CD4⁺ T Cells Elicit Host Immune Responses to MHC Class II⁻ Ovarian Cancer through CCL5 Secretion and CD40-Mediated Licensing of Dendritic Cells

T cells are the only leukocyte subset in the ovarian cancer microenvironment known to exert spontaneous immune pressure against tumor progression, such that the infiltration of tumor islets by CD3⁺ T cells was definitively associated with a better outcome in a large cohort of patients (1). Interestingly, subsequent studies confirming the protective role of T cells in ovarian cancer suggest that CD4⁺ T cells actually have an unfavorable effect on prognosis, restricting the beneficial mediators of antitumor immune pressure to CD8⁺ T cells (2).

CD8⁺ lymphocytes have the ability to recognize and directly kill tumor cells and can be readily isolated from certain tumors, activated, and expanded ex vivo for reinfusion into cancer patients to achieve varying degrees of responses (3–7). Thus, CD8⁺ T cells have been the focus in generating potent adoptive immunotherapies (8). Yet, studies dating back more than two decades indicate that in the absence of CD8⁺ T cells, CD4⁺ T cells directly act to eradicate both solid and hematologic malignancies (9, 10). Since then, several reports have confirmed the contributory role of CD4⁺ T cells in antitumor immunity, culminating with a report in 2008 by Hunder et al. (11) of a single case of complete response attained by a patient receiving adoptive immunotherapy with CD4⁺ T cell clones specific to the NY-ESO-1 Ag, demonstrating the compelling prospects of CD4⁺ T cells in adoptive immunotherapy against other tumors. Lastly, successful adoptive T cell administration protocols that have induced impressive clinical responses against other tumors have used a mixture of CD4⁺ and CD8⁺ T cells (4).

The beneficial or detrimental effect of including expanded CD4⁺ T cells in adoptive immunotherapies specifically against ovarian cancer remains unclear. To our knowledge, evidence is limited to a recent clinical report describing a therapeutic endogenous boost in antitumor immunity in two out of four patients with ovarian cancer treated with i.p.-infused peripheral blood Th1 cells expanded against the tumor Ag MUC1 (12). Because the expression of MHC class II (MHC-II) on these tumors is unknown, the mechanisms mediating this potentially Th1-dependent therapeutic effect need to be clarified. Confirming the therapeutic potential of CD4⁺ T cells and defining their mechanisms of antitumor activity are necessary for designing expansion protocols that maximize the phenotypic attributes required for their efficacy.

Conventional thought has limited the effects of the CD4⁺ T cell population in antitumor immunity to a role of merely providing additional and beneficial yet nonessential stimuli for supporting the maintenance of the antitumor CD8⁺ T cell population (13, 14). CD4⁺ Th cells may achieve this goal through the production of cytokines like IL-2 and IFN-γ that both regulate the responses of APCs and function in the differentiation, expansion, and maintenance of cytolytic CD8⁺ T cells (15, 16). CD4⁺ T cells also contribute to the maturation of APCs through interactions between CD40 on the APC and its cognate ligand CD40L/CD154 on the T cell, resulting in the licensing of the APC. The hallmarks for this licensing process are the upregulation of costimulatory molecules, subsequent secretion of IL-12, and corresponding ability of the licensed APC to prime naive T cells against Ag (17–19) .

We recently demonstrated that the adoptive transfer of briefly primed, tumor Ag-reactive, polyclonal T cells, coupled with the depletion of tumor-associated immunosuppressive CD11c⁺ dendritic cells (DCs), depending on the tumor model, induced the rejection or significantly delayed the progression of established ovarian cancer (20). CCL5 was required for these therapeutic benefits, although its source remained undetermined, as long-term protection was directly mediated by host (endogenous) immune cells. The studies presented in this paper aimed to elucidate the suitability of including tumor Ag-reactive CD4⁺ T cells in adoptive transfer protocols against ovarian cancer. Based on our results, we have defined certain phenotypic attributes required for maximizing their observed therapeutic effectiveness. We demonstrate that CCL5, which recruits APCs to the tumor site, is primarily produced by adoptively transferred CD4⁺ T cells. CD4⁺ T cells are also necessary for activating these recruited APCs at tumor locations through CD40–CD40L interactions and thereby stimulating therapeutically relevant host-derived antitumor CD8⁺ T cells. Therefore, CD4⁺ T cells may be critical for the efficacy of future adoptive (T) cell therapies (ACTs) against ovarian cancer. Our results provide a mechanistic rationale for including CCL5-secreting, CD40L-expressing CD4⁺ T cells as a beneficial and pertinent part of the antitumor armament of adoptive immunotherapy, particularly against lethal epithelial ovarian cancer.

