitro remains to be determined. Nonetheless, the findings in murine TDLN are compatible with a linear model of T cell differentiation in which tumor-reactive Tc1 and Th1 pre-effector/effector cells synthesized high levels of selectin ligands in vivo.

The cultured Plighigh cells contained several potential effector populations, including the CD8 and CD4 subsets of {alpha}{beta} T cells, cells expressing NK epitopes, and {gamma}{delta} T cells. CD19-positive B cells were also detected in the adoptively transferred populations. However, the paramagnetic depletions indicated that 95–97% of the antitumor activity resided in cells expressing CD8 or CD4 epitopes. The subsets made independent contributions to tumor suppression because depletion of both diminished activities significantly more than depletion of either one alone. The CD8 fraction was 5-fold larger than the CD4 fraction and contained most of the antitumor activity. However, if one adjusts for its small size, the CD4 subset equaled or exceeded the activity of the CD8 subset on a per cell basis. Finally, neither NK-T nor true NK cells contributed to tumor suppression because complete removal of cells expressing the NK 1.1 and DX-5 epitopes had no impact on tumor suppression. Consequently, the CD8 and CD4 subsets of {alpha}{beta} T cells mediated the increased antitumor activity of cultured Plighigh cells.

The cultured Plighigh cells secreted substantially more IFN-{gamma} than either the unfractionated or Pliglow populations. Previous studies with cultured TDLN and TIL cells indicated that tumor-elicited IFN-{gamma} secretion by the adoptively transferred cells was a major factor in the suppression of MCA sarcomas (4, 10). The antitumor activities of IFN-{gamma} include direct inhibition of sarcoma growth (26), induction of macrophage-dependent delayed-type hypersensitivity (27, 28), and inhibition of angiogenesis (29). Therefore, the tumor-specific IFN-{gamma} secretion by the cultured Plighigh cells is likely to account, in part, for their increased potency against pulmonary MCA sarcoma micrometastases in vivo.

The depletion of TDLN cells expressing P-selectin ligands before culture reduced, but did not eliminate antitumor activity. As noted above, small numbers of T cells expressing selectin ligands remained in the Pliglow fractions. These cells expressed low number of P-selectin binding sites. The time course for selectin-ligand synthesis during T cell differentiation is not known, but a gradual increase in ligand density as T cells mature is consistent with the multistep process required for selectin-ligand synthesis (7, 12, 14, 30, 31, 32). In keeping with this hypothesis, Thoma and colleagues (33) found that both Plighigh and Pliglow CD4 cells produced IFN-{gamma} in vivo, with the former population showing higher activity than the latter. Therefore, T cells with low levels of P-selectin ligands may account for the residual antitumor activity in the Pliglow population. In addition, the Pliglow population may contain Th2 precursors with antitumor activities such as those reported in responses to B16 melanomas (34) and OVA-transduced A20 tumors (35). Regardless of the cell type(s) responsible for the residual activity of cultured Pliglow cells, the Plighigh subset of TDLN clearly developed the most active tumor-specific {alpha}{beta} T1 effector cells after expansion in vitro.

At the time of infusion, the cultured Plighigh population invariably contained more T cells expressing P-selectin ligands than the cultured unfractionated or cultured Pliglow populations. However, this factor did not contribute to the observed differences in therapeutic activity in vivo. Wild-type cultured Plighigh cells suppressed MCA 205 pulmonary micrometastases equally well in wild-type and E-/P-selectin deletion mutants. This outcome is consistent with immunocytochemistry, showing minimal expression of P- and E- selectin on the microvasculature of tumor-bearing lungs (not shown). Thus, the therapeutic activity of cultured Plighigh cells does not require selectin expression in or near lung micrometastases.

A recent report by Plautz and colleagues (36) concluded that adoptively transferred cultured TDLN must enter metastatic lesions to suppress growth. How, then, did the cultured Plighigh cells enter tumor emboli in the lung if selectin-mediated recruitment is not involved? The age of the metastases used in this study and the existence of redundant recruitment mechanisms suggested several possibilities. In previous studies, the first step in lung colonization after i.v. infusion was the arrest of individual tumor cells and clumps in the pulmonary microvasculature (37). Some tumor emboli eroded and destroyed the occluded vessels within 48–72 h (38). Others grew within the lumen or in a subendothelial location, causing focal narrowing of the involved vessel at early time points (39). Assuming similar events occurred following infusion of MCA 205 cells, T lymphoblasts administered 3 days later could interact directly with tumor cells in partially occluded blood vessels. They could also extravasate into lesions through damaged vessels, thus bypassing the usual recruitment process. Alternatively, one or more other tethering receptors may be involved, including CD44 (40, 41), the {alpha}4 integrins (42, 43), or VAP-1 (44). Finally, adhesion-receptor independent arrest and transmigration of circulating leukocytes can occur in the lung (45, 46). Consequently, circulating T lymphoblasts have multiple, selectin-independent mechanisms for accessing the small tumor emboli used in the current study.

However, the high levels of selectin ligands on the cultured Plighigh cells may augment their clinical activity in other settings. Trafficking studies in the skin (6, 8), lung (47, 48), and peritoneal cavity (9) showed clearly that selectin ligands increased the recruitment of circulating T lymphoblasts when target lesions expressed P- and/or E-selectin on the local microvasculature. In malignant tumors, selectin expression on the microvasculature was highly variable. High levels were detected in some murine (49) and human malignancies (50), frequently at the invasive margin (51, 52). In advanced melanomas, very little expression was detected (53). In contrast, radiation therapy increased selectin expression in the microvasculature and increased leukocyte entry into experimental tumors (54). In addition, infusion of TNF-{beta} and leukotriene B4 into tumor vessels of Lewis-lung carcinomas increased selectin-mediated tethering and {beta}2 integrin-mediated transmigration into the tumor mass (52). Therefore, the potency of cultured Plighigh cells may increase further when selectins are either spontaneously expressed or induced on the microvasculature in metastatic lesions.

In brief, this study demonstrated that the Ag-driven synthesis of P-selectin ligands observed in inflammatory and OVA-transgenic models occurred during tumor Ag-driven T cell responses in lymph nodes as well. Paramagnetic sorting with an IgM-CD62P chimera concentrated the Plighigh T cells without diminishing their growth potential. Ag-independent expansion of the subset, without added IL-12, enhanced tumor-specific IFN-{gamma} synthesis and markedly improved the therapeutic impact of adoptive immunotherapy. We conclude that {alpha}{beta} T cells primed for differentiation into tumor-reactive Tc1 and Th1 effector cells express high levels of P-selectin ligands in TDLN.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01 CA73059 (to L.M.S.) and a Gillson Longenbaugh Foundation award (to A.E.C.). Back

2 K.T., N.T., and R.A.C. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Lloyd M. Stoolman, University of Michigan, M4224 Medical Sciences Building 1, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address: stoolman{at}umich.edu Back

4 Abbreviations used in this paper: TDLN, tumor-draining lymph node; AP/CS, Ag-presenting and costimulatory; DPBS, Dulbecco’s PBS; MLM, mouse lymphocyte culture medium; TLE, T lymphocyte enriched; T1, Tc1 and Th1. Back

Received for publication November 20, 2000. Accepted for publication June 11, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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