Further characterization of in vivo effective and impaired CD8+ T cells.

To investigate the mechanisms underlying the lack of antitumor effectiveness of highly differentiated T cells, we extended our characterization of early effector and intermediate effector T cells using microarray analysis. RNA was extracted from early effector and intermediate effector T cells and indirectly labeled after a single round of linear amplification. The labeled samples were combined and hybridized overnight on a murine 22,000-gene oligonucleotide array. All raw data sets have been deposited, and processed values and complete gene lists are available (GEO series id GSE2348; http://www.ncbi.nlm.nih.gov/geo/).

Microarray analysis confirmed that impaired cells differentially expressed genes associated with highly differentiated effector T cells. The expression of many genes involved in effector functions such as granzymes, perforin, IFN-γ, and fas-ligand were strongly upregulated in impaired cells compared with in vivo effective cells (Table ​(Table1).1). Furthermore, intermediate effector cells overexpressed eomesodermin, a transcription factor recently described as a master regulator for CD8⁺ effector functions (17). Conversely, early effector cells showed a more naive profile, expressing higher levels of the lymphoid-homing molecules cd62l, integrin-α_E, and ccr7, the costimulatory molecule cd27 and IL-7 receptor α (il-7rα). However, highly differentiated T cells overexpressed a number of genes that may prejudice their survival capability, such as the proapoptotic molecules bid, bad, and fas-ligand (18–20). Intermediate effector cells also expressed higher levels of molecules associated with replicative senescence such as killer cell lectin-like receptor G1 (klrg1) (21, 22) and the mortality factor mrgx (23), which suggests that the proliferative potential of these cells may be decreased. Microarray data were validated by flow cytometry, Western blot, and functional analyses (Figure1, A and B; Figure ​Figure2,2, A and B). Interestingly, according to the Western blot analysis, it was the truncated, active, C-terminal products of caspase-3 and -8 cleavage of BID (tBID) that were present at higher levels in impaired cells (18, 20). It should be noted that it was the p13 product — usually a minor form exclusively associated with the mitochondria — that was strongly elevated (>3-fold by densitometry) in intermediate effector T cells (20). Thus, the Western blot confirmed and further refined the results of the analysis of global gene expression.

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Figure 2

Cytofluorometry and Western blot analysis of effective and impaired CD8⁺ T cells. Pmel-1 CD8⁺ T cells at progressive stages of differentiation were generated using multiple in vitro stimulations with 1 μM hgp10025–33 and 30 IU/ml of rhIL-2. (A) Flow cytometry for the surface expression of IL-7Rα and KLRG-1. Results after gating for CD8⁺ cells are shown. (B) Detection of BID and its activated, truncated C-terminal cleavage products (tBID) p15 and p13 and BAD by Western blot analysis.

Table 1

Microarray analysis of early effector and intermediate effector T cells

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T cell differentiation is associated with progressive impairment of proliferative capacity.

The analysis of global gene expression suggested that fully differentiated effector T cells may be less “fit” (24) and more proapoptotic. We sought to determine the relative proliferative capacity of effector T cell subpopulations in vitro and in vivo. We evaluated the proliferative capability in vitro with a CFSE-based assay. Four days after antigen stimulation in IL-2, early effector cells had proliferated vigorously — only 15% of the starting population failed to dilute the CFSE label to levels comparable to those of unlabeled cells (data not shown). By sharp contrast, 55% of the intermediate effector and 59% of the effector T cells failed to fully dilute the label, which indicates that the differentiation state was inversely related to T cell proliferative capacity (Figure ​(Figure33A).

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Figure 3

The differentiation state of CD8⁺ T cells is inversely related to their proliferative capacity. Pmel-1 CD8⁺ T cells at progressive stages of differentiation were generated using multiple in vitro stimulations with 1 μM hgp10025–33 and 30 IU/ml of rhIL-2. (A) In vitro proliferation of effector subgroups. Early effector, intermediate effector, and effector T cells were labeled with CFSE and were cultured with 1 μM hgp10025–33 peptide–pulsed on irradiated splenocytes in the presence of 30 IU/ml rhIL-2. Cells were harvested after 4 days, and their CFSE content was determined. Results after gating on Vβ13⁺ population are shown. This experiment was performed twice with similar results. (B) In vivo proliferation of effector subgroups. Absolute numbers of adoptively transferred pmel-1 cells (CD8⁺Vβ13⁺) in the spleens of tumor-bearing, sublethally irradiated WT mice. Mice were treated with rFPhgp100, rhIL-2 (36 μg per dose), and 1 × 10⁶ pmel-1 early effector, intermediate effector, and effector T cells. Data shown are the mean of 2 mice per group. This experiment was performed twice, with similar results.