C57BL/6 (B6), CD45.1 (B6-Ly5.2), and FVB mice were purchased from the National Cancer Institute (Frederick, MD).

CCL5-deficient (#005090), CD40L-deficient (#002770), and CD40-deficient (#002928) mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Experiments were conducted in accordance with the Dartmouth Medical School (Lebanon, NH) guidelines. Lewis lung carcinoma, MT2, ID8-Defb29/Vegf-a cultured T cells and DCs were maintained in RPMI 1640 medium containing 10% FBS.

To develop ovarian tumors, mice were injected i.p. with 1.5 × 10⁶ ID8-Defb29/Vegf-a cells. The ID8 ovarian cancer model was originally derived from the spontaneous transformation of C57BL6 mouse surface epithelial cells after rounds of in vitro passaging (21). The ID8-Defb29/Vegf-a ovarian cancer model was formed from the stable transfection of the original ID8 cell line with Defb29 and VEGF-a, resulting in accelerated tumor progression and ascite formation compared with the parental cell line, which recapitulates the microenvironment of human solid ovarian carcinomas in a more aggressive and faithful manner (22–26). Lewis lung carcinoma cells (American Type Culture Collection, Manassas, VA) and MT2 ovarian cancer cells (a kind gift from S. Orsulic, Cedars-Sinai Medical Center, Los Angeles, CA) (27, 28) were i.p. injected (1.0 × 10⁶ in 0.2 ml PBS). Treatments were always administered to mice bearing established tumors (at least 7 d). For protection experiments, mice were s.c. challenged with 15 × 10⁶ ID8-Defb29/Vegf-a in Matrigel (BD Biosciences, San Jose, CA).

Anti-mouse Abs specific for CD45.1 (A20), CD45.2 (104), CD3 (145.2C11), CD4 (GK1.5), CD8 (Ly-2), CD11c (N418), MHC-II (M5/114.15.2), Gr-1 (RB6-8C5), CD14 (rmC5-3), CD80 (B7-1), CD86 (GL1) CD40 (HM40-3), CD40L (MR-1), CD70 (FR70), and CCR5 (7A4) were all obtained from eBioscience (San Diego, CA), BD Biosciences, or Biolegend (San Diego, CA).

Sorted CD4⁺ and CD8⁺, unsorted CD3⁺ T cells, and CD40L^−/− T cells that had been activated with tumor-pulsed DCs were incubated at a concentration of 1.0 × 10⁶ cells/ml in complete media overnight. A total of 100 μl supernatant obtained from these cultures were then plated on a 96-well microplate coated with the CCL5 capture Ab from the mouse CCL5 Duoset ELISA development kit (R&D Systems, Minneapolis, MN). The ELISA was conducted as per the manufacturer’s instructions. IL-12 (p70) ELISA was performed with 100 μl supernatant obtained from wild-type (wt) or CD40^−/− DCs cultured with wt or CD40L^−/− T cells for 24 h. IL-12 (p70) ELISA was performed according to manufacturer’s directions (eBioscience).

Bone marrow-derived DCs were produced (20), then pulsed overnight (10:1) with irradiated (10,000 rad) and UV-light exposed ID8-Defb29/Vegf-a. Approximately 2.5 × 10⁸ splenocytes were harvested from CD45.1⁺ mice and cultured at a concentration of 2 × 10⁶ cells/ml for 7 d with ID8-Defb29/Vegf-a–pulsed DCs and 10 U/ml recombinant human IL-2 (PeproTech, Rocky Hill, NJ). Six- to 8-wk-old mice were injected i.p. with 1.5 × 10⁶ ID8-Defb29/Vegf-a cells and treated on days 7 and 14 with i.p. adoptive transfer of 1.5 × 10⁶ in vitro-activated splenic T cells. Lymphopenia was induced by sublethal TBI (300 rad) of tumor-bearing mice on day 7, 5 h prior to ACT. DCs were eliminated by i.p. administration of 10 μg/mouse anti-CD11c immunotoxin (IT) (25) on the day prior to T cell transfer (day 6) and three times weekly for 2 wk thereafter.

