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Adoptive Immunotherapy of Advanced Tumors with CD62 L-Selectin^low Tumor-Sensitized T Lymphocytes Following Ex Vivo Hyperexpansion¹

Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06510

	Abstract

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Tumor-draining lymph nodes (TDLN) contain sensitized T cells with the phenotype CD62 L-selectin^low (CD62L^low) that can be activated ex vivo with anti-CD3 mAb and IL-2 to acquire potent dose-dependent effector function manifested upon adoptive transfer to secondary tumor-bearing hosts. In this study advanced tumor models were used as a stringent comparison of efficacy for the CD62L^low subset, comprising 5–7% of the TDLN cells, vs the total population of TDLN cells following culture in high dose IL-2 (100 U/ml). During the 9-day activation period the total number of CD8⁺ T cells increased 1500-fold, with equivalent proliferation in the CD62L^low vs the total TDLN cell cultures. Adoptive transfer of activated CD62L^low cells eliminated 14-day pulmonary metastases and cured 10-day s.c. tumors, whereas transfer of maximally tolerated numbers of total TDLN cells was not therapeutic. Despite their propagation in a high concentration of IL-2, the hyperexpanded CD62L^low subset of TDLN cells functioned in vivo without exogenous IL-2, and CD8⁺ T cells demonstrated relative helper independence. Moreover, the anti-tumor response was specific for the sensitizing tumor, and long term memory was established. The facile enrichment of tumor-reactive TDLN T cells, based on the CD62L^low phenotype, circumvents the need for prior knowledge of the relevant tumor Ags. Coupling the isolation of pre-effector T cells with rapid ex vivo expansion to >3 logs could overcome some of the shortcomings of active immunotherapy or in vivo cytokine treatment, where selective robust expansion of effector cells has been difficult to achieve.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Successful T cell immunotherapy of cancer is the outcome of a multistep process that is dependent on successful Ag priming, numerical amplification of rare Ag-specific precursors, and infiltration of tumors in all metastatic sites by effector T cells. A deficiency at any step along this chain of events can dampen the aggregate antitumor response to a subtherapeutic level; consequently, efforts have been directed toward optimizing each step (1, 2, 3). The existence of specific T cells with therapeutic potential that were naturally primed in the face of progressive human tumor was first documented using tumor-infiltrating lymphocytes (4, 5) and was confirmed in PBL (6). Recently more sophisticated studies in patients with several types of malignancies have confirmed the existence of T cells reactive against peptides derived from putative tumor Ags before exogenous priming (7, 8, 9, 10, 11, 12). This has provided the theoretical foundation for active immunotherapy using optimized peptide vaccines from defined tumor Ags to amplify the endogenous response, with evidence for increased precursor frequency following vaccination (13, 14, 15, 16). While encouraging, the increases in precursor frequency following Ag vaccination detected in peripheral blood have typically been <10-fold and are far below what is observed in response to viral infection. Moreover, it has been difficult to account for effector cell redistribution and loss through apoptosis (17) in assessing the total numerical expansion mediated by active immunotherapy. More importantly, in some instances the resultant T cells exhibited functional defects, and this is echoed by less than optimal clinical responses against advanced tumors (15, 18). This raises questions about whether the magnitude of numerical expansion or gain of effector function induced by vaccination alone will be sufficient to impact macroscopic tumor burden and whether this can be augmented by ex vivo expansion followed by adoptive transfer.

There is strong preclinical evidence that tumor-specific T cells need to be brought to a heightened state of activation to mediate regression of established tumors. Because the effector requirements to achieve regression of established tumor are more stringent than other measures of T cell function, it is important to assay for tumor regression rather than use surrogate measures of efficacy. As an example of the relative resistance of tumors to effector cells, it was demonstrated that transgenic T cells reactive against minor Ags were not depleted during progressive tumor growth and were competent to reject simultaneous skin grafts, yet failed to protect against tumor (19). This may in part be due to the production of immunosuppressive molecules by tumor cells and/or stromal cells that prevent the development of effector function (20, 21, 22). Thus, an important consequence of ex vivo activation is that culture conditions can be optimized to eliminate tumor-induced suppressive effects. Our previous studies demonstrated that freshly isolated tumor-draining lymph nodes (TDLN)³ T cells had defects in TCR-mediated signal transduction and were not immediately competent in adoptive transfer models (23, 24). Importantly, ex vivo activation of TDLN cells using anti-CD3 mAb and a low concentration of IL-2 (4 U/ml) reversed defects in TCR-mediated signal transduction and resulted in acquisition of antitumor effector function; however, the total numerical expansion of lymph node (LN) cells was typically 3- to 5-fold, representing 6- to 10-fold T cell expansion.

