CD4⁺ Th1 cells promote CD8⁺ Tc1 cell survival, memory response, tumor localization and therapy by targeted delivery of interleukin 2 via acquired pMHC I complexes

Abstract

The cooperative role of CD4⁺ helper T (Th) cells has been reported for CD8⁺ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4⁺ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8⁺ T cell proliferation and activation. In this study, we used CD4⁺ Th1 and CD8⁺ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4⁺ Th1 cell's help effects on active CD8⁺ Tc1 cells and the molecular mechanisms involved in CD8⁺ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4⁺ Th1 cells with acquired pMHC I by OVA-pulsed DC (DC_OVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8⁺ Tc1 cells in vitro, and promoting CD8⁺ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8⁺ Tc1 with CD4⁺ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4⁺ Th1’s help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8⁺ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.

Introduction

CD8⁺ cytotoxic T (Tc) lymphocytes which are a major immunological effector cell population mediating resistance to cancer can eradicate the growth and metastasis of malignant tumor cells¹. Effective cancer immunotherapy with adoptively transferred tumor-sensitized Tc cells has been well documented in animal models^2,3. CD8⁺ Tc cells can be polarized to effector subsets with distinct cytokine production profiles (Tc1 cells producing IFN-γ and Tc2 cells secreting IL-4 and IL-5)⁴. Dobrzanski et al. previously reported that tumor-specific Tc1 cells were relatively more effective reduction of lung metastasis of OVA-transfected B16 melanoma than Tc2 cells⁵. Recently, they have further shown that these Tc1 and Tc2 can also cure intradermally transplanted OVA-transfected B16 melanomas, but only in their palpable sizes⁶. In clinical trials, only a limited number of patients have responded to T-cell therapy^7,8 partly due to lacking of T cell helper arm and/or only very small fractions of transferred T cells accumulating in tumors^9,10.

CD4⁺ T helper (Th) cells can also be subdivided into Th1 cells producing IL-2 and IFN-γ and Th2 cells secreting IL-4, IL-5 and IL-10. It has been reported that Th2 cells responsible for humoral immunity¹¹ neither enhanced nor suppressed antitumor CD8⁺ cytotoxic T lymphocyte (CTL) responses¹² whereas Th1 cells essential for cellular immunity play an important role in priming CTL-mediated antitumor responses⁹. The traditional explanation is that CD4⁺ Th1 cells provide IL-2 or helper to CD8⁺ Tc cells¹³. CD4⁺ Th cells have also been shown to have another role in induction of CD8⁺ Tc cell responses through DC activation by CD40/CD40L interactions¹⁴. Recently, it has also been reported that CD4⁺ Th cells are required in determining the magnitude and persistence of CTL responses¹⁵ and for CD8⁺ T cell infiltration of tumors¹⁶. However, the underlying immune mechanisms of these CD4⁺ Th's effects in adoptive CD8⁺ Tc-cell therapy are still largely unknown.

Stimulation of T cells by antigen-presenting cells (APCs) involves at least two signaling events: one elicited by T cell receptor (TCR) recognition of major histocompatibility complex/peptide (pMHC) complexes and the other one by costimulatory molecule signaling (e.g. T cell CD28/APC CD80)¹⁷. A consequence of such Ag-specific T cell–APC interactions is the formation an immunological synapse, comprising a central cluster of TCR-pMHC complexes and CD28–CD80 interactions surrounded by rings of engaged accessory molecules (e.g. complexed LFA-1-CD54)^18,19. One important feature of synapse physiology is that APC-derived surface molecules are transferred to the Th cells during the course of their TCR internalization followed by recycling^20,21. Recently, we have demonstrated that naive CD4⁺ T cells acquire DC molecules by DC activation and act as Th-APCs. These Th-APCs with acquired pMHC complexes and costimulatory molecules can stimulate naïve CD8⁺ T cell proliferation in vitro and in vivo and induce CTL responses and antitumor immunity²². However, the molecular mechanisms responsible for the functional effects of Th-APCs have not been well elucidated, and the critical role the acquired pMHC I complexes play in targeting CD4⁺ Th's effects to CD8⁺ T cells in vivo has not been clearly defined due to lacking the appropriate control cells such as CD4⁺ Th(pMHC I^–/–) cells used in this study.

In this study, we developed a model system with a defined tumor antigen OVA using the OVA-transfected EG7 tumor cells and the OVA-specific TCR transgenic OT I and OT II mice with class I and II specificities, respectively,²³. Based upon this model system, we investigated the help effects of OT II CD4⁺ Th1 cells in active CD8⁺ Tc1-cell immunotherapy of established solid EG7 tumors. We found that CD4⁺ Th1 cells prolonged active OT I CD8⁺ Tc1 cell survival and promoted active OT I CD8⁺ Tc1 cell tumor localization and memory responses. We further elucidated the molecular mechanisms responsible for their help effects in CD8⁺ Tc1 cell immunotherapy and disclosed the critical role of acquired pMHC I complexes in delivery of CD4⁺ T help effects to CD8⁺ Tc1 cells in vivo by using the recently established control CD4⁺ Th(pMHC I^–/–) cells.