CD45.1⁺ mice bearing ID8-Defb29/Vegf-a tumors were treated with wt, CD40L^−/−, or CCL5^−/− T cells along with three weekly injections of an anti-CD11c cell-depleting IT. On day 28 of tumor progression, when transferred T cells are no longer detectable (20), host CD3⁺ T cells were obtained from the spleens of treated mice by FACS. CD45.2⁺ mice that would become the recipients of the sorted host (CD45.1⁺) T cells were irradiated (300 γ) at least 5 h pretransfer. A total of 3 × 10⁶ host T cells from the mentioned groups were transferred i.v. into irradiated recipient mice. Two days posttransfer, mice were challenged with s.c. flank injections of 15 × 10⁶ ID8-Defb29/Vegf-a tumor cells. Cell sorting was performed with an FACSAria (BD Biosciences). CD3 expression was confirmed by flow cytometry on an FACSCanto (BD Biosciences). Tumor growth was measured over time and reported in units of volume.

CD40L^−/− or wt T cells were expanded with DCs pulsed with irradiated tumor cells and IL-2 as described above (T cell expansion, adoptive immunotherapy, and DC depletion), for 7 d. CD45.2⁺CD11c⁺MHC-II⁺ cells were obtained from the peritoneum of 7-d-old ID8-Defb29/Vegf-a tumor-bearing CD40^−/− and wt mice by cell sorting, then cocultured with expanded T cells at a ratio of 1 DC:2 T cells. At least 10⁵ DCs were cultured per condition. The expression of the maturation markers CD70, CD80, CD86, and MHC-II was then determined by flow cytometry at the indicated time points.

T cells from wt or CD40L-deficient T cells were activated for 7 d against tumor Ag as described above (T cell expansion, adoptive immunotherapy, and DC depletion). wt or CD40-deficient DCs sorted from tumor-bearing mice were cocultured for 24 h with activated wt or CD40L-deficient T cells, then admixed with tumor-infiltrating CD8⁺ T cells for 48 h in ELISPOT analyses. Flat-bottomed, 96-well nitrocellulose-lined plates (Millipore, Bedford, MA) were coated with IFN-γ mAb (AN-18; eBioscience) or granzyme B mAb (R&D Systems) and incubated overnight at 4°C. After washing with washing buffer (0.05% Tween 20 in PBS), plates were blocked with RPMI 1640 supplemented with 10% FBS for 2 h at room temperature or blocking buffer (R&D Systems). Bone marrow-derived DCs preciously primed for 24 h with T cells as indicated were cultured with sorted 10⁵ CD8⁺ T cells from tumor-bearing mice at a 10 T cell:DC ratio for 48 h in 10% FBS RPMI 1640 medium. Postincubation, the plates were washed with washing buffer, and after addition of biotinylated secondary IFN-γ mAb (R4-6A2; eBioscience) or granzyme B mAb (R&D Systems), were left to incubate for 24 h at 4°C. Plates were washed and developed with avidin-HRP (eBioscience) or streptavidin-AP (R&D Systems) for 2 h at room temperature. After washing, fresh substrate (3-amino-9-ethyl carbazole, Sigma-Aldrich, St. Louis, MO) or BCIP/NBT (R&D Systems) was added and the plates incubated for ∼45 min.

At indicated time points, peritoneal lavages were performed on mice. Upon RBC lysis, cells were enumerated by trypan blue exclusion, then analyzed by flow cytometry for expression of CD3, CD4, CD11c, MHC-II, CD80, and congenic markers, CD45.2 (host) and CD45.1 (transferred). Absolute cell numbers were determined by multiplying the percentage of the relevant population (as determined by flow cytometry) by the total cell count per organ.

Tumor survival curves were compared using the log rank test (Prism, GraphPad, San Diego, CA). Statistical differences between numbers of spots in ELISPOT assays, proportions of cells in flow cytometry, total number of cells, or tumor volumes were determined by Mann-Whitney U test (Prism, GraphPad). All p values <0.05 were considered statistically significant.