Tumor regression mediated by adoptive transfer of T cells is dose dependent, and ex vivo anti-CD3/IL-2 activation provides the opportunity to numerically expand TDLN cells to large numbers by using high concentrations of IL-2. Anti-CD3 and high dose IL-2 stimulation is not selective for the infrequent tumor-reactive T cells among TDLN cells, and in previous experiments the use of either 100 or 1000 U/ml IL-2 during a 5-day culture activation resulted in abrogation of in vivo efficacy against 3-day established pulmonary metastases (24). Such studies were performed only on unfractionated TDLN T cells. However, our subsequent studies showed that CD62 L-selectin^low (CD62L^low) TDLN cells were enriched for tumor-reactive T cells, whereas the reciprocal CD62L^high population had no therapeutic activity (25, 26). Therefore, in this report we isolated cells with the CD62L^low phenotype from TDLN before culture activation with anti-CD3 mAb and high dose IL-2 (100 U/ml) and compared their therapeutic activity to that of the total population of TDLN cells. The CD62L^low subset underwent rapid and extensive proliferation, with preservation of in vivo therapeutic activity. The antitumor response retained features of a classical T cell response to tumor-specific transplantation Ag-type Ags, including specificity and development of long-term memory. Interestingly, although the T cells were derived using a high concentration of IL-2, they did not require the administration of exogenous IL-2 to the host to function or persist.

	Materials and Methods

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Animals

Female B6 (C57BL/6) mice, 6–8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained in a specific pathogen-free environment and fed ad libitum according to National Institutes of Health guidelines.

Tumor

MCA 205 H12 was derived from the 3-methylcholanthrene-induced fibrosarcoma MCA 205 by limiting dilution cloning. MCA 207 G11 was similarly derived from MCA 207 by limiting dilution cloning. Tumor cell lines were maintained by serial passage in vitro in conditioned medium (CM): RPMI 1640 supplemented with 10% heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 50 µg/ml amphotericin B (all obtained from Life Technologies, Grand Island, NY) and 5 x 10^-5 M 2-ME (Sigma-Aldrich, St. Louis, MO) as previously described (27).

Flow cytometry and mAbs

Freshly harvested LN cells or culture-activated cells were incubated with anti-CD4, anti-CD8, anti-CD62L, or anti-TCR or isotype control mAb (all obtained from BD PharMingen, San Diego, CA) and analyzed using CellQuest software.

Activation of LN T cells

Inguinal lymph nodes draining 12-day s.c. MCA 205 H12 tumors were removed, mechanically teased apart, and prepared as a single-cell suspension in MACS buffer. A portion of the LN cells was incubated with anti-CD62L microbeads (Miltenyi Biotec, Auburn, CA) using 100 µl microbeads/10⁸ LN cells in 1 ml, according to the manufacturer’s instructions. The cells were applied to Midi-MACS columns, and the nonadherent (CD62L^low) fraction was collected. Total LN cells or the CD62L^low fraction were suspended in CM at 2 x 10⁶/ml and activated with plate-bound anti-CD3 mAb (145-2C11) for 48 h at 37°C in 5% CO₂ in 24-well plates. Activated cells were resuspended at a concentration of 5 x 10⁴/ml in CM with IL-2 (2 or 100 U/ml) for 3 days, then diluted to a concentration of 10⁵/ml in CM with IL-2 for an additional 4 days in gas-permeable culture bags (Baxter Healthcare, Deerfield, IL). In one experiment, LNs or spleens from mice cured of s.c. tumor were harvested 117 days after initial adoptive transfer and activated as described above.