Materials and methods

Antibodies, cytokines, cell lines and animals

Biotin-conjugated antimouse MHC class I (H-2K^b) and II (Ia^b), CD4, CD8, CD11c, CD25, CD69 and Vβ5·1,5·2 TCR antibodies (Abs) were obtained from BD Pharmingen Inc. (Mississauga, Ontario, Canada). The FITC-conjugated avidin was obtained from Bio/Can Scientific (Mississauga, Ontario, Canada). PE-labeled H-2K^b/OVA_257−264 (OVA I) tetramer and FITC-labeled anti-CD8 Ab were obtained from Beckman Coulter, Missisauga, Ontario, Canada. The anti-IL-2, -IL-4, -IFN-γ Abs, and the recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF), IL-2, IL-12 and interferon (IFN)-γ were purchased from R & D Systems (Minneapolis, MN). The anti-H-2K^b/OVA I (pMHC I) Ab was obtained from Dr R. Germain, National Institute of Health, Bethesda, MD²⁴. The mouse B cell hybridoma cell line LB27 expressing Ia^b, thymoma cell line EL-4 and its derivative OVA-transfected cell line EG7 were obtained from American Type Culture Collection (ATCC), Rockville, MD. OVA I (SIINFEKL) and OVA II (ISQAVHAAHAEINEAGR) peptides were synthesized by Multiple Peptide Systems (San Diego, CA). Female C57BL/6 mice and OT I and OT II mice having transgenic Vα2Vβ5 TCRs specific for OVA_257−264 (OVA II) epitope in the context of H-2K^b and OVA_323−339 epitope in the context of Ia^b22,23, respectively, and H-2K^b, IL-2 and IFN-γ gene knockout (KO) mice on C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, Maine). Homozygous OT II/H-2K^b–/–, OT II/IL-2^–/– and OT II/IFN-γ^–/– mice were generated by backcrossing the designated gene KO mice onto the OT II background for three generations; homozygosity was confirmed by polymerase chain reaction (PCR) according to Jackson laboratory's protocols. All mice were maintained in the animal facility at the Saskatoon Cancer Center and treated according to Animal Care Committee guidelines of University of Saskatchewan.

Preparation of dendritic cells

Bone marrow-derived dendritic cells (DCs) were generated using GM-CSF and IL-4 as described previously²⁵. To generate OVA protein-pulsed DCs, DCs derived from wild-type C57BL/6 mice were pulsed overnight at 37° with 0·1 mg/ml OVA protein (Sigma, St. Louis, MO) and termed as DC_OVA. DC_OVA were capable of stimulating both OT II CD4⁺ and OT I CD8⁺ T cell proliferation in vitro²⁶ indicating that OVA-pulsed DC_OVA express both pMHC II and pMHC I complexes, respectively. DC_OVA derived from C57BL/6 mice with H-2K^b gene KO were termed as (K^b–/–)DC_OVA.

Preparation of active T cell subsets

OVA-specific CD4⁺ T and CD8⁺ T cells were isolated from the spleens and lymph nodes of OT-II and OT-I TCR-transgenic mice, enriched by passage through nylon wool columns (C & A Scientific Inc, Manassas, VA), and then, the CD4⁺ and CD8⁺ T cells were further fractionated by negative selection using antimouse CD8 (Ly2) and CD4 (L3T4) paramagnetic beads (DYNAL Inc., Lake Success, NY), respectively, according to the manufacturer's protocols. The OVA-specific T cell subsets (Th1 and Tc1 cells) were further generated by culturing naïve CD4⁺ and CD8⁺ T cells (3 × 10⁵ cells/well) with irradiated (4000 rad) DC_OVA (2 × 10⁵ cells/well) in 96-well plate in the presence of IL-2 (20 U/ml), IL-12 (5 ng/ml) and anti-IL-4 Ab (5 µg/ml)²², respectively. In vitro-activated Th1 and Tc1 cell subsets were harvested after 4 days' culture and purified using Ficoll-Paque (Sigma, St. Louis, MO) density gradient centrifugation²⁶ and followed by using CD8- and CD4-microbeads (Milttenyi Biotec, Auburn, CA), respectively²². These in vitro (K^b–/–)DC_OVA-activated wild-type OT II CD4⁺ T cells were termed as Th1(pMHC I^–/–), whereas wild-type DC_OVA-activated CD4⁺ T cells from designated gene-deleted OT II (OT II/K^b–/–, OT II/IL-2^–/– and OT II/IFN-γ^–/–) mice under the same culture conditions were termed as CD4⁺ Th1(K^b–/–), Th1(IL-2^–/–) and Th1(IFN-γ^–/–), respectively. Con A-stimulated OT II CD4⁺ T (Con A-OT II) cells were generated and purified as previously described²².