We previously demonstrated that a mixed population of naive T cells shortly primed against tumor Ags was able to induce antitumor effects, leading to delayed progression of tumors in preclinical models of established ovarian cancers. The transfer of these tumor-specific T cells was found to evoke host (endogenous) antitumor responses, which were paramount for the observed therapeutic effects and involved the upregulation of CCL5 (20). To demonstrate that CCL5 secreted by transferred lymphocytes (and not by the host [endogenous] immune cells activated by treatment) is required for inducing therapeutic immunity against our aggressive i.p. ID8-Defb29/Vegf-a ovarian cancer model (20, 22, 24, 25), we first administered equal numbers of CCL5-deficient or wt tumor-primed total T cells into mice growing established tumors. Restricting the requirement of CCL5 to the transferred T cell compartment, tumor-primed CCL5^−/− lymphocytes administered with or without depleting immunosuppressive DCs with an anti-CD11c IT (20) induced no significant survival increase compared with tumor DC depletion alone (Fig. 1A, 1B). In contrast, equal numbers of identically primed wt T cells with similar CD4:CD8 ratios of 2:3 significantly increased the survival of tumor-bearing mice, and their therapeutic benefits synergized with the concurrent elimination of tumor-associated immunosuppressive DCs, supporting our previous report (20) (Fig. 1A, 1B). The therapeutic effects elicited by the administration of wt tumor-reactive T cells were associated with a stronger accumulation of activated (CD80⁺) DCs expressing CCR5 (Supplemental Fig. 1A) at tumor (peritoneal) locations compared with the transfer of CCL5^−/− T cells (Fig. 1C). In contrast, the absence of CCL5 within the host population had no effect on the therapeutic benefits elicited by the adoptive transfer of wt tumor-reactive T cells in conjunction with the elimination of CD11c⁺ regulatory DCs (Fig. 1D). Thus, ID8-Defb29/Vegf-a tumors progressed similarly in untreated CCL5^−/− mice as in wt mice, whereas treatments induced comparable survival increases (Fig. 1D). Taken together, these data indicate that CCL5 is required within the transferred T cell compartment but not within the host compartment to obtain the therapeutic benefits of ACT in established preclinical ovarian cancer.

We and others have demonstrated that the long-term antitumor immunity elicited by transferred (exogenous) tumor-reactive T cells is ultimately elicited in ovarian cancer by T cells of host (endogenous) origin (12, 20). We therefore investigated whether CCL5 specifically produced by adoptively transferred T cells was required for the mobilization of endogenous memory T cells that provide sustained protection. For that purpose, we depleted immunosuppressive DCs from mice bearing established i.p. tumors and treated them with congenically labeled (CD45.1⁺) T cells, briefly primed against ID8-Defb29/Vegf-A-tumor, from either wt or CCL5-deficient mice. After 28 d of tumor progression, splenic T cells of host origin (CD45.2⁺) were obtained from both groups and from mice that did not receive T cell transfer but were treated with PBS only and transferred (3 × 10⁶/mouse) into irradiated naive mice, which were challenged 48 h later with flank ID8-Defb29/Vegf-A tumors (Experimental design; Supplemental Fig. 1B). As shown in Fig. 2 and Supplemental Fig. 1C, host-derived T cells transferred from tumor-bearing mice previously treated with (exogenous) wt tumor-reactive T cells elicited significant protection against ID8-Defb29/Vegf-A tumors compared with endogenous T cells transferred from mice treated with either (exogenous) CCL5-deficient T cells or PBS (p < 0.01 for both comparisons, Mann-Whitney U test). Taken together, these results suggest that CCL5 specifically secreted by adoptively transferred T cells mediates the recruitment of activated (CCR5⁺) DCs to the tumor microenvironment (20) and demonstrate that CCL5 is essential for inducing persistent changes in host (endogenous) T cells that mediate sustained antitumor protection.