Adoptive immunotherapy

B6 mice were inoculated s.c. in the hind flank with 1.5 x 10⁶ tumor cells to initiate tumors. Ten days later mice received 5 Gy total body irradiation (TBI) from a ¹³⁷Cs source, followed by injection with the indicated number of activated T cells or HBSS through the tail vein. Tumor size was measured in two perpendicular dimensions three times per week with Vernier calipers (Bel-Art Products, Pequannock, NJ). Pulmonary metastases were established byi.v. inoculation of 3 x 10⁵ tumor cells. Fourteen days later mice received 5 Gy TBI, followed byi.v. transfer of the indicated number of T cells. Mice were sacrificed 21 days after tumor inoculation, lungs were insufflated with india ink, and metastases were enumerated using a dissecting microscope. CD4⁺ or CD8⁺ cells were depleted by i.v. administration of 100 µg anti-CD4 (GK 1.5) or anti-CD8 (2.43), respectively.

IFN- production assay

Culture-activated T cells were incubated with an irradiated single-cell digest of MCA 205 H12 tumor or MCA 207 G11 tumor or with PHA (3 µg/ml), or were unstimulated for 4 h in the presence of monensin and stained with anti-IFN- and anti-CD8. Secreted IFN- was determined by incubating 2 x 10⁶ culture activated T cells with 4 x 10⁵ irradiated tumor cells for 24 h at 37°C and storing supernatants at -70°C. Concentrations of IFN- were measured by ELISA using paired mAb purchased from BD PharMingen.

Statistical analysis

A t test was performed on paired samples, and p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Total TDLN cells and the CD62L^low subset each undergo rapid proliferation ex vivo

MCA 205 H12 is a weakly immunogenic fibrosarcoma that grows progressively in B6 mice. LN draining 12-day s.c. tumors were harvested as a source of sensitized T cells, and a portion was subjected to magnetic bead enrichment of the CD62L^low subset. In a series of experiments the recovery of CD62L^low cells consistently ranged between 5 and 7% of the input cells. FACS analysis of freshly harvested LN cells pooled from five mice demonstrated that 11% of the LN cells were TCR⁺ and CD62L^low, whereas the majority (55%) were TCR⁺ CD62L^high (Fig. 1A). The CD62L^low subset was 31% TCR⁺ (Fig. 1C). The composition of the CD62L^low T cells showed a predominance of CD4⁺ cells at 25%, with only 6% CD8⁺ cells (Fig. 1G), whereas the TDLN had a reversed CD8:CD4 ratio, with 16% CD4⁺ and 22% CD8⁺ cells (Fig. 1E). After activation for 2 days on plate-bound anti-CD3 mAb, most cells developed lymphoblast morphology; however, there was a 50% decrease in the total cell count in the CD62L^low cultures due to loss of TCR-negative cells. Activation of TDLN cells (Fig. 1B) or CD62L^low cells (Fig. 1D) in the presence of rIL-2 (100 U/ml) for 7 additional days stimulated rapid proliferation of TCR⁺ cells that uniformly became CD62L^low. The CD8⁺ cells proliferated more rapidly than the CD4⁺ cells and predominated the cultures after 9 days, comprising 86% of the TDLN culture and 69% of the CD62L^low culture, but CD4⁺ cells were still present (Fig. 1, F and H). The activated T cells did not express NK1.1 (data not shown). The stimulation with anti-CD3 and a high concentration of IL-2 had equivalent effects on the numerical expansion of the CD8⁺ subset in the total TDLN cultures (1470-fold) and the CD62L^low-enriched cells (1680-fold). Evidently the strong mitogenic signal provided by anti-CD3 and high concentration of IL-2 was sufficient to drive rapid proliferation in CD8⁺ TDLN cells with a naive (CD62L^high) phenotype; thus, there was no gain in proliferative potential for T cells of the CD62L^low effector/memory phenotype. The peak of proliferation was reached on day 10 of culture, and subsequently the number of cells declined.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Phenotype of TDLN and the CD62Llow subset at isolation and at time of adoptive transfer. Inguinal LNs draining progressive 12-day MCA 205 H12 fibrosarcomas were prepared as a single-cell suspension (TDLN). The CD62Llow subset was collected by magnetic bead depletion of CD62Lhigh cells, yielding 5.7% of the input cells. Total TDLN cells or the CD62Llow subset were independently activated with anti-CD3 mAb for 2 days, followed by IL-2 (100 U/ml) for 7 days. Cells were incubated with the indicated Ab pairs and analyzed by FACS. FITC-conjugated anti-CD62L and PE-conjugated anti-TCR paired Abs for TDLN day 0 (A), TDLN day 9 (B), CD62Llow day 0 (C), and CD62Llow day 9 (D) are shown. FITC-conjugated anti-CD8 and PE-conjugated anti-CD4 paired Abs for TDLN day 0 (E), TDLN day 9 (F), CD62Llow day 0 (G), and CD62Llow day 9 (H) are shown.