Phenotypic characterization of active T cell subsets

The active T cell subsets were stained with a panel of antibodies and analyzed by flow cytometry²⁷. Isotype-matched monoclonal Abs with irrelevant specificity were used as controls.

Cytokine secretion

Tc1 and Th1 subsets were re-stimulated by culturing T cells (0·5 × 10⁶ cells/well) in flat-bottom 96-well plates (Costar Corp, Cambridge, MA) with irradiated (6000 rad) EG7 (0·6 × 10⁵ cells/well) and OVAII-pulsed irradiated (4000 rad) LB27_OVAII (0·6 × 10⁵ cells/well), respectively²⁶. The culture supernatants were harvested at 1 day for measurement of IFN-γ, IL-4, and IL-2 secretion by using enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Woburn, MA). The results were normalized to the recombinant cytokine standard curves.

PMHC I complex transfer assay

Naïve CD4⁺ T cells (3 × 10⁵ cells/well) derived from OT II mice with H-2K^b gene KO were cultured with irradiated (4000 rad) DC_OVA (2 × 10⁵ cells/well) in the presence of IL-2 (20 U/ml), IL-12 (5 ng/ml) and anti-IL-4 Ab (5 µg/ml) for 4 days. The active OT II T cells were analyzed using the antipMHC I Ab by flow cytometry.

Naïve CD8⁺ T cell proliferation assay

A constant number of naïve OT I CD8⁺ T cells (0·5 × 10⁵ cells/well) were cultured with irradiated (4000 rad) stimulators including CD4⁺ Th1, Th1(pMHC I^–/–), Th1(k^b–/–) cells and DC_OVA (0·4 × 10⁵ cells/well), respectively, and their 2-fold dilutions. After 48 hr, all wells were pulsed for 12 h with 1 µCi of [³H]-thymidine (Amersham, Arlington Heights, IL) and then harvested onto glass fiber filters, respectively. Thymidine incorporation in each well was determined by liquid scintillation counting²⁷.

Active Tc1 cell survival analysis

To examine CD8⁺ Tc1 cell survival in absence of IL-2 stimulation, in vitro-activated CD8⁺ Tc1 cells (0·4 × 10⁵ cells/well) were incubated with or without irradiated (4000 rads) active CD4⁺ Th1 cells (0·4 × 10⁵ cells/well) in RPMI 1640 plus 10% FCS in flat-bottom 96-well plates. To assess CD8⁺ Tc1 cell apoptosis formation, T cells were harvested after 4 days, stained with FITC-Annexin V (BD Pharmingen) and PE-anti-CD8 Ab, and analyzed by flow cytometry. In some experiments, each of a panel of neutralizing reagents (anti-IL-2 and -IFN-γ Abs) (each 15 µg/ml) was added to the culture system. To examine the effect of acquired pMHC I complexes on CD8⁺ Tc1 cell survival, Th1(pMHC I^–/–) cells without acquired pMHC I complexes were used as control cells.

Tc1 cell tumor infiltration

Detection of tumor infiltration of transferred active CD8⁺ Tc1 cells in vivo was performed as previously described²⁷. Briefly, in vitro DC_OVA-activated CD8⁺ Tc1 cells (5 × 10⁶ cells/mouse) together with or without in vitro DC_OVA-activated CD4⁺ Th1, Th1(pMHC I^–/–), Th1(K^b–/–), Th1(IL-2^–/–) and Th1(IFN-γ^–/–) cells (2 × 10⁶ cells/mouse) were i.v. injected into C57BL/6 mice bearing EG7 tumors with ∼5 mm in diameter, respectively. At different days subsequent to T cell injection, tumors were removed. Cell suspensions were prepared from these EG7 tumors by mincing them into small pieces and pressing them through a fine mesh¹⁶. Red cells were lysed by using 0·84% ammonium chloride. T cells were then purified from these cell suspensions by using the CD3 microbeads (Mittenyi Biotec), stained using PE-labeled H-2K^b/OVA I tetramer (PE-tetramer) and FITC-labeled anti-CD8 Ab (FITC-CD8) for flow cytometric analysis.

Adoptive Tc1 cell immunotherapy model

Mice (10 per group) received s.c. injections of 1 × 10⁶ EG7 tumor cells in their thighs. At 10–12 days postinoculation, tumors became around 5 mm in diameter. To study the help effect of CD4⁺ Th1 cells, tumor-bearing mice were injected i.v. with 5 × 10⁶ CD8⁺ Tc1 cells alone, or in conjunction with different amounts of CD4⁺ Th1 cells, respectively. To study the molecular mechanism of CD4⁺ Th1’s help effect in CD8⁺ Tc1-cell therapy, tumor-bearing mice were also injected i.v. with 5 × 10⁶ of CD8⁺ Tc1 cells in conjunction with 2 × 10⁶ CD4⁺ Th1(pMHC I^–/–), Th1(K^b–/–), Th1(IL-2^–/–) and Th1(IFN-γ^–/–) cells, respectively. Animal mortality and tumor growth or regression were monitored daily for up to 10 weeks; for humanitarian reasons, all mice with tumors that achieved a size of 1·5 cm in diameter were sacrificed.