To test whether the in vitro findings could predict the proliferative capacity of T effector subsets upon adoptive transfer, we directly quantified the absolute number of CD8⁺Vβ13⁺ pmel-1–Tg T cells at multiple time points in the blood and in the spleen. The differences observed in vivo were more pronounced than those observed in vitro. Each of the effector subsets was transferred (1.0 × 10⁶) with altered peptide ligand vaccine and IL-2. The progeny of all effector subgroups reached a peak number between days 4 and 6 after transfer. However, in the spleen alone, we found that 39.5 × 10⁶ progeny derived from early effectors but only 5.7 × 10⁶ and 1.8 × 10⁶ pmel-1 T cells derived from intermediate effector and effector cells, respectively (Figure ​(Figure3B).3B). A similarly pronounced difference was observed in the blood (data not shown). Thus, progressive acquisition of antitumor effector function led to deterioration of the numbers of surviving T cells in vivo.

Further subfractionation of early effector T cells by expression of CD62L identifies CD8+ T cells with superior in vivo antitumor properties.

In our experiments using the highly effective/early effector population, we observed significant homogeneity with regard to most differentiation markers. However, in the early effector subpopulation there remained significant heterogeneity in the expression of CD62L, which was lost with further T cell differentiation and failure of antitumor function in vivo (Figure ​(Figure11A).

We sought to understand whether CD62L was functionally important or merely a marker of the state of differentiation. We further subfractionated the early effector T cell population by sorting cells into CD62L^high and CD62L^low subsets (Figure ​(Figure4A).4A). The CD62L^low population appeared slightly more differentiated on the basis of CD25 and CD69 expression, but all cells in the CD62L^high and CD62L^low subgroups demonstrated an “antigen-experienced” phenotype. Most phenotypic markers were expressed at a similar level, including CD27, CD44, and IL-7Rα (Figure ​(Figure4B).4B). As KLRG-1 — a sensitive marker for replicative senescence — was virtually absent in both CD62L^high and CD62L^low subsets, neither of the 2 groups had entered into a senescence state (Figure ​(Figure4B).4B). Functionally, CD62L^high and CD62L^low subsets showed similar cytolytic activity and IFN-γ production. Importantly, no deficit in IL-2 release was seen in either population (Figure ​(Figure4C).4C). Furthermore, no differences in proliferation were observed when CFSE-labeled CD62L^high and CD62L^low populations were stimulated in vitro with antigen and IL-2 (Figure ​(Figure44D).

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Figure 4

Subfractionated CD62L^high and CD62L^low early effector cells possess similar properties in vitro. (A and B) Phenotypic characterization of CD62L^high and CD62L^low subsets. Five days after the initial stimulation with antigen and IL-2, CD62L^high and CD62L^low populations were sorted by MACS CD62L-positive selection column and analyzed by flow cytometry on day 6 for the expression of IL-7Rα; lymphoid-homing molecules β7 integrin and CCR7; the costimulatory molecule CD27; activation/effector markers CD44, CD69, CD25, and granzyme-B; and the senescence marker KLRG-1. MFI values after gating on CD8⁺Vβ13⁺ cells are shown. The MFI was determined for all groups at the same time under the same cytoflourometric settings using the same reagents and isotype-matched controls. Data shown are representative of 2 independent experiments. (C) CD62L^high and CD62L^low subsets have similar functional qualities. ⁵¹Cr, IFN-γ, and IL-2 release against hgp100_25–33–pulsed MCA-205 targets. MCA-205 cells plus influenza nucleoprotein peptide (np) were used as control. Where error bars are not visible, it is because they are obscured by the symbols used. Data shown are representative of 2 independent experiments. (D) CD62L^high and CD62L^low subsets have similar proliferative capacity. CD62L^high and CD62L^low cells were labeled with CFSE and were cultured with hgp100_25–33 peptide–pulsed on irradiated splenocytes in the presence of 30 IU/ml of rhIL-2. Cells were harvested from day 1 to day 4, and their CFSE content was determined. Results after gating on CD8⁺Vβ13⁺ population are shown. This experiment was performed twice, with similar results.

Despite these similarities, when transferred in conjunction with an altered ligand vaccine and IL-2, the CD62L^high subset had superior antitumor function (P = 0.009; Figure ​Figure5A).5A). This was associated with pronounced in vivo expansion of the CD62L^high subset in the spleen (Figure ​(Figure5B).5B). Similar results were observed in tumor (Figure ​(Figure5C)5C) and blood (data not shown). Thus, these differences were not the result of selective accumulation in lymphoid tissues mediated by CD62L. However, no statistical difference was observed when high or low subsets were transferred in the absence of vaccination (P > 0.05; Figure ​Figure5A).5A). To definitively assess whether CD62L was functionally important, we crossed pmel-1–Tg mice with CD62L^–/– mice. Splenocytes from pmel-1-CD62L^–/– and age-matched pmel-1-CD62L^+/+ mice possessed similar phenotypic and functional attributes in vitro after activation (data not shown). By contrast, when adoptively transferred in WT mice bearing B16 tumors established for 10 days, pmel-1-CD62L^–/– cells were less effective in treating tumors compared with pmel-1-CD62L^+/+ (P = 0.014; Figure ​Figure5D).5D). Thus, CD62L not only represents a marker of T cell differentiation state but also plays an important functional role in this adoptive immunotherapy model of large, established tumors.

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Figure 5

CD62L-selectin identifies CD8⁺ T cells with superior in vivo antitumor properties. (A) CD62L^high subset possesses superior in vivo antitumor properties. WT mice bearing 10-day-old established subcutaneous B16 tumors were sublethally irradiated and left untreated as control or received adoptive transfer of 1 × 10⁶ of pmel-1 CD62L^high or CD62L^low cells with exogenous rhIL-2 (36 μg per dose) with or without rFPhgp100 vaccination. Results for tumor area are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 3 independent experiments. (B and C) CD62L^high subset expands vigorously in vivo. (B) Absolute numbers of adoptively transferred CD8⁺ pmel-1cells in the spleen of tumor-bearing, sublethally irradiated WT mice. Mice were treated with FPhgp100, rhIL-2 (36 μg per dose), and 1 × 10⁶ of pmel-1 CD62L^high or CD62L^low cells. Data shown are the mean of 2 mice per group. This experiment was performed twice, with similar results. (C) Percentage of CD8⁺ pmel-1cells in the tumor. Mice were treated with FPhgp100, rhIL-2 (36 μg per dose), and 1 × 10⁶ of pmel-1 CD62L^highLy5.1 or CD62L^lowThy1.1 cells. Data shown are the mean of 2 mice per group. This experiment was performed twice with similar results. (D) CD62L expression is required to maximize the antitumor efficacy of adoptively transferred T cells. WT mice bearing 10-day-old established s.c. B16 tumors were sublethally irradiated and left untreated as control or received adoptive transfer of 1 × 10⁶ one-week-cultured pmel-1 CD62L^+/+ cells or pmel-1 CD62L^–/– cells in conjunction with rFPhgp100 vaccination and exogenous rhIL-2 (36 μg per dose). Tumor area results are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 3 independent experiments.

Tumor antigen presentation by BM-derived APCs is required for successful tumor treatment.

Differences between CD62L^high and CD62L^low population were observed only in the presence of vaccination. Consistent with findings of others (25, 26), we found that CD62L^high T cells preferentially homed to lymph nodes. This finding was obtained using confocal laser scanning microscopy (data not shown). Taken together, these data suggested that the lymphoid homing of CD62L^high T cells could result in a more effective interaction with APCs.

In order to evaluate the role of APCs in this model, we generated a variety of chimeras by transferring BM cells from β₂ microglobulin^–/– (β₂m^–/–) and from WT mice into lethally irradiated β₂m^–/– and WT mice (Figure ​(Figure6A).6A). Chimeras were analyzed for reconstitution with the appropriate donor marrow after 8 weeks (data not shown) and subsequently challenged subcutaneously with B16 melanoma. Adoptive transfer of early effector cells, in combination with an altered ligand vaccine and exogenous IL-2, was unable to treat B16 tumors in β₂m^–/–/β₂m^–/– chimeras (Figure ​(Figure6B).6B). As previously reported in studies using other models (27), self/tumor antigen presentation by B16 melanoma was insufficient to efficiently stimulate adoptively transferred T cells, and no significant differences in tumor growth were seen compared with untreated mice (P > 0.05). However, transfer of CD8⁺ T cells into β₂m^–/– mice that had been engrafted with BM cells derived from WT mice treated B16 melanoma as efficiently as did pmel-1 cells adoptively administered to WT/WT chimeras (P > 0.05; Figure ​Figure6D).6D). Tumor antigen presentation by BM-derived APCs (presumably professional APCs) appear to be critical for successful treatment: adoptively transferred T cells in vivo activated by parenchymal cells, in β₂m^–/–/WT chimeras, were less effective in controlling tumor growth (P = 0.016; Figure ​Figure6C).6C). Thus, our findings suggest that in lymphoid tissues, adoptively transferred T cells utilize CD62L to target professional APCs expressing antigen as a result of the vaccination and that the downregulation of CD62L, due to progressive differentiation, impairs their in vivo activation, their proliferation, and consequently their antitumor activity.

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Figure 6

Antigen presentation by BM-derived APCs is critical for tumor treatment. (A) Generation of BM chimeras. We generated chimeras by transferring 5 × 10⁶ BM cells from β₂m^–/– and WT mice into lethally irradiated WT or β₂m^–/– mice. (B–D) Results from BM chimeras indicate that BM-derived APCs expressing MHC class I are required for tumor treatment. Chimeras bearing 10-day-old established s.c. B16 tumors were sublethally irradiated and left untreated as controls or received adoptive transfer of 1 × 10⁶ pmel-1 cells cultured for 1 week in conjunction with rFPhgp100 vaccination and exogenous rhIL-2 (36 μg per dose). Results for tumor area are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 2 independent experiments.

Generation of BM chimeras.

BM cells were obtained from femurs and tibias of β₂m^–/– and C57BL/6 mice (Taconic). Recipients were irradiated with 10 Gy and injected i.v. with 5 × 10⁶ BM cells. PBMC chimeras were analyzed at 8 weeks.

In vitro activation, cytokine release, and cytolytic assays.

Pmel-1 splenocytes were isolated as described previously (13) and cultured in the presence of 1 μM human gp100_25–33 (hgp100_25–33) and CM containing 30 IU/ml of recombinant human IL-2 (rhIL-2) (Chiron Corp.) or 10 ng/ml rhIL-15 (PeproTech Inc.). Cells were restimulated weekly with 1 μM hgp100_25–33–pulsed, irradiated splenocytes. Pmel-1 cells were frozen after each restimulation. Prior to adoptive transfer, pmel-1 lymphocytes were thawed and kept overnight in IL-2 culture medium at 37°C. Cells were concurrently used for flow cytometry analysis, recognition, or cytolytic assays. When naive cells were used, pmel-1 splenocytes were depleted of non-CD8⁺ T cells using a MACS negative selection column (Miltenyi Biotec). CD62L^high and CD62L^low populations were sorted the day before adoptive transfer using a MACS CD62L-positive selection column (Miltenyi Biotec). Cytokine release assays and cytolytic assays were performed as previously described (29).

Adoptive cell transfer, vaccination, and cytokine administration.

Female C57BL/6 mice (Jackson Laboratory) at 6–12 weeks of age were injected s.c. with 2 × 10⁵ to 5 × 10⁵ B16 melanoma cells. Mice (n = 5 for all groups) were treated 10–14 days later with i.v. adoptive transfer of pmel-1 CD8⁺ T enriched splenocytes or in vitro activated splenocytes, as indicated. Lymphopenia was induced by sublethal irradiation (500 cGy) of tumor-bearing mice on the day of treatment. Mice were vaccinated with 2 × 10⁷ PFU of a previously described recombinant fowlpox virus expressing human gp100 (rFPhgp100; Therion Biologics) (53) and rhIL-2 at 600,000 IU (36 μg per dose) were administered by i.p. injection twice daily for a total of 6 doses. Calipers were used to measure tumors, and the products of the perpendicular diameters were recorded. All experiments were performed in a blinded, randomized fashion (the investigator who performed the measurements had no knowledge of the experimental group) and performed independently at least twice with similar results. All animal experiments were approved by the NCI Animal Ethics Committee of the NCI.

Enumeration of adoptively transferred cells.

On the days indicated, mice were bled by tail vein into heparin-containing microcentrifuge tubes (Marsh Bio Products) and sacrificed. We harvested spleen and tumor and homogenized them into a single-cell suspension using the rubber end of a 3-cc syringe and a 40-μM filter cup. Samples were enumerated in our laboratory using trypan blue exclusion for splenocyte count, and in the NIH Clinical Laboratory for peripheral blood analysis, and were analyzed by flow cytometry for CD8 and Vβ13, Ly5.1, or Thy.1 expression by cells. We calculated the absolute number of pmel-1 cells/μl by multiplying the splenocyte count or ALC by the ratio CD8⁺ Vβ13⁺/lymphocyte gate, CD8⁺ Ly5.1⁺/lymphocyte gate, or CD8⁺ Thy1.1⁺/lymphocyte gate.

In vitro CFSE proliferation assay.

Pmel-1 cells were labeled with 1 μM CFSE (Invitrogen Corp.) and restimulated with 1 μM hgp100_25–33–pulsed, irradiated splenocytes in CM containing 30 UI/ml of rhIL-2. From day 1 to day 4 after restimulation, cells were harvested and analyzed by flow cytometry for CFSE, CD8, and Vβ13 expression.

Flow cytometry and antibodies.

All antibodies were purchased from BD Biosciences except goat polyclonal antibody against CCR7 (Abcam Inc.), donkey anti-goat–PE (Jackson ImmunoResearch Laboratories Inc.), KLRG-1–PE (2F1) (SouthernBiotech), and anti-human granzyme B–PE (GB11) (CALTAG Laboratories). Samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson).

Detection of BID and BAD protein expression by Western blot analysis.

Cells were lysed in RIPA buffer (Boston BioProducts Inc.) with 1 mM of phenylmethylsulfonyl fluoride (Sigma-Aldrich). Then 10 μg protein from each lysate was loaded to a well of a 12% PAGE gel (PAGEgel Inc.) and run in reducing conditions for an hour. After transfer, the membrane was blocked with 10% nonfat milk in wash buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl (Quality Biological Inc.), and 0.1% Tween-20 (Fisher Scientific) overnight. The membrane was incubated with 1 μg/ml rat anti-mouse BID (R&D Systems), 0.5 μg/ml mouse anti-mouse BAD (BD Biosciences), and 0.3 μg/ml mouse anti-mouse GAPDH (Chemicon International) for 1 hour and subsequently washed 3 times. We used 1 μg/ml of HRP-conjugated anti-mouse Ig (Amersham Biosciences) or anti-rat IgG (SouthernBiotech) as secondary antibodies. The membrane was developed in SuperSignal West Pico Chemiluminescent Substrate (Pierce) and photographed by LAS-1000 luminescent image analyzer (FUJIFILM Medical Systems).

Microarray analysis.

RNA was isolated from CD8⁺-enriched pmel-1 cells after 1 and 2 stimulations using RNeasy columns (QIAGEN) and indirectly labeled via a single round of linear amplification with Amino Allyl MessageAmp reagents (Ambion). The labeled samples were combined and hybridized overnight to 22,000-gene oligonucleotide arrays supplied by the Laboratory of Molecular Technology (NCI). We obtained data image files using a GenePix 4000B scanner (Axon Instruments) and imported them into GeneSpring version 6.2 (Silicon Genetics).

The authors would like to thank A. Mixon and S. Farid of the Flow Cytometry Unit (Surgery Branch of the NCI) for flow cytometry analysis and P.A. Antony, C.M. Paulos, C.S. Hinrichs, and A. Ranganathan for critical review of the manuscript.