The function of the culture-activated T cells was assessed in vitro and in vivo. Each of the cell populations, total TDLN and the CD62L^low subset, had minimal spontaneous production of IFN-, but a large response to PHA stimulation (Fig. 2, B and F). In contrast, Ag-driven stimulation provided by a single-cell suspension of MCA 205 H12 tumor resulted in production of IFN- by 4% of the CD62L^low culture compared with 0.15% of the TDLN culture (Fig. 2, C and G). As might be anticipated for an Ag-specific immune response, the tumor-reactive cells were a minority even in the enriched CD62L^low subset. There were far fewer CD8⁺ T cells (0.65%) that reacted with the MCA 207 fibrosarcoma with background levels in the total LN T cell cultures (Fig. 1, D and H). Although the intracellular IFN- assay provides information on the percentage of responding T cells, it does not provide quantitative information on secreted IFN-. A simultaneous IFN- ELISA assay using a separate aliquot of the activated cells demonstrated that total TDLN cells produced 60 pg/ml spontaneously, 200 pg/ml in response to MCA 205, and 55 pg/ml in response to MCA 207 tumor digest. In contrast, the CD62L^low cultures produced 90 pg/ml spontaneously, 7200 pg/ml in response to MCA 205, and 400 pg/ml in response to MCA 207 tumor digest. Similar results were observed in an identically designed experiment for both the intracellular IFN- and ELISA assays.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Production of IFN- by activated T cells in response to PHA or tumor cells. TDLN cells or the CD62Llow subset were culture activated with anti-CD3 for 2 days, followed by IL-2 (100 U/ml) for 7 days. T cells (106) were incubated for 4 h without additional stimulation in the presence of PHA, with a single-cell digest of an MCA 205 H12 tumor (2 x 105 cells), or with a single-cell digest of MCA 207 G11 tumor (2 x 105 cells). T cells were harvested, incubated with FITC-conjugated anti-CD8 and PE-conjugated anti-IFN-, and analyzed by FACS. A, CD62Llow no stimulation control; B, CD62Llow cells stimulated with PHA; C, CD62Llow cells stimulated with MCA 205 H12 tumor; D, CD62Llow cells stimulated with MCA 207 G11 tumor; E, TDLN no stimulation control; F, TDLN stimulated with PHA; G, TDLN stimulated with MCA 205 H12 tumor; H, TDLN stimulated with MCA 207 G11 tumor.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Adoptive immunotherapy of 10-day s.c. MCA 205 H12 tumors using culture-activated total TDLN or CD62Llow cells. B6 mice (n = 5/group) were inoculated with 1.5 x 106 MCA 205 H12 tumor cells in the flank to initiate tumor. Ten days after inoculation mice received 5 Gy TBI from a 137Cs source followed by transfer of 1 ml HBSS, 4 x 107 TDLN cells, or 4 x 107 CD62Llow cells via tail vein injection. Tumors were measured in two perpendicular dimensions by Vernier calipers three times per week and were plotted as the mean ± SEM. Comparison of tumor growth by t test: TDLN vs HBSS, p = 0.84; CD62Llow vs HBSS, p < 0.001; TDLN vs CD62Llow, p < 0.001. All mice treated with CD62Llow cells had complete resolution of palpable tumor and were rechallenged with 1.5 x 106 tumor on day 49. No tumor recurrence at the original site or at the challenge site was observed beyond 100 days.