Assessment of CD8⁺ Tc1 cell memory responses

Naïve C57BL/6 mice were i.v. injected with active CD8⁺ Tc1 cells (5 × 10⁶ cells) alone or in conjunction with CD4⁺ Th1 or Th1(pMHC I^–/–) cells (2 × 10⁶ cells). Tetramer staining assay was performed to examine the presence of OVA-specific CD8⁺ memory T (Tm) cells in mouse peripheral blood 3 months after the adoptive Tc1 cell transfer. The mouse tail blood samples were incubated with PE-tetramer, FITC-CD8 and ECD-anti-CD44 (ECD-CD44) Abs (Beckman Coulter, Mississauga, Ontario, Canada). The erythrocytes were then lysed using lysis/fixed buffer (Beckman Coulter) and the samples were analyzed by flow cytometry according to the company's protocol. In one set of experiments, the immunized mice were boosted with i.v. injection of irradiated (4000 rad) DC_OVA (1 × 10⁶ cells). Four days after the boost, the mouse tail blood samples were tested for tetramer staining as described above. In another set of experiments, the immunized mice were challenged with s.c. injection of 1 × 10⁶ or 3 × 10⁶ EG7 tumor cells. Animal mortality and tumor growth or regression were monitored daily for up to 10 weeks as described above.

Results

Characterization of active CD4⁺ Th1 and CD8⁺ Tc1 cells

Two different T cell subsets (CD4⁺ Th1 and CD8⁺ Tc1 effector cells) were generated in vitro from OVA-specific TCR transgenic OT II and OT I mice, respectively, as described in Materials and Methods. Both CD4⁺ and CD8⁺ T cells displayed their T cell subset marker (CD4 or CD8), Vα2Vβ5⁺ TCR and active T cell markers CD25 and CD69 (Fig. 1a), indicating that they are OVA-specific active CD4⁺ and CD8⁺ T cells. They also expressed some MHC class II and CD80 molecules, which may be acquired from DC through synapse-composed molecule internalization/recycling^18,19,22. In addition, there was no CD11c-positive DC population existing in these purified active T cells (Fig. 1a). This is because that any survival of irradiated DC_OVA cells and potential small amount of contamination of spleen DC or B cells with the original naïve OT II CD4⁺ and OT II CD8⁺ T cell preparations, which might have picked up OVA peptides from irradiated DC_OVA in the culture, would be eliminated by the killing activity of these activated Th and Tc cells expressing perforin^22,27,28. These activated Th and Tc cells secreted abundant IFN-γ (2·5 ng/ml/10⁶ cells/24 h and 3·2 ng/ml/10⁶ cells/24 hr) and IL-2 (1·9 ng/ml/10⁶ cells/24 h and 0·9 ng/ml/10⁶ cells/24 hr), respectively, but very little IL-4 (∼50–60 pg/ml/10⁶ cells/24 hr) in their culture supernatants (Fig. 1b), indicating that they are type 1 CD4⁺ Th1 and CD8⁺ Tc1 cells, respectively.

Figure 1.

Phenotypic characterization of in vitro DC_OVA-activated CD4⁺ Th1 and CD8⁺ Tc1 cells.(a) The in vitro DC_OVA-activated CD4⁺ Th1 and CD8⁺ Tc1 cells as described in Materials and Methods were stained using a panel of Abs for analysis of CD4, CD8, CD11c, CD25, CD69, CD80, Ia^b and Vβ5·1TCR (solid lines). The isotype-matched irrelevant Abs were used as controls (dotted lines).(b) The supernatants of these CD4⁺ Th1, Th1(pMHC I^–/–) and CD8⁺ Tc1 cells were assayed for IFN-γ, IL-4 and IL-2 secretion by ELISA, respectively. The values presented represent the means of triplicate cultures from three distinct experiments.(c) Transfer of H-2K^b and pMHC I molecules onto CD4⁺ Th1 cells by DC activation. The active CD4⁺ Th1 or CD4⁺ Th1(K^b–/–) cells (solid lines) derived from activation of naïve OT II CD4⁺ T cells or CD4⁺ T(K^b–/–) cells of OT II/H-2K^b–/– mice by DC_OVA, and the original naïve CD4⁺ T or CD4⁺ T(K^b–/–) cells (thick dotted lines) as well as the active CD4⁺ Th1(pMHC I^–/–) cells (solid lines) derived from activation of naïve OT II CD4⁺ T cells by (K^b–/–)DC_OVA, and the original naïve OT II CD4⁺ T cells (thick dotted lines) were stained with a panel of Abs for H-2K^b and pMHC I and analyzed by flow cytometry. The isotype-matched irrelevant Abs were used as controls (thin dotted lines). One representative experiment of three in the above different experiments is shown.