To dissect the mechanisms whereby individual T cell subsets contribute to the observed therapeutic effect, CD45.1⁺ T cells were primed as previously described (20), then CD4⁺ and CD8⁺ T cells were separated by FACS sorting and transferred individually into established ovarian cancer-bearing mice depleted of tumor-associated immunosuppressive DCs through the i.p. administration of an anti-CD11c IT (20). As expected, CD8⁺ T cells were independently able to significantly enhance the survival of mice bearing aggressive orthotopic ID8-Defb29/Vegf-a tumors by 30.1% (Fig. 3A). Surprisingly, identical numbers of CD4⁺ T cells alone induced comparable therapeutic benefits in multiple experiments (25.3% increase in survival; Fig. 3A), although the transfer of a combination of CD4⁺ and CD8⁺ T cells (CD3) had the greatest impact on survival (53% increase in survival; Fig. 3A). Corresponding effects were observed in the absence of regulatory DC depletion, however, supporting our previous findings (20, 25) that the therapeutic benefit elicited by adoptively transferred tumor-primed T cells alone was significantly weaker (Supplemental Fig. 1D).

As ID8-Defb29/Vegf-a tumor cells do not express MHC-II in culture or in vivo (Supplemental Fig. 1E), they are not directly cleared by CD4⁺ T cells. Interestingly, we found that the individual transfer of CD4⁺ T cells resulted in 32% more CCR5⁺CD11c⁺ DCs at the tumor (peritoneal) site compared with the individual transfer of CD8⁺ T cells (Supplemental Fig. 1F). This was not due to in situ differentiation of immature progenitors into DCs because >95% of tumor-associated leukocytes in both groups were either: 1) bona fide CD3⁺ T cells; 2) already differentiated CD11c⁺ DCs; or 3) committed F4/80⁺ macrophages at the time of adoptive transfer, supporting the concept that these were newly recruited DCs. Correspondingly, adoptively transferred CD4⁺ T cells were found to produce four times more CCL5 than the same number of CD8⁺ T cells (Fig. 3B), suggesting that transferred (exogenous) CD4⁺ T cells represent the main source of this chemokine. In addition, there was a significantly higher proportion of CD80⁺MHC-II⁺CD11c⁺ DCs in mice receiving adoptively transferred CD4⁺ versus CD8⁺ T cells (Fig. 3C).

To confirm that the effects specifically triggered by CD4 T cells were mediated at least in part through their superior production of CCL5, negatively immunopurified CCL5-deficient and wt CD4⁺ T cell splenocytes were simultaneously activated against tumor Ag and then transferred into wt mice growing established ID8-Defb29/Vegf-a ovarian tumors. Supporting the importance of CCL5 for the therapeutic effectiveness of adoptively transferred lymphocytes, CD4⁺ T cells that were deficient in CCL5 were impaired in their ability to delay tumor progression in comparison with wt CD4⁺T cells (Fig. 3D). Correspondingly, fewer total DCs and thus fewer mature (MHC-II⁺CD80⁺) DCs were present at the tumor site upon adoptive transfer of CCL5^−/− CD4⁺ T cells compared with wt CD4⁺ T cells (Fig. 3E). These data suggest that CD4⁺ T cells not only induce the recruitment of greater numbers of host-derived DCs, but also play a role in their activation at tumor locations and that CCL5 within the CD4 compartment is necessary for these effects.

Although the ID8-Defb29/Vegf-a model recapitulates the massive recruitment of CD11c⁺DEC205⁺CD8a⁺ leukocytes found in most solid human ovarian carcinoma specimens better than any other orthotopic model (22, 23, 25), we next aimed to rule out the unlikely event that the transduction of this aggressive cell line could obscure any homeostatic host events. Supporting the general applicability of our findings, mice with advanced Lewis lung peritoneal carcinomatosis treated with T cells expanded to tumor Ag survived significantly longer than untreated mice (Fig. 4A). The transferred T cells secreted levels of CCL5 comparable to T cells activated against ID8-Defb29/Vegf-a tumors (Fig. 4B), which was required for their therapeutic effect, as transferred CD4⁺ T cells deficient in CCL5 were unable to induce the same delay in tumor progression as wt CD4⁺ T cells (Fig. 4C), suggesting a similar mechanism possibly playing a role. Furthermore, FVB mice challenged with the MT2 strain of ovarian carcinoma also presented increased numbers of DCs expressing higher levels of CD80 and CD70 in mice treated with tumor-activated T cells (that also secrete CCL5; Fig. 4B) than their untreated counterparts (Fig. 4D, 4E). Therefore, the induction of host immunity to peritoneal carcinomatosis through ACT may be independent of genetic and haplotype differences and relevant to the natural diversity observed within patients with peritoneal tumors of different types.

As the expanded (CD4⁺) T cells express CD40L (Fig. 5A), we hypothesized that they could activate newly recruited host DCs through CD40 signaling. Supporting this proposition, transferred T cells that were deficient in CD40L recruited similar numbers of leukocytes to the tumor site, but this population contained only half as many mature host CD80⁺MHC-II⁺CD11c⁺ DCs as in wt treated mice (Fig. 5B). Correspondingly, tumor-bearing mice depleted of tumor-associated regulatory DCs survived for significantly shorter periods upon adoptive transfer of CD40L-deficient tumor-reactive T cells than wt T cells (Fig. 5C). In agreement with our previous findings, similar effects were observed without eliminating immunosuppressive DCs (20, 25), although the therapeutic responses observed after the individual administration of tumor-primed T cells were not as dramatic (data not shown). Importantly, this reduced antitumor effect was not caused by impaired secretion of the chemokine CCL5, as CD40L^−/− T cells secreted as much CCL5 as wt T cells (Fig. 5D) and show identical CD4:CD8 ratios (Fig. 5E) and activation markers (Fig. 5F, 5G) postexpansion, suggesting that the inability of the (CD4⁺) T cells to signal through CD40 on newly recruited DCs prevented their maturation in situ.

Additionally, host-derived (endogenous) T cells obtained from regulatory DC-depleted, CD40L-deficient T cell-treated mice failed to protect mice challenged with flank ID8-Defb29/Vegf-a tumors (Fig. 5H, Supplemental Fig. 2). Taken together, these data suggest that transferred T cells recruit APCs to the tumor site through the secretion of CCL5 (predominantly secreted by the CD4⁺ T cell subset). In the presence of tumor Ag (presumably released by transferred CD8⁺ T cells) (20), adoptively transferred CD4⁺ T cells signal through CD40 on the newly recruited host DCs to trigger their maturation. The mature DCs then activate tumor-specific host-derived (endogenous) T cells to control tumor progression.

To confirm that the activating function of adoptively transferred T cells is indeed mediated by CD40 stimulation on host DCs recruited to tumor locations, we briefly primed T cells against tumor Ag and cultured them with DCs sorted from wt or CD40-deficient mice bearing ovarian tumors. As expected, wt tumor-derived DCs significantly upregulated expression of the costimulatory molecules CD70, CD86, and CD80, as well as MHC-II, after culture with wt T cells compared with unstimulated DCs (Fig. 6). In addition, these DCs also secreted significantly higher levels of IL-12 than their unstimulated counterparts (Fig. 7A). In contrast, DCs sorted from the peritoneal cavity of tumor-bearing CD40-deficient mice cultured with wt T cells primed in an identical manner expressed significantly reduced levels of CD70, CD86, CD80, and MHC-II (Fig. 6) and also secreted significantly less IL-12 than wt DCs (Fig. 7A). Further supporting the requirement of interactions between CD40 (on host DCs) and CD40L (on adoptively transferred T cells) for the maturation of newly recruited tumor DCs, CD40L-deficient T cells briefly primed to tumor Ags were incapable of inducing the maturation and secretion of IL-12 by cultured tumor-derived DCs to the extent observed by identically primed wt T cells (Figs. 6, 7A). Interestingly, differences in DC activation were not as strong when wt DCs were cultured with CD40L^−/− T cells as seen when DCs lacking CD40 were cultured with wt T cells, suggesting that although these ID8-Defb29/Vegf-a tumor-primed T cells act to mature DCs through CD40 signaling, there probably exists other mechanisms through which they additionally act to mature DCs. Nevertheless, together, these data confirm that adoptively transferred (exogenous), briefly primed (CD4⁺) T cells induce the maturation of tumor-associated DCs of host (endogenous) origin, suggesting that these DCs are licensed in situ at tumor locations to induce the expansion and activation of host-derived (endogenous) tumor-specific T cells.

We next sought to confirm the functional and thus potential therapeutic relevance of our findings that expanded T cell license tumor-associated DCs to activate endogenous antitumor CTLs. For that purpose, we FACS-sorted CD11c⁺MHC-II⁺ DCs from the peritoneal cavity of untreated mice growing established i.p. ovarian cancer (Experimental design; Supplemental Fig. 3). As we reported, these cells are strongly immunosuppressive (26). When these tumor-derived DCs were incubated for 24 h with adoptive transfer-ready wt T cells from healthy mice (20), they were able to induce the secretion of both cytolytic granzyme B and IFN-γ by high numbers of subsequently added CD3⁺CD8⁺ CTLs sorted from tumor (peritoneal) locations in ELISPOT analysis (Fig. 7B, 7C). In contrast, when tumor-derived CTLs were added to cultures in which tumor-associated DCs had been incubated with CD40L-deficient T cells primed in an identical manner, the number of lymphocytes producing granzyme B and IFN-γ was significantly decreased (Fig. 7B, 7C). These results indicate that adoptively transferred (CD4⁺) T cells transform tumor-associated immunosuppressive DCs into effective APCs, and, as no additional antigenic stimulus was added, the data confirm the notion that tumor-associated DCs spontaneously engulf tumor materials (26).

To reciprocally confirm that the capacity of licensed tumor DCs to activate (rather than inhibit, as they spontaneously do) (20) tumor-infiltrating CTLs was dependent on CD40 signaling, we challenged CD40-deficient mice with i.p. ovarian tumors and first confirmed that their ascitic fluid contained the same numbers and proportions of microenvironmental cells as wt mice (data not shown). When tumor-associated (CD40-deficient) DCs sorted from these mice were incubated with either wt or CD40L-deficient T cells de novo primed against tumor Ags, the number of tumor-derived CTLs producing granzyme B or IFN-γ was again significantly decreased compared with wt DCs (Fig. 7B, 7C). Together, these data indicate that adoptively transferred polyclonal T cells briefly primed de novo against tumor Ags can license host-derived tumor-associated DCs through CD40 to become mature DCs capable of activating host (endogenous) tumor-associated T cells to induce protection against subsequent challenge with the same tumor.

CD4⁺ T cells have been associated with a negative outcome in patients with ovarian cancer (2), and the therapeutic potential of adoptively transferring tumor-reactive Th1 cells to ovarian cancer patients demands further investigation (12).

We previously reported that the transfer of a mixed population of polyclonal T cells briefly primed de novo against tumor Ags promotes the regression of established ovarian cancer or significantly delays the progression of a more aggressive model of ovarian cancer when coupled with the depletion of tumor-associated immunosuppressive CD11c⁺ DCs (20). In this current study, we demonstrate that CD4⁺ T cells are necessary for maximum effectiveness. We further elucidated the mechanisms of the contributions of such CD4⁺ T cells as being 2-fold initially through the recruitment of host immune cells to the tumor site via CCL5 secretion and, more importantly, by directly interacting with and licensing host DCs to become capable of activating potent antitumor CD8⁺ T cells.

Most adoptive immunotherapy regimens focus on enhancing the effect of CD8⁺ T cells, partly due to their ability to eradicate highly prevalent MHC-I⁺ tumor cells. Yet, most methods currently employing the individual transfer of CD8⁺ T cells have not been met with much success (29–31). This may be due to the fact that although these CD8⁺ T cells may initially induce strong tumorlytic effects, they eventually succumb to the paralytic tumor microenvironment and become tolerized and thus incapable of eliciting sustained antitumor effects. Developing strategies that awaken and permit host immune cells to overcome such debilitating effects constitute the future for ACTs. With this intention, and given the fact that the earlier the activation phenotype of the transferred T cells, the better they fare, we aimed to develop a clinically translatable technique of activating T cells. A potential source of nonparalyzed T cells for activation and infusion may be aphaeresis samples from patients with ovarian cancer. We recapitulate this stage wherein there are few Ag-experienced T cells by utilizing splenic T cells from healthy unchallenged mice, which we previously demonstrated are as effective as those from tumor-bearing mice at inducing enhanced survival of tumor-bearing mice (20).

With the understanding that there are several immunosuppressive networks at play in the context of ovarian cancer, we initially sought to establish the contribution of individual T cell subsets to ACT with the hypothesis that CD4⁺ T cells would prove to be required for maximal therapeutic benefits by providing help for CD8⁺ T cells. In fact CD4⁺ T cells were found to be the major producers of CCL5, which was required for the accumulation of CCR5⁺ activated DCs and antitumor T cells of host origin at the tumor site. This is consistent with the results of Dobrzanski et al. (12) that attribute the therapeutic benefits triggered by adoptively transferred Th1 cells to subsequently activated CCR5⁺ T cells of host origin. CCL5 secretion is also pertinent for the long-term antitumor protective effects, as activated host T cells mediated protection against secondary tumor challenge only when CCL5 was produced by the transferred T cell compartment.

Importantly, the protective effects mediated by endogenous T cells reactivated upon ACT against a secondary tumor challenge were also abrogated by the elimination of CD40 signaling in the transferred T cells. Thus, adoptively transferred wt but not CD40L^−/− (CD4⁺) T cells were capable of licensing DCs newly recruited to tumor locations. DCs licensed by transferred CD4⁺ T cells were then capable of activating tumor-reactive CD8⁺ T cells of host origin. Endogenous CD8⁺ T cells primed in this fashion had a greater capacity for secreting the tumorlytic factors IFN-γ and granzyme B and also displayed increased capacity for long-term persistence and antitumor immunity. This translated to their capacity to induce sustained host immune responses such that their infusion into naive mice was protective against a secondary challenge with s.c. tumors of the same strain. Because T cells deficient in CD40L were unable to induce such effects, it was apparent that this mechanism of DC licensing, completely independent of the direct cytotoxic activity recently found in nonepithelial tumor models (32, 33), was absolutely necessary for the prolonged CTL effector functions and later memory/protective responses.

These studies have revealed that adoptively transferred CD4⁺ T cells license DCs to prime antitumor CD8⁺ T cells. We have previously demonstrated that within 3 d of adoptive T cell transfer and CD11c⁺ regulatory DC depletion, we observe increased maturation of returning DCs, which remain in this state up to 7 d after T cell transfer (20). Upon administration of the CD11c⁺ IT, ∼70% of CD11c⁺ regulatory DCs are eliminated within the peritoneum, but there is a rapid turnover such that within 48 h, there is an almost complete repopulation (25). Furthermore, transferred T cells do not persist for long periods after T cell transfer. This implies that the T cell licensing occurs within a short span of T cell transfer, as confirmed by our in vitro data in which activated T cells induce DCs to upregulate activation markers after 24 h in culture. Importantly, the transmission of immunity from transferred T cells to the host population through licensing of host DCs, though rapid, is long-lasting, supported by the ability of host T cells to protect against secondary tumor challenge.

Traditionally, licensing of DCs is considered to occur through the interactions with CD4⁺ T cells in the lymph node and to be followed with the subsequent priming of circulating CD8⁺ T cells there. However, reports present evidence for the in situ licensing of DCs and their further in situ priming of resident CD8⁺ T cells (34, 35). As such, because CD4⁺ T cells, CD40 signaling, and CCL5 were all required for successful ACT, we propose a mechanism whereby CD4⁺ T cells may, upon administration in the peritoneum, secrete CCL5, initiating the recruitment of immune cells to the tumor site. CD8⁺ T cells administered concurrently may begin killing tumor cells, whereas the CD4⁺ T cells primed against tumor Ags pretransfer and from the repeated exposure to Ag produced by the CD8⁺ T cell-mediated tumor cell death act upon endogenous tumor-resident DCs to license them to further prime host CD8⁺ T cells also present or recruited to the tumor site. These newly awakened host CD8⁺ T cells can now additionally function to fight against the tumor.

Collectively, the data presented in this study show that CD4⁺ T cells contribute positively and significantly to the beneficial effects of ACT against ovarian cancer and define the phenotypic attributes that can maximize their effectiveness. These results emphasize the need for consistently studying methods of achieving optimal effects in ACTs and advance the field’s understanding of the mechanisms at play in the context of ovarian cancer.

We thank the Irradiation Shared Resource (Lebanon, NH) for irradiating mice and cells and the Englert Cell Analysis Laboratory (Lebanon, NH) for sorting cells. We also thank S. Orsulic (Cedars-Sinai Medical Center, Los Angeles, CA) for generously authorizing the use of the MT2 ovarian cancer cell line.

Disclosures The authors have no financial conflicts of interest.