The antitumor reactivity of the CD62L^low subset of LN cells was tested in another stringent model, 14-day pulmonary metastases. As demonstrated in Fig. 4, adoptive transfer of 20 x 10⁶ total TDLN cells had no effect on the number of pulmonary metastases (p = 0.637 vs HBSS control). In marked contrast, the CD62L^low subset displayed high therapeutic activity. A dose of 5 x 10⁶ cells completely eliminated lung metastases, and 1.25 x 10⁶ cells significantly reduced the number of metastases (p < 0.001 vs HBSS control). Comparison of the relative therapeutic efficacy showed that 16-fold fewer cells from the CD62L^low subset had superior activity to a dose of the total TDLN cells that was not yet in a therapeutic range. Because the CD62L^low subset initially comprised one-sixth of the total TDLN T cells (Fig. 1A), this result suggests that T cells with regulatory activity were present in the CD62L^high fraction of the total TDLN cultures. Although CD62L^low cells maintained in cultures with a low IL-2 concentration (2 U/ml) demonstrated superior therapeutic activity at the lowest dose tested (1.25 x 10⁶), a greater number of cells was derived in the high dose IL-2 cultures. The concentration of IL-2 present in the cultures was the major factor determining the proliferation of T cells. Overall, there was a 20-fold greater proliferation of CD8⁺ cells and a 10-fold greater proliferation of CD4⁺ cells in the CD62L^low cultures maintained in 100 U/ml IL-2 compared with those in 2 U/ml IL-2.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Adoptive immunotherapy of 14-day MCA 205 H12 pulmonary metastases using activated TDLN or CD62Llow cells. B6 mice (n = 5/group) were injected with 3 x 105 MCA 205 H12 tumor cells via tail vein to initiate pulmonary metastases. Fourteen days later mice received 5 Gy TBI from a 137Cs source, followed by transfer of HBSS (A), 1.25 x 106 CD62Llow cells cultured in 2 U/ml IL-2 (B), 1.25 x 106 CD62Llow cells cultured in 100 U/ml IL-2 (C), 5 x 106 CD62Llow cells cultured in 100 U/ml IL-2 (D), 5 x 106 TDLN cultured in 100 U/ml IL-2 (E), and 20 x 106 TDLN cultured in 100 U/ml IL-2 (E) via tail vein injection. Mice were sacrificed on day 21, lungs were insufflated with india ink, and the metastatic lesions were enumerated. Groups B–D vs group A, p < 0.001 for each; group A vs E, p = 1; group A vs F, p = 0.637.

Lymphocytes maintained in culture with a high concentration of IL-2 often lose their Ag specificity. One of the definitive characteristics of total TDLN or the CD62L^low subset in previous studies employing low concentrations of IL-2 during ex vivo activation was retention of specificity for the tumor that provided the in vivo sensitization (25, 27). We investigated the Ag specificity of CD62L^low T cells derived from MCA 205 H12 draining LNs and cultured in high dose IL-2 (100 U/ml) by adoptive transfer to recipients bearing 10-day MCA 205 H12 or MCA 207 G11 tumors. As demonstrated in Fig. 5, there was no cross-reactivity against the MCA 207 G11 tumor. This indicates that the antitumor response is directed against tumor-specific transplantation Ags rather than tissue-restricted or shared tumor Ags or tumor endothelium.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Specificity of antitumor response mediated by CD62Llow T cells. B6 mice (n = 5/group) were inoculated s.c. with 1.5 x 106 MCA 205 H12 or 1.5 x 106 MCA 207 G11 tumor cells. Ten days later they received 5 Gy TBI from a 137Cs source, followed by adoptive transfer of 5 x 106 CD62Llow T cells derived from an MCA 205 H12 TDLN and activated with anti-CD3 and IL-2 (100 U/ml). Tumor size was measured in two perpendicular dimensions and is plotted the mean ± SEM. Significant difference in tumor size was observed for MCA 205 HBSS vs cell transfer (p = 0.004). There was no difference in size for MCA 207 HBSS vs cell transfer (p = 0.52). There was a significant difference between MCA 205-treated and MCA 207-treated groups (p = 0.005).