CD4⁺ Th1 cells acquire pMHC I complexes by DC activation

We have previously shown that active CD4⁺ Th cells can acquire DC molecules such as MHC I and II and costimulatory molecules by DC activation²². In this study, we demonstrated that active CD4⁺ Th1 cells expressed large amount of H-2K^b and some pMHC I complexes detected by the antipMHC I Ab (Fig. 1c). To confirm the acquisition of pMHC I complexes, we used CD4⁺ T cells derived from OT II mice with H-2K^b gene KO to rule out the possibility of its endogenous H-2K^b picking up OVA I peptide released from DC_OVA in the culture. As shown in Fig. 1(d), naïve CD4⁺ Th1(K^b–/–) cells themselves did not display any endogenous H-2K^b expression, but the active CD4⁺ Th1(K^b–/–) cells derived from naïve CD4⁺ Th1(K^b–/–) cells activated by DC_OVA did express both H-2K^b and pMHC I. As a control, the active CD4⁺ Th1(pMHC I^–/–) cells derived from activation of naïve OT II CD4⁺ T cells with (K^b–/–)DC_OVA did express H-2K^b, but did not express pMHC I (Fig. 1c), indicating that the pMHC I complexes on active CD4⁺ Th1 cells are acquired from DC_OVA, but not derived from uptaking the DC_OVA-derived OVA protein and self-loading OVA I peptide onto the self H-2K^b molecules. In addition, the active CD4⁺ Th1(pMHC I^–/–) cells with acquired pMHC I complexes displayed a similar cytokine profiles as the active CD4⁺ Th1 cells with acquired pMHC I complexes (Fig. 1b). Therefore, these two types of CD4⁺ Th1 cells became useful reagents in studying the role of acquired pMHC I complexes in delivery of CD4⁺ Th1’s effect to CD8⁺ Tc1 cells in vivo.

CD4⁺ Th1 cells stimulate naïve CD8⁺ T cell proliferation via acquired pMHC I complexes

We have previously shown that active CD4⁺ Th cells with acquired DC molecules can stimulate naïve CD8⁺ T cell proliferation and activation²². To confirm that the stimulation of naïve CD8⁺ T cells is mediated via acquired pMHC I on CD4⁺ Th1 cells, we repeated T cell proliferation assays using CD4⁺ Th1(pMHC I^–/–) cells with similar cytokine profiles [secreting IFN-γ (∼2·6 ng/ml/10⁶ cells/24 hr) and IL-2 (∼2·2 ng/ml/10⁶ cells/24 hr)] as CD4⁺ Th1 cells, but without acquired pMHC I complexes. As shown in Fig. 2(a), the positive control DC_OVA strongly induced OT I cell proliferation. Active CD4⁺ Th1 cells with acquired DC molecules also stimulated proliferation of naïve OT I CD8⁺ T cells, but in a less extent, possibly due to its less costimulatory molecules compared with DC_OVA. Interestingly, CD4⁺ Th1(pMHC I^–/–) cells without acquired pMHC I complexes failed in stimulation of naïve CD8⁺ T cell proliferation, whereas CD4⁺ Th1(K^b–/–) cells lacking self H-2K^b, but with acquired pMHC I complexes had similar effect as CD4⁺ Th1 cells, thus confirming that activation of naïve CD8⁺ OT I T cells is critically mediated via acquired pMHC I complexes.

Figure 2.

Functional characterization of in vitro DC_OVA-activated CD4⁺ OT II Th1 cells.(a) Naïve OT I CD8⁺ T cell proliferation assay. Varying numbers of stimulators including the irradiated CD4⁺ Th1, Th1(K^b–/–), Th1(pMHC I^–/–) cells and DC_OVA were cocultured with a constant number of naïve OT I CD8⁺ T cells. After two days, the proliferative responses of the CD8⁺ T cells were determined by ³H-thymidine uptake assays. (b) Apoptosis formation assay. Active CD8⁺ Tc1 cells were harvested 4 days after in vitro DC_OVA activation, and cultured with or without CD4⁺ Th1 cells in the medium without IL-2 for 4 days. T cells were harvested, stained with FITC-Annexin V and PE-anti-CD8 Ab, and analyzed by flow cytometry. In some experiments, each of a panel of neutralizing reagents (anti-IL-2 and -IFN-γ Abs) (each 15 µg/ml) was added to the culture system. Th1(pMHC I^–/–) cells without acquired pMHC I complexes were used as control cells in the above assay. *P < 0·05 (Student t-test) vs. cohorts of Th1 plus Tc1 cells. One representative experiment of two in the above different experiments is shown.

CD4⁺ Th1 cells prolong active CD8⁺ Tc1 cell survival in vitro via IL-2 secretion

T cell death or apoptosis formation can be derived from lymphokine withdrawal²⁹. As shown in Figs 2(b) and 95% of active CD8⁺ Tc1 cells expressed Annexin V (early apoptosis marker) in medium without IL-2 after 4 days incubation. The CD8⁺ Tc1 cell apoptosis formation dramatically dropped to only 32% in presence of CD4⁺ Th1 cells, indicating that CD4⁺ Th1 cells can inhibit apoptosis formation of active Tc1 cells. CD4⁺ Th1(pMHC I^–/–) with similar level of IL-2 secretion, even without acquired pMHC I, can still prolong CD8⁺ Tc1 survival in vitro. Interestingly, our data showed that Th1’s protection was significantly reduced (P < 0·05) in presence of anti-IL-2 Ab, but not in presence of anti-IFN-γ Ab, indicating that Th1’s help effect is mediated via its IL-2 secretion.

CD4⁺ Th1 cells promote CD8⁺ Tc1 cell tumor infiltration in vivo

We next tested whether CD4⁺ Th1 cells can promote CD8⁺ Tc1 cell tumor infiltration in vivo by flow cytometry. Our data showed that the amount of transferred CD8⁺ Tc1 cells reached peak at 3 days in tumors, but dramatically dropped to a minimal level of detection at 5 days subsequent to T cell injection (Fig. 3a). At day 3 subsequent to Tc1 cell injection, the transferred OVA-specific CD8⁺ Tc1 cells detected in tumors of mice with injection of CD8⁺ Tc1 and CD4⁺ Th1 cells is 0·52% of the total CD8⁺ T cells, which is around 3-folds more than that (0·16%) detected in tumors of mice with injection of CD8⁺ Tc1 cells alone (Fig. 3b). These results clearly indicate that OVA-specific CD4⁺ Th1 cells can also promote tumor infiltration of OVA-specific CD8⁺ Tc1 cells in vivo. In addition, the numbers of OVA-specific CD8⁺ Tc1 cells detected in EG7 tumors reduced by nearly 42% and 60% in the mice with cotreatment of Th1(IL-2^–/–) and Th1(pMHC I^–/–) cells, respectively, but were not affected with Th1(IFN-γ^–/–) and Th1(K^b–/–) cells (Fig. 3c). Our data thus indicate that the degree of CD8⁺ Tc1 cell tumor infiltration is greatly affected by the helper cytokine IL-2, but not IFN-γ, and critically dependent on the OVA-specific delivery to CD8⁺ Tc1 cells in vivo via acquired pMHC I complexes on active CD4⁺ Th1 cells.

Figure 3.

Enumeration of OVA-specific CD8⁺ Tc1 cells in tumors. Active CD8⁺ Tc1 cells (5 × 10⁶ cells/mouse) alone or together with in vitro-activated Th1 cells (2 × 10⁶ cells/mouse) were i.v. injected into EG7 tumor-bearing C57BL/6 mice.(a) Tumors were removed for preparation of cell suspensions as described in Materials and Methods at different days subsequent to T cell injection. T cells were purified from the cell suspensions by using anti-CD3 Ab-coated magnetic beads and analyzed by flow cytometry using PE-tetramer and FITC-labeled anti-CD8 Ab.(b) The tumor-bearing mice were injected i.v. with 5 × 10⁶ Tc1 cells alone or in conjunction with 2 × 10⁶ Th1 cells. Tumors were removed for preparation of cell suspansions 3 days after T cell injection. Plots display the percentages of FITC-CD8 and PE-tetramer positive CD8⁺ Tc1 cells in the total CD8⁺ T cell population.(c) The tumor-bearing mice were injected i.v. with 5 × 10⁶ Tc1 cells in conjunction with 2 × 10⁶ Th1, Th1(IL-2^–/–), Th1(IFN-γ^–/–), Th1(pMHC I^–/–) and Th1(K^b–/–) cells. Tumors were removed for preparation of cell suspensions 3 days after T cell injection. *P < 0·05 vs. cohorts of injection of Tc1 plus Th1 cells (Student's t-test). Data shown are representative of three separate experiments with three to five mice per experimental group.

CD4⁺ Th1 cells enhance adoptive CD8⁺ Tc1 cell therapy in vivo

To assess CD4⁺ Th1's help effects in vivo, we conducted animal studies using in vitro-activated CD8⁺ Tc1 cells for the passive transfers in combination with different amounts of in vitro-activated CD4⁺ Th1 cells. As shown in Fig. 4, CD4⁺ Th1 cells alone did not induce any tumor growth reduction. A combined adoptive T-cell therapy of CD8⁺ Tc1 (5 × 10⁶ cells per mouse) with CD4⁺ Th1 (2 × 10⁶ cells per mouse) resulted in regression of all 10 established EG7 tumors (∼5 mm in diameter), indicating that OVA-specific CD4⁺ Th1 cells can greatly enhance CD8⁺ Tc1-cell immunotherapy. The therapeutic efficiency is dose-dependent, since CD8⁺ Tc1 cells (5 × 10⁶ cells per mouse) in conjunction with less CD4⁺ Th1 cells (1 × 10⁶ and 0·5 × 10⁶ cells per mouse) only cured 7/10 (70%) and 4/10 (40%) tumor bearing mice, respectively.

Figure 4.

Impact on tumor growth and mortality rates of cotransfer of Tc1 with Th1 cell subset in adoptive T-cell immunotherapy of established tumors. Mice bearing established (i.e. ∼5 mm in diameter) EG7 tumors were given i.v. injections of 5 × 10⁶ CD8⁺ Tc1 in conjuction with different amounts of CD4⁺ Th1 cells. (a) Tumor growth was monitored and the tumor size (diameter) measured daily using an engineering caliper. The evolution of the tumors in individual mice is depicted, as are the fractions of mice in each treatment group that were tumor-free at 60 days post-treatment. (b) The readout in this figure represents the long-term mortality among the animals, as determined by daily assessments across 60 days post-treatment. The data closely mirror that in (a) with a conclusion that the combined CD4⁺ Th1 and CD8⁺ Tc1 cell immunotherapy provides a significant therapeutic advantage over the single use of Tc1 cells alone. One representative experiment of two is shown.

Targeted delivery of Th1’s IL-2 to CD8⁺ Tc1 cells in vivo via acquired pMHC I

To study the mechanism of Th1's effect, mice bearing tumors were i.v. injected with 5 × 10⁶ Tc1 cells in conjunction with 2 × 10⁶ CD4⁺ Th1(IL-2^–/–) and CD4⁺ Th1(IFN-γ^–/–) cells with respective cytokine deficiency. As shown in Fig. 5, the Th1’s help effect was substantially lost in mice with administration of CD4⁺ Th (IL-2^–/–) cells, but not affected in mice injected with CD4⁺ Th1(IFN-γ^–/–) cells, indicating that the Th1’s help effect is mainly dependent upon its IL-2, but not IFN-γ. CD4⁺ Th1(K^b–/–) cells with acquired pMHC I complexes had similar help effect as CD4⁺ Th1 cells. Interestingly, the CD4⁺ Th1’s help effect was completely lost in mice with administration of CD4⁺ Th1(pMHC I^–/–) cells with similar cytokine profiles as CD4⁺ Th1, but without acquired pMHC I complexes, indicating the critical importance of acquired pMHC I complexes in targeted delivery of CD4⁺ Th1's helper cytokine IL-2 to CD8⁺ Tc1 cells in vivo.

Figure 5.

Animal study using CD4⁺ Th1 cells with respective gene deficiency. The mice bearing tumors were injected i.v. with 5 × 10⁶ of Tc1 cells in conjunction with 2 × 10⁶ Th1, Th1(IL-2^–/–), Th1(IFN-γ^–/–), Th1(K^b–/–) and Th1(pMHC I^–/–) cells. Tumor growth was monitored and the tumor size (diameter) measured daily using an engineering caliper. The evolution of the tumors in individual mouse is depicted, as are the fractions of mice in each treatment group that were tumor-free at 60 days post-treatment.

CD4⁺ Th1 cells promote CD8⁺ Tc1 cell memory responses in vivo

We next assessed Th1's effect on CD8⁺ Tm cell responses by tetramer staining assays. Active CD8⁺ Tc1 cells can become long-lived memory Tm cells after adoptive transfer in vivo³⁰. Three months after adoptive transfer of 5 × 10⁶ active CD8⁺ Tc1 cells, there were 1·32% OVA-specific CD8⁺ T cells with CD44 expression (Tm marker)³¹ in the total host CD8⁺ T cell population (Fig. 6a), indicating that they became CD8⁺ Tm cells. The number of detected OVA-specific CD8⁺ Tm cells increased nearly by 3-folds to 3·86% in mice with transfer of both CD8⁺ Tc1 and CD4⁺ Th1 cells, indicating that CD4⁺ Th1 cells greatly promote CD8⁺ Tc1 cell memory responses. In the mice with transfer of CD8⁺ Tc1 and CD4⁺ Th1(pMHC I^–/–) cells, however, the number of detected OVA-specific CD8⁺ Tm cells remained the same as that in the mice with transfer of CD8⁺ Tc1 cells alone, indicating that the acquired pMHC I complexes play an important role in targeting Th1’s effect to the promotion of CD8⁺ Tc1 cell memory responses in vivo. These CD8⁺ Tm cells still remained functional because they could be stimulated for expansion (∼10 folds) by DC_OVA stimulation (Fig. 6b). Interestingly, transfer of CD8⁺ Tc1 cells alone can completely protect 8/8 mice from a challenge of 1 × 10⁶ EG7 tumor cells, but only 4/8 mice from a challenge of 3 × 10⁶ EG7 tumor cells (Fig. 6c). However, all (8/8) mice with transfer of both CD8⁺ Tc1 and CD4⁺ Th1 cells survived even with a challenge of 3 × 10⁶ EG7 tumor cells, indicating that CD4⁺ Th1 cells can promote CD8⁺ Tc1 cell memory responses leading to an enhanced antitumor immunity.

Figure 6.

CD4⁺ Th1 cells promote active CD8⁺ Tc1 cell memory responses.(a) In vitro DC_OVA-activated CD8⁺ Tc1 cells can become long-lived memory T cells. In vitro DC_OVA-activated CD8⁺ Tc1 cells (5 × 10⁶ cells/mouse) alone or in conjuction with active CD4⁺ Th1 or Th1(pMHC I^–/–) cells (2 × 10⁶ cells/mouse) were i.v. injected into C57BL/6 mice. Three months later, tail blood samples were taken from the mice, and a triple staining for PE-K^b/OVA tetramer, FITC-anti-CD8 and ECD-anti-CD44 Abs were conducted to determine the percentage of OVA-specific triple staining-positive CD8⁺ T cells in the total CD8⁺ population indicated in each right upper plot. The T cell population with PE-tetramer and FITC-CD8 double positivity was also the respective population with PE-tetramer and ECD-CD44 double positivity by flow cytometric analysis.(b) Three months later, the immunized mice were boosted by i.v. injection of 1 × 10⁶ irradiated DC_OVA. Four days after the boost, tail blood samples were taken from the boosted mice, and a double staining for PE-K^b/OVA tetramer and FITC-anti-CD8 Ab were conducted to determine the percentage of OVA-specific double staining-positive CD8⁺ T cells in the total CD8⁺ population indicated in each right upper plot. Naive C57BL/6 mice were also boosted with DC_OVA and used as control.(c) Three months later, the immunized mice were also s.c. challenged with 1 × 10⁶ or 3 × 10⁶ EG7 tumor cells. Mouse survival was monitored daily. The results presented are representative of two separate experiments with 4 mice (a & b) or 8 mice (c) per group.

Discussion

The cooperative role of CD4⁺ T cells has been extensively reported for CD8⁺ CTLs in tumor eradication in animal models^11,15,16. However, the potential immune mechanisms underlying enhancement of CD4⁺ Th1 cells in adoptive CD8⁺ Tc1-cell therapy have not been well elucidated. In this study, we developed a model system with a defined tumor Ag OVA using the OVA-transfected EG7 tumor cells and the OVA-specific TCR transgenic OT I and OT II mice with class I and II specificities, respectively^23,24. These transgenic mice provide a virtually monoclonal source of T cells with known specificity, where the respective role of OVA-specific CD4⁺ Th1 cells in OVA-specific CD8⁺ Tc1 cell-mediated antitumor immunity can thus be feasibly evaluated. In previous animal tumor models using TCR transgenic CD8⁺ T cells, the treatment of only lung tumor metastasis or solid tumors in small palpable size has been investigated^5,6. In clinical settings, however, patients usually have well-established tumors. In this study, we demonstrated that a combined adoptive transfer of CD8⁺ Tc1 cells with CD4⁺ Th1 cells resulted in regression of well-established tumors (5 mm in diameter), which more mimics the clinical situations, in all 10/10 mice, compared to only 40% curing rate when using adoptive transfer of CD8⁺ Tc1 cells alone, indicating the critical help effect of CD4⁺ Th1 cells in CD8⁺ Tc1-cell immunotherapy of established solid tumors. Active CD8⁺ Tc1 cells can become long-lived CD8⁺ Tm cells after adoptive transfer in vivo³⁰. In this study, we also demonstrated that CD4⁺ Th1 cells greatly promoted CD8⁺ Tc1 cell memory responses by 3-folds. Interestingly, mice with transfer of CD8⁺ Tc1 cells alone can only protect 50% (4/8) of them from a challenge of 3 × 10⁶ EG7 tumor cells, whereas all (8/8) mice with transfer of both CD8⁺ Tc1 and CD4⁺ Th1 cells survived, indicating that CD4⁺ Th1 cells can promote CD8⁺ Tc1 cell memory responses leading to an enhanced antitumor immunity.

Interleukin-2 (IL-2) support of transferred Tc cells in vivo can be critical to T-cell therapeutic efficiency. IL-2 administration after T cell transfer was shown to augment the antitumor therapy and increase transferred Tc cell persistance^32,33. The potential mechanism by which IL-2 treatment may augment adoptive Tc cell therapy in vivo by promoting the survival and proliferation of transferred Tc cells^34,35. Although the systemic use of IL-2 could enhance T-cