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Arming Tumor-Reactive T Cells with Costimulator B7-1 Enhances Therapeutic Efficacy of the T Cells

¹ Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, Illinois; ² Department of Histology and Embryology, Second Military Medical University, Shanghai, China; and ³ Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania

Requests for reprints: Aoshuang Chen or Guoxing Zheng, Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, 1601 Parkview Avenue, Rockford, IL 61107. Phone: 815-395-5680; Fax: 815-395-5666; E-mail: aoshuang{at}uic.edu or guoxingz{at}uic.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cells ectopically expressing costimulators are pathogenic and contribute to autoimmunity against self-antigens. Given that tumor antigens are often self-antigen or mutated self-antigens, we hypothesize that neoexpressing a costimulator on tumor-reactive T cells may likewise enhance their reactivity to tumor. To test this hypothesis, we have expressed B7-1 on OT-1 CD8⁺ T-cell receptor transgenic T cells via protein transfer (or protein "painting"). Naïve OT-1 T cells, after being painted with B7-1, can self-costimulate themselves, elicit enhanced proliferative and CTL responses to E.G7-ovalbumin tumor cells (expressing a cognate antigen), and become resistant to CD4⁺CD25⁺ regulatory T-cell-mediated suppression. Importantly, these T cells, when coimplanted with E.G7-ovalbumin tumor cells into a syngeneic host, are three to nine times more potent than are control T cells (mock painted with human IgG) in inhibiting tumor growth. Further, on transfer into mice bearing established E.G7-ovalbumin tumors, B7-1-painted ex vivo–amplified OT-1 T cells induced complete tumor regression in 65% of treated mice, whereas the control T cells did so in only 28% of treated mice. Finally, on transfer into mice bearing less immunogenic 4T1 breast tumors, B7-1-painted tumor-reactive CD8⁺ T cells improved the survival of treated mice to a greater extent than did the control T cells. Hence, this study establishes that arming tumor-reactive T cells with a costimulator can enhance their antitumor efficacy. (Cancer Res 2006; 66(13): 6793-9)

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The idea of adoptive T-cell therapy for cancer is simple: autologous tumor-reactive T cells can be enriched from tumor-infiltrating lymphocytes or draining lymph nodes of a patient, activated and amplified ex vivo, and reinfused back to the patient to eradicate tumors (1). In practice, however, the efficacy of such therapy is often limited by the weak reactivity of the T cells to tumors (2). Moreover, on adoptive transfer, the T cells can be functionally compromised by the host immune mechanisms that are nonsupportive (i.e., the lack of antigenic, costimulatory, and/or proinflammatory signals; refs. 3, 4) and even suppressive [i.e., presence of suppressive cytokines and/or regulatory T (Treg) cells; refs. 5, 6]. Thus, additional strategies are needed to improve the efficacy of these T cells.

Costimulation is one of the key mechanisms by which T cells are regulated. Costimulation is required for T-cell activation; in the absence of costimulation, antigenic stimulation of T cells results in T-cell anergy (7). In addition, costimulation augments T-cell effector responses, including cytotoxicity, to weak antigens, such as self-antigens and tumor antigens (8–10). Further, there is evidence that strong costimulation, such as signaling via the CD28 axis (11–13), can enhance the resistance of T cells to suppression mediated by CD4⁺CD25⁺ Treg cells.

Normally, costimulators are expressed on activated antigen-presenting cells (APC), including dendritic cells. T cells that ectopically express a high level of costimulators, such as B7-1 (14, 15), CD40 (16), or LIGHT (17), are pathogenic and contribute to autoimmune diseases. Elevated autoimmune propensity, however, can be desirable for tumor-reactive T cells (18). This is because most tumor antigens are self-antigens or mutated self-antigens (19); to attack tumors effectively, tumor-reactive T cells must likewise be able to breach the barrier against autoimmunity.

Based on these notions, we hypothesize that arming tumor-reactive T cells with costimulator(s) may enhance their antitumor efficacy. By colocalizing a costimulator with its cognate receptor on same T cells, we enable the T cells to "self-costimulate" themselves in an autocrine/paracrine-like mode and bypass the costimulator-mediated mechanism(s) that limit T-cell activation and effector function, thereby eliciting enhanced antitumor responses.

To test this hypothesis, in this study, we have neoexpressed the costimulator B7-1 on tumor-reactive CD8⁺ T cells and evaluated the functional capacities of the T cells so engineered both in vitro and in vivo. The study establishes that arming tumor-reactive T cells with a costimulator enhances their antitumor efficacy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and tumor cell lines. BALB/c and C57BL/6 mice (3- to 5-week old) were purchased from The Jackson Laboratory (Bar Harbor, ME). OT-1 T-cell receptor (TCR) transgenic mice (20) in the C57BL/6 background were bred at the animal facility of the University of Illinois College of Medicine at Rockford (Rockford, IL) using hemizygous males and wild-type females. To screen for transgenic offspring, 1 µL blood was collected from the tail, immunostained with FITC-labeled anti-V2 and allophycocyanin-labeled anti-CD8 monoclonal antibodies (mAb), and analyzed by flow cytometry. Mice with nearly all of their CD8⁺ T cells stained positive for V2 were selected. The animals were maintained in a pathogen-free facility and used in accordance with the institutional guidelines for animal care. E.G7-ovalbumin, EL4, 4T1, and L5178Y tumor cell lines were purchased from the American Type Culture Collection (Manassas, VA) and maintained as per supplier's recommendation.

Antibodies. FITC-labeled rat anti-mouse V2 mAb (B20.1) was from Caltag Laboratories (Burlingame, CA). Rat anti-mouse CD3 mAb (TK3) was purchased from Serotec Ltd. (Oxford, United Kingdom). Rat anti-mouse CD16/32 mAb (clone 93), FITC-labeled rat anti-mouse B7-1 mAb (1G10), FITC-labeled rat anti-mouse B7-2 mAb (GL1), PE-labeled or nonlabeled hamster anti-mouse CD28 mAb (37.51), allophycocyanin-labeled rat anti-mouse CD8 mAb, PE-labeled rat anti-mouse CD62L mAb (MEL-14), and various isotype controls were from eBioscience (San Diego, CA). Anti-mouse CD28 Fab was generated from the corresponding mAb (37.51) using the ImmunoPure Fab preparation kit (Pierce Biotechnology, Rockford, IL). Control Fab was purchased from the Jackson ImmunoResearch Laboratories (West Grove, PA).

T-cell isolation. All T cells were purified by magnetically activated cell sorting using various isolation kits (all from Miltenyi Biotec, Auburn, CA). OT-1 T cells were purified from bulk splenocytes via negative selection using a mouse CD8⁺ T-cell kit. CD4⁺CD25⁺ T cells were isolated from lymph nodes by the use of a CD4⁺ T-cell kit (negative selection) followed by the use of a CD25⁺ cell kit (positive selection). T-cell purity of >97% was achieved as determined by flow cytometry.

Flow cytometry. Cell samples were first blocked with rat anti-mouse CD16/32 (1 µg per 10⁶ cells) and subsequently stained with mAb(s) as per manufacturers' instructions. Flow cytometry was done with a FACSCalibur (BD Biosciences, San Jose, CA). Live cells were identified and gated by forward and side scatter and, if necessary, by staining with 7-aminoactinomycin D (7-AAD; Invitrogen, Carlsbad, CA). Data were analyzed using the CellQuest Pro software (BD Biosciences).

Protein transfer. T cells were suspended to 2 x 10⁷/mL in DMEM/0.1% bovine serum albumin (BSA)/50 µmol/L ß-mercaptoethanol (transfer buffer). Palmitated protein A was generated as we described previously (21) and added to the cells at 30 µg/mL. Cells were incubated at 37°C for 15 minutes and then at 42°C for 5 minutes. Cells were washed and resuspended at 4 x 10⁷/mL in transfer buffer, and Fc₁-derivatized mouse B7-1 (B7-1-Fc; ref. 22) or human IgG (hIgG; Sigma-Aldrich Corp., St. Louis, MO) was added to cells at 30 µg/mL. Cells were incubated at 4°C for 15 minutes. Unbound proteins were removed by washing.

Carboxylfluorescein diacetate succinimidyl ester labeling. Cells were thoroughly washed with and suspended (at 2.5 x 10⁶/mL) in PBS containing Ca²⁺ and Mg²⁺. Carboxylfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) was added to cells at 1 µmol/L. The reaction was allowed to last for 5 minutes at room temperature and terminated with the addition of 0.5 volume of FCS.

In vitro proliferation assay. Tumor cells (serving as APC) were suspended at 2 x 10⁶/mL in DMEM/2% FCS and mitotically inactivated with mitomycin C (Sigma-Aldrich) added to 100 µg/mL. Cells were treated at 37°C for 1 hour and then thoroughly washed before use. CFSE-labeled OT-1 T cells were seeded in triplicate wells (5 x 10⁴ per well) in RPMI 1640/10% FCS/15 mmol/L HEPES/50 µmol/L ß-mercaptoethanol (R10 medium) in a U-bottomed 96-well plate together with mitomycin C–treated E.G7-ovalbumin or EL4 tumor cells (5 x 10⁴ per well). After 48 or 72 hours, cells were harvested and stained with allophycocyanin-labeled anti-CD8 mAb (to allow us to gate on the OT-1 T cells) and analyzed by flow cytometry.

In vivo proliferation assay. CFSE-labeled OT-1 T cells were painted with B7-1 or hIgG, mixed with E.G7-ovalbumin tumor cells at 1:1 ratio, and injected (0.5 x 10⁶ – 1 x 10⁶ total cells per injection) into the footpads of the hind legs of syngeneic C57BL/6 mice (two – three mice per group for each experiment). The cell mixture containing the B7-1-painted T cells was injected into the right footpads (test); that containing the hIgG-painted T cells, into the contralateral left footpads (control). The recipient mice were maintained in the dark for 4 days and then sacrificed. Cell suspensions were prepared from pooled popliteal lymph nodes draining the same injection site, stained with allophycocyanin-labeled anti-CD8 mAb and 7-AAD, and analyzed by flow cytometry (gated on the CD8⁺ population).

Detection of cytokines. OT-1 T cells were cocultured with tumor cells for 48 hours, and the conditioned media were analyzed by flow cytometry using a cytometric bead array kit for Th1/Th2 cytokines (BD Biosciences) as per manufacturer's protocol.

CTL assay. Effector T cells were seeded in triplicate wells of a U-bottomed 96-well plate together with CFSE-labeled, mitomycin C–inactivated tumor cells (target) at different E:T ratios. At 12 hours, the cultures were mixed with an equal volume of PBS containing 2 µg/mL 7-AAD. Cells were analyzed by flow cytometry. CFSE-labeled cells (target) were gated. Raw data were converted into specific lysis by the formula: specific lysis = (x – c)/(100 – c), where x is the percentage of 7-AAD⁺ target cells in the presence of effector cells and c is that in the absence of effector cells (spontaneous lysis).

Analysis of antitumor titer in vivo. The antitumor activity of OT-1 T cells was titered by the Winn assay (23). Briefly, naïve OT-1 T cells painted with B7-1 or hIgG were serially diluted and mixed with 1 x 10⁶ E.G7-ovalbumin tumor cells to reach various OT-1 to E.G7-ovalbumin cell ratios. Each mixture of a defined OT-1 to EG.7-ovalbumin ratio was tested in a group of C57BL/6 mice (four mice per group); mixtures were each injected i.d. into mice. As a negative control group, mice were injected with the tumor cells alone. Growth of tumors in all groups was measured weekly.

Adoptive immunotherapy. For immunotherapy in the E.G7-ovalbumin model, OT-1 T cells were prepared. Naïve OT-1 T cells were preactivated for 2 days with anti-CD3 (plate bound at 6 µg/mL) and interleukin-2 (IL-2; 100 IU/mL) and amplified for 4 days with IL-2; subsequently, the T cells were painted with hIgG or B7-1 as described earlier. For treatment, EG.7-ovalbumin tumors were established by injecting i.d. 1 x 10⁶ E.G7-ovalbumin tumor cells into the right flank of C57BL/6 mice (day –5). Before treatment, the mice were divided into two groups (five mice per group) and individually ear marked within each group. On days 0 (tumors were 25 mm² in size) and 1, each group of mice was injected intratumorally (1 x 10⁶ T cells per injection) with the hIgG-painted or B7-1-painted OT-1 T cells. Tumor sizes were measured weekly after the treatment. Cured mice were rechallenged with 1 x 10⁶ E.G7-ovalbumin tumor cells injected i.p. 2 to 3 months after the complete tumor regression.

For immunotherapy in the 4T1 tumor model, 4T1 tumor-reactive T cells were prepared. To that end, BALB/c mice were immunized twice with mitomycin C–treated 4T1 tumor cells. The immunized mice were then challenged with live 4T1 tumor cells on both sides of the flank; the mice that showed partial resistance to this challenge were used as the donors for 4T1 tumor-reactive CD8⁺ T cells. The CD8⁺ T cells isolated from tumor-draining lymph nodes in the donor mice were preactivated for 2 days with anti-CD3 (plate bound at 6 µg/mL) and IL-2 (100 IU/mL), amplified for 4 days with IL-2, and painted with hIgG or B7-1. For treatment, tumors were established in BALB/c mice by injecting i.d. 3 x 10⁴ 4T1 tumor cells on the right flank of BALB/c mice (day –4). On day –1, the mice were given an i.p. injection of cyclophosphamide (2.5 mg in 100 µL water) to slow down tumor growth (24). Before the T-cell therapy, mice were divided into three groups (five mice per group). On day 0, each group of mice was injected intratumorally (3 x 10⁶ cells per injection) with medium (DMEM/0.1% BSA) or the 4T1 tumor-reactive CD8⁺ T cells painted with either IgG or B7-1 (described above). Tumor sizes were measured weekly. Mice were euthanized when they became moribund or when their tumors exceeded 400 mm² in size.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expressing B7-1 on primary T cells via protein transfer. OT-1 TCR transgenic CD8⁺ T cells, expressing a TCR specific for the MHC I–restricted ovalbumin peptide antigen (ovalbumin_257-264), are a well-established system for studying antigen-specific CD8⁺ responses. Naïve OT-1 T cells express a high level of costimulatory receptor CD28 but not its ligand B7-1 or B7-2 (data not shown). To neoexpress costimulator B7-1 on OT-1 T cells, we used the protein transfer (or protein painting) method we developed previously (21). In that method, protein A, after being chemically derivatized with palmitate in a simple reaction, is first incorporated into cell membranes; in turn, this membrane-anchored palmitated protein A serves as a "trap" for secondarily added Fc fusion proteins. With its simplicity in expressing defined amounts of proteins and in coexpressing more than one protein at a time (21, 22, 25), the method provides an alternative means to gene transfer for expressing proteins on cells, especially on those primary cells that are resistant to gene transfer. Here, as shown in Fig. 1A , naïve primary OT-1 T cells, after being painted with palmitated protein A and murine B7-1 Fc fusion protein (B7-1-Fc), were double positive for both CD28 and B7-1.

View larger version (12K): [in this window] [in a new window]   Figure 1. Self-costimulation by B7-1-painted OT-1 T cells. A, nonpainted or B7-1-painted OT-1 T cells were double stained for B7-1 and CD28 and analyzed by flow cytometry. B, CFSE-labeled OT-1 T cells, painted with hIgG or B7-1, were mixed (1:1) with E.G7-ovalbumin (OVA) or control EL4 tumor cells. Cells (1 x 105 per well) were incubated for 48 hours in the presence or absence of 10 µg blocking anti-CD28 or control Fab, stained for CD8, and analyzed by flow cytometry. Histograms, CFSE from CD8-gated (OT-1) population. Peaks, subpopulations of OT-1 T cells, which shift leftward as they divide. C, B7-1 painting augments the proliferation of OT-1 T cells in response to tumor cells in vivo. Mixtures of E.G7-ovalbumin tumor cells and CFSE-labeled OT-1 T cells that had been painted with either hIgG (control mixture) or B7-1 (test mixture) were injected into C57BL/6 mice. Control mixtures were injected into left footpads; test mixtures, into the contralateral right footpads. Four days later, total cell contents from draining lymph nodes (popliteal) on each side were analyzed by flow cytometry. CD8+ T cells were identified by immunostaining and gated. Bottom right quadrant, live OT-1 donor T cells, which are CFSE+7-AAD–, are located. Vertical bands, subpopulations of OT-1 T cells, which shift leftward as they divide. Numbers, numbers of cell divisions. Data were obtained from one of two to four independent experiments.

To verify our finding, we determined the effect of B7-1 in vivo. CFSE-labeled OT-1 T cells were mixed with E.G7-ovalbumin tumor cells and adoptively transferred into syngeneic C57BL/6 mice via footpad injection. Subsequently, the proliferation of the donor OT-1 T cells in the draining lymph nodes (popliteal) was analyzed by flow cytometry. The majority of the B7-1-painted OT-1 T cells divided two to three times following adoptive transfer, whereas the majority of hIgG-painted control T cells divided only once or stayed undivided (Fig. 1C).

In summary, these data indicate that the expression of B7-1 on OT-1 T cells can augment the proliferation of the T cells in response to tumor cells both in vitro and in vivo.

B7-1 painting enhances antitumor effector function of OT-1 T cells in vitro. Having documented the effect of B7-1 painting on the proliferation of OT-1 T cells in response to tumor cells, we wanted to substantiate our finding and determine whether B7-1 expression also has an effect on effector responses of the T cells, particularly Th1 cytokines and CTL responses, against the tumor cells. After stimulation by EG.7-ovalbumin tumor cells, B7-1-painted OT-1 T cells produced higher levels of Th1 cytokines, especially IL-2 and IFN-, than did hIgG-painted control T cells (Fig. 2A ). Further, B7-1-painted OT-1 T cells, on a per cell basis, displayed stronger CTL activity against EG.7-ovalbumin tumor cells than did hIgG-painted T cells (Fig. 2B and C). Together, these results show that the expression of B7-1 on OT-1 T cells can enhance antitumor effector responses of the T cells in vitro.

View larger version (10K): [in this window] [in a new window]   Figure 2. B7-1-painted OT-1 T cells produce enhanced antitumor effector responses. OT-1 T cells painted with hIgG or B7-1 were cocultured with E.G7-ovalbumin tumor cells for 48 hours; conditioned media and activated OT-1 T cells were collected. A, cytokine content in the conditioned medium was quantified by a flow cytometry-based cytometric bead array assay. Results were from one of two experiments of similar results. B and C, CTL responses of OT-1 to tumor cells were determined. CFSE-labeled and mitomycin C–treated E.G7-ovalbumin tumor cells (as target, 5 x 104 per well) were incubated for 12 hours with the preactivated OT-1 T cells at indicated E:T ratios. Cell mixtures were then stained with 7-AAD and analyzed by flow cytometry. 7-AAD staining of CFSE-gated target cells is shown. B, dose-dependent killing of target cells depicted by the conversion from being 7-AAD negative to positive. C, as a negative control, target cells incubated alone were 7-AAD negative. Percentage killing of target cells is indicated. Similar results were obtained in two independent experiments.

View larger version (12K): [in this window] [in a new window]   Figure 3. B7-1-painted OT-1 T cells are resistant to suppression mediated by CD4+CD25+ Treg cells. CFSE-labeled OT-1 T cells were painted with hIgG or B7-1 and mixed (in 1:1 ratio, 1 x 105 per well) with either E.G7-ovalbumin tumor cells (solid line) or control EL4 tumor cells (dashed line). Treg cells isolated from lymph nodes draining progressively growing E.G7-ovalbumin tumors were added to the wells at indicated Treg to OT-1 ratios. After 48 hours, OT-1 T-cell proliferations were analyzed by flow cytometry as described in Fig. 1. Similar results were obtained in two independent experiments.

View larger version (13K): [in this window] [in a new window]   Figure 4. B7-1 painting increases antitumor titer of OT-1 T cells in vivo. E.G7-ovalbumin tumor cells (as target, 1 x 106) were injected i.d. into C57BL/6 mice (four mice per group) either alone () or together with hIgG-painted or B7-1-painted naïve OT-1 T cells at indicated OT-1 to target ratio (). Growth of tumors was measured weekly. Similar results were obtained in three independent experiments.

View larger version (12K): [in this window] [in a new window]   Figure 5. B7-1 painting increases therapeutic efficacy of ex vivo–amplified OT-1 T cells against established E.G7-ovalbumin tumors. A, B7-1 painting of ex vivo–amplified OT-1 T cells. OT-1 T cells were cultured for 6 days with IL-2, painted with hIgG (dashed line) or B7-1 (solid line), and analyzed by immunostaining and flow cytometry. B, E.G7-ovalbumin tumors were established in C57BL/6 mice (five mice per group) by i.d. inoculation of 1 x 106 tumor cells (day –5). On days 0 and 1, hIgG-painted or B7-1-painted OT-1 T cells were injected intratumorally into the mice (1 x 106 per dose). Tumor sizes were measured weekly after the treatment and graphed for individual, ear-marked animals. Lines, tumor growth curve of a single animal. C, 2 to 3 months after complete tumor regression, a lethal dose (1 x 106) of E.G7-ovalbumin tumor cells was inoculated i.p. into mice (four or five mice per group) cured by B7-1-painted OT-1 T cells (solid line) or naïve mice (dotted line). Mice were monitored for appearance of tumor weekly. Results are representative of three independent experiments.

In summary, these data show that B7-1 painting improves the therapeutic antitumor efficacy of ex vivo–amplified/activated OT-1 T cells against established tumors.

B7-1 painting increases antitumor efficacy of 4T1 tumor-reactive CD8⁺ T cells. Thus far, we have shown in the previous figures the effects of B7-1 painting on antitumor efficacy of OT-1 TCR transgenic CD8⁺ T cells, a well-defined immunologic system for studying antigen-specific responses. Next, we wanted to further substantiate our findings in T cells that are of high heterogeneity and with relatively poor reactivity to tumors, a scenario resembling more closely to clinical settings. To that end, we determined the effects of B7-1 painting on CD8⁺ T cells harvested from mice bearing the murine mammary carcinoma 4T1, a model for human stage IV breast cancer (27). Compared with the E.G7-ovalbumin tumors, the 4T1 tumors are less immunogenic and more metastatic.

First, to generate 4T1 tumor-reactive CD8⁺ T cells, syngeneic BALB/c mice were immunized twice with mitotically inactivated 4T1 tumor cells and subsequently inoculated with live 4T1 tumor cells on both sides of the flank. Although the prior immunization failed to prevent tumor occurrence completely, retarded tumor growth was noted in about half of the immunized animals (data not shown). Hence, the tumor-draining lymph nodes from the mice that showed retarded tumor growth were analyzed by flow cytometry. The total count of CD8⁺ T cells was 22% (Fig. 6A ), which was similar to that of corresponding lymph nodes from naïve mice (data not shown). However, 18% (4% divided by 22%) of these CD8⁺ T cells were CD62L^low (suggestive of their previous encounter with antigens; Fig. 6A) compared with 11% of CD8⁺ T cells in corresponding lymph nodes from naïve mice (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 6. B7-1 painting enhances antitumor efficacy of 4T1 tumor-reactive CD8+ T cells. A, total lymph node cells were isolated from the lymph nodes draining 4T1 tumors, immunostained for CD8 and CD62L, and analyzed by flow cytometry. B, CD8+ T cells isolated from the draining lymph nodes were ex vivo amplified for 6 days with IL-2. Subsequently, the T cells (effector) were incubated with 4T1 (specific target; ) or L5178Y (nonspecific target; ) tumor cells at indicated E:T ratios, and CTL activities were determined as described in Fig. 2. C, ex vivo–amplified T cells were painted with hIgG (dashed line) or B7-1 (solid line), immunostained, and analyzed by flow cytometry. D, 4T1 tumors were established in BALB/c mice (five mice per group) by i.d. inoculation of 3 x 104 tumor cells (day –4). On day 0, each group of mice was injected intratumorally with medium (solid line) or ex vivo–amplified T cells (3 x 106 per injection) painted with either hIgG (dotted line) or B7-1 (broken line). The difference between the group treated with B7-1-painted T cells and that treated with hIgG-painted T cells is statistically significant (P < 0.05). Similar results were obtained in three independent experiments.

Next, we determined whether B7-1 painting can increase the antitumor efficacy of these T cells on intratumoral transfer into mice bearing 4-day established 4T1 tumors. The treatment did not cure any of the animals. However, as depicted in Fig. 6D, a single injection of 3 x 10⁶ B7-1-painted T cells significantly prolonged the survival of the treated mice, whereas the injection of the same number of hIgG-painted T cells was less effective. These results show that B7-1 painting improves the therapeutic effect of natural, heterogeneous T cells originated from the tumor-draining lymph nodes, thus further solidify our findings made in the OT-1 T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we used the protein transfer method to paint APCs and tumor cells with costimulators in an effort to improve the immunogenicity of these cells (21, 22). In the present study, we apply the same method to tumor-reactive T cells to enhance their antitumor efficacy. Our data show that neoexpression of B7-1 via protein transfer on naïve OT-1 TCR transgenic CD8⁺ T cells enables the T cells to self-costimulate themselves. Such self-costimulating T cells produce enhanced proliferative and CTL responses to EG.7-ovalbumin tumor cells expressing a cognate antigen and become resistant to Treg cell-mediated suppression. Significantly, when cotransplanted with the EG.7-ovalbumin tumor cells into syngeneic animals, these B7-1-painted OT-1 cells show three to nine times higher antitumor titer than do hIgG-painted control T cells. Moreover, in both E.G7-ovalbumin (highly immunogenic) and 4T1 (poorly immunogenic) tumor models, B7-1 painting of ex vivo–amplified tumor-reactive CD8⁺ T cells improves their therapeutic efficacy against established tumors. Together, these data support our hypothesis that antitumor T cells can be functionally enhanced by arming them with a costimulator.

Until now, adoptive T-cell immunotherapies have largely relied on ex vivo amplification to increase the number of therapeutic T cells. Our present study suggests that arming tumor-reactive T cells with a costimulator may further enhance the in vivo antitumor efficacy of these T cells on reinfusion into the patient. Thus, engineering self-costimulating T cells may provide an alternative means to improve the efficacy of adoptive immunotherapies.

With its simplicity in efficiently anchoring premanufactured recombinant costimulators onto cell surfaces, protein transfer is particularly suited for engineering tumor-reactive T cells and other immune cells. Moreover, from the safety standpoint, protein transfer has an advantage over gene transfer. It permits temporary modification of immune cells because the painted proteins will "wear off" over time, likely due to metabolism and/or shedding. Thus, protein transfer is unlikely to cause long-term side effect, such as the induction of autoimmune disease. Furthermore, after the initial proof of principle of protein transfer obtained via the glycosylphosphatidylinositol modification (28–30), several protein transfer methods, including the method used in this study, have been developed (reviewed in refs. 31, 32). These protein transfer methods, combined with the expanding repertoire of costimulators, now provide an enlarging set of options for engineering T cells and other immune cells for treating cancer and autoimmune diseases.

Mechanistically, the enhanced antitumor efficacy of self-costimulating T cells can be attributed to the involvement of costimulation in the priming and/or the effector stages of T-cell function. For the B7-1-painted naïve OT-1 T cells, the enhanced antitumor titer may result, at least partially, from direct priming of the T cells by the tumor cells, which may otherwise be suppressed (by the Treg cells and/or other factors) or be ineffective in the absence of B7-1. This interpretation is consistent with both the data in this study (Figs. 2 and 3) and a recent finding that priming of naïve antitumor T cells can occur within a tumor with enforced costimulation and lead to tumor regression (33). For the ex vivo–amplified T cells, however, their enhanced antitumor efficacy may derive from the costimulation of effectors (8–10); in the present study, the need for costimulation from the tumor target or tumor-infiltrating APCs is bypassed by directly painting the effector T cells with B7-1.

Liu and Janeway (34) showed previously that antigen and B7 need to be expressed on the same accessory cells to achieve maximal costimulation. It is unclear whether self-costimulation by B7-1-painted T cells can meet such spatial requirement. A spatial separation between antigenic and costimulatory signals is inevitable if the B7-1 can only function in a paracrine mode, through T-cell–T-cell interaction. On the other hand, if the B7-1 can also function in an autocrine mode, it may satisfy the spatial requirement by aligning closely with antigen, in a manner similar to what achieved by colocalizing B7 and antigen on the same accessory cells. Only future experiments will resolve this issue.

Our finding that B7-1-painted T cells are resistant to Treg-mediated suppression apparently contradicts the previous finding by Paust et al. (35) that B7 expression on target T cells facilitates their suppression by Treg cells. This discrepancy may be due to the difference in the level of B7 on T cells, which can either promote suppression or abolish it. The endogenous expression of B7 by activated T cells is rather limited (35), and a low-level B7 may favor suppression by preferentially engaging CTLA-4, a high-affinity but low-abundance receptor. In comparison, much more B7-1 can be exogenously incorporated onto T cells via protein transfer, which may promote the triggering of CD28, a lower affinity but more abundant receptor. Other studies have shown that, when B7 is abundant (11) or CD28 is selectively triggered with an antibody (12, 13), suppression is overcome. Taken together, these prior findings may suggest that the B7-CD28 interaction must be enforced to overcome suppression, which is what we have accomplished here by painting T cells with B7-1.

Finally, as the present study was intended for establishing proof of principle, there is a need for optimization. For example, the efficacy of self-costimulating T cells may be further enhanced by copainting the T cells with multiple costimulators, including B7-1, 4-1BBL, OX40L, GITRL, etc. Moreover, these T cells may be painted with other immunostimulatory molecules, such as dendritic cell activators and cell-cell adhesion molecules, to promote their interaction with host dendritic cells and to redirect their in vivo distribution. Thus, it is worthwhile to determine in the future whether various molecules copainted onto the same T cells can indeed synergize in enhancing the antitumor efficacy of the T cells.

	Acknowledgments

 
Grant support: Illinois Department of Public Health grant (G. Zheng) and NIH grant CA92243 (A. Chen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank John Javaherian for providing care for the animals as well as breeding the transgenic mice.

Received 2/ 3/06. Revised 4/15/06. Accepted 4/25/06.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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