During Ag sensitization in tumor-draining LN both CD4⁺ and CD8⁺ T cells are generated. Our previous studies have demonstrated that each of these subsets is able to independently mediate tumor regression (26, 28). Although tumor regression did not require exogenous IL-2, there was the possibility that CD4⁺ cells sustained CD8⁺ T cell viability in vivo through local production of IL-2. The activation conditions ex vivo using high dose IL-2 might also favor the generation of effector CD8⁺ cells with IL-2 dependence. Therefore, anti-CD4 or anti-CD8 mAb was administered to tumor-bearing hosts in addition to the sublethal TBI before T cell transfer to eliminate host T cells and the relevant transferred T cell subset. Sentinel mice harvested 1 day after adoptive transfer demonstrated complete elimination of the relevant subset. As shown in Fig. 6, depletion of CD4⁺ cells caused a delay in the onset of tumor regression, but retention of a therapeutic effect. In contrast, depletion of CD8⁺ cells resulted in prolonged inhibition of tumor progression, but no regression. Likewise, advanced pulmonary tumors were eliminated in the face of CD4 depletion, but not with CD8 depletion, thus demonstrating relative helper-independent function (Table I).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. The CD8 subset of CD62Llow T cells is sufficient to mediate regression of advanced s.c. tumors. B6 mice (n = 5/group) were inoculated with 1.5 x 106 MCA 205 H12 tumor cells s.c. The indicated groups were injected i.v. with anti-CD4 or anti-CD8 mAb on day 9 and received 5 Gy TBI on day 10 before T cell transfer. Tumor size is displayed as the mean ± SEM. Differences between the HBSS control group and the CD62Llow cell and anti-CD4+ cell groups were significant (p < 0.001). The difference between the HBSS and anti-CD8+ cell groups was not significant (p = 0.11).

The effector/memory response differs from the primary response in magnitude, time course, and costimulation requirements (29, 30, 31). Two types of memory cells have been characterized: central memory cells that preferentially migrate to secondary lymphoid tissue, and effector/memory cells that infiltrate peripheral tissues at sites of inflammation. The activation conditions, using high dose IL-2, and the phenotype of uniform low expression of CD62L on transferred T cells were characteristic of effector/memory cells (32). The capacity of transferred effector cells to persist long term following tumor regression and then respond to a second course of extensive proliferation ex vivo was determined. Peripheral LNs and spleens were harvested from mice cured of established tumor 117 days earlier by transfer of CD62L^low cells. The CD62L^low fraction was purified and subjected to culture activation with anti-CD3 for 2 days and IL-2 (100 U/ml) for 7 days. Groups of five mice bearing 3-day pulmonary metastases were treated by adoptive transfer of 10⁶ LN cells or 2 x 10⁶ spleen cells. The reactivated LN or spleen memory T cells completely eliminated the pulmonary metastases, whereas HBSS-treated control mice had >250 lung metastases (p < 0.001 for each group).

	Discussion

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These findings demonstrate that T cells are sensitized to tumor Ags during progressive tumor growth and can be activated ex vivo under conditions that generate large numbers of effectors yet retain the ability to function against advanced tumors. Several features of this approach are noteworthy. We isolated T cells from hosts bearing advanced, progressively growing, weakly immunogenic tumors as well as treated recipients bearing advanced tumors. This is a more challenging test of therapeutic efficacy than vaccination/challenge models or treating hosts with microscopic tumor burdens. Advanced established tumor models were investigated because they may help elucidate tumor sensitization and effector mechanisms under conditions that are more relevant to human cancer patients with metastatic disease. With regard to sensitization, adoptive transfer studies have established that TDLNs are an enriched source of tumor-sensitized T cells compared with spleen, and that there was an undetectable level of activity in non-draining LNs (24). Similarly, for patients with malignant melanoma, Melan-A/MART-1 tetramer-reactive T cells displaying CD45RO, an Ag-experienced phenotype, were identified in tumor-involved LN, but not in uninvolved LN or PBL (33). Thus, TDLN or, alternatively, vaccine-primed LNs are an ideal source from which to isolate sensitized T cells. For therapeutic purposes, vaccine-primed LNs may offer advantages over tumor-infiltrated LNs because the kinetics of the reaction can be controlled such that T cells are harvested at the peak of local accumulation. There is also the possibility that tumors releasing locally immunosuppressive cytokines could blunt the response in tumor-involved LNs compared with tumor-free vaccine-primed LNs. The TDLNs in this tumor model are analogous to vaccine-primed LNs in that they are removed at the peak of the primary response and because MCA 205 H12 tumor cells do not metastasize to LNs at a detectable level. For these studies we used whole tumor cells as a vaccine to sensitize draining LNs because the relevant tumor Ags have not been defined. This approach would not necessarily preclude other forms of tumor Ag vaccines that potentially are more efficacious than whole tumor cells. The route of vaccine delivery impacts the location and type of response, and intradermal delivery of peptide or dendritic cell preparations may similarly result in sensitization, predominantly within draining LNs.

Tumor regression is dependent on the number of Ag-specific effector T cells transferred relative to the tumor burden. Therefore, the frequency of Ag-specific effectors in the adoptively transferred T cell population is as important as the total number of T cells generated. Despite enrichment of sensitized T cells in TDLN compared with other sites, it is evident that without further purification a sufficient dose to treat advanced, weakly immunogenic tumors could not be achieved. There is a limit to the total number of activated T cells that can be accommodated by the recipient. Immediate toxicity from leukostasis and pulmonary emboli became dose-limiting at T cell doses higher than those reported here when the cells were activated with high concentrations of IL-2. In contrast, regression of advanced tumors occurred with far fewer CD62L^low cells well within tolerability. It is important to point out that the anti-CD3 mAb/IL-2 stimulation does not selectively amplify the tumor-reactive T cells relative to irrelevant T cells. By eliminating irrelevant T cells present in the CD62L^high subset by magnetic bead separation, the frequency of specific effector T cells was increased such that a tolerable total cell dose could treat advanced tumors. The CD62L^low subset is enriched, but is not completely purified, for tumor-reactive T cells, as reflected by the IFN- response to tumor stimulation in only 4% of the transferred cells. It remains to be seen whether other strategies for T cell purification based on binding to tumor-Ag/MHC tetramer complexes or IFN- production will effectively purify tumor-reactive T cells (33, 34, 35, 36).

A highly purified, tumor-reactive T cell subset is likely to initially contain a relatively small number of cells. However, purification provides an opportunity to achieve massive expansion of cell numbers while retaining the desired repertoire diversity through the use of Ag-independent stimuli such as anti-CD3 mAb. In this study we used high concentrations of IL-2 to drive proliferation of effector CD8⁺ T cells to >1000-fold over 9 days while retaining significant numbers of CD4⁺ T cells. The power of ex vivo expansion to augment immunotherapy is illustrated by the fact that the quantity of CD62L^low cells obtained from a single LN expanded to provide a sufficient dose to treat 20 mice bearing 14-day pulmonary metastases. It was not implicit from earlier studies that T cells would function so well in vivo after growth in high dose IL-2. In studies performed soon after the identification of anti-CD3 mAb and the availability of human rIL-2, it was demonstrated that splenocytes stimulated with anti-CD3 and high concentrations of IL-2, termed anti-CD3-induced killer cells, could be hyperexpanded >1000-fold in short-term culture (37, 38). While these initial studies showed far greater proliferation for anti-CD3/IL-2-stimulated cultures compared with IL-2-stimulated lymphokine-activated killer cells, the in vivo antitumor reactivity was relatively modest, and a memory response was not generated.

Another aspect of these experiments that has relevance for clinical application is that the transferred T cells functioned in vivo without administration of adjunct cytokine. Typically, adjunct IL-2 has been an integral component of T cell adoptive immunotherapy, particularly for strategies such as tumor-infiltrating lymphocytes that incorporate high concentrations of IL-2 for ex vivo propagation (39, 40). However, our previous studies have demonstrated an inhibitory effect of IL-2 on the efficacy of T cell immunotherapy for tumors in certain anatomic locations, including the brain and s.c. tumors (27, 41). This may be due in part to an altered capacity of T cells to infiltrate s.c. or intracranial tumors in the presence of exogenous IL-2 (42). IL-2 plays a critical, time-dependent role in the development of CD8⁺ effector cells. Tumor models that examined in vivo efficacy of adoptively transferred naive OT-1 TCR-transgenic T cells challenged with OVA expressing EL-4 thymoma have identified activation-induced nonresponsiveness of CD8⁺ T cells as a critical impediment to tumor eradication (43). Exogenous IL-2 exposure at a critical time following Ag stimulation induces long-term signaling alterations in CD8⁺ T cells that permits their subsequent proliferation upon Ag exposure (44). In a similar fashion, provision of IL-2 during a critical period following ex vivo Ag stimulation generates better CD4⁺ effector T cell function (45). In our experimental system the IL-2 is provided as T cells are differentiating to an effector/memory phenotype; thus, at the time of adoptive transfer they may have passed a critical period for IL-2 stimulation (30, 46, 47).

The rationale for investigating the effector response in advanced tumor models is that it may reveal unique requirements not apparent through in vitro assays or therapy of microscopic tumors. Recent studies have demonstrated the inefficiency of extralymphatic tumor to stimulate effector T cell responses (19, 48, 49). In addition, the threshold for immune rejection appears to be much higher for tumors containing established stroma than for normal tissue or for injected single-cell suspensions of tumor (21). One possible effect of stroma in advanced tumors is that it may limit the infiltration of effector T cells from the vasculature into tumors, a requirement for regression to occur (50). A second possibility is that a sufficient local concentration of immunosuppressive cytokines is retained when tumors reach a sufficient size or have recruited fibroblasts and other accessory cells (20). The effectiveness of therapy with CD62L^low cells against advanced s.c. tumors indicates that their physical characteristics or the presence of stroma is not an insurmountable obstacle to T cell immunotherapy. The pattern of slow regression, lack of hemorrhagic necrosis, and, most importantly, specificity indicates that tumor cells, rather than tumor vasculature, were the target of the response.

Despite significant recent progress in defining human tumor Ags and understanding the process of Ag presentation, immunotherapy for cancer patients has not yet advanced to a reliable durable therapy. Generation of a sufficient dose of cells that have acquired competence to perform effector function within tumor tissue may be one limiting factor, particularly in patients with advanced disease. The data presented here indicate that enrichment of tumor-sensitized precursors followed by ex vivo activation in conditions that achieve rapid proliferation can preserve subsequent in vivo effector function. This strategy has the potential to augment the aggregate potency of cancer immunotherapy in situations where the number of effector cells is a limiting factor.

	Acknowledgments

 
We thank Drs. Peter Cohen and Suyu Shu for their helpful and critical reviews of this manuscript.

	Footnotes

 
¹ This work was supported by a grant from the James S. McDonnell Foundation (to G.E.P.).

² Address correspondence and reprint requests to Dr. Gregory E. Plautz, Department of Pediatrics, Yale University School of Medicine, LMP 4083, 333 Cedar Street, New Haven, CT 06510. E-mail address: gregory.plautz{at}yale.edu

³ Abbreviations used in this paper: TDLN, tumor-draining lymph node; CD62L, CD62 L-selectin; CM, conditioned medium; LN, lymph node; TBI, total body irradiation.

Received for publication February 21, 2002. Accepted for publication July 18, 2002.

	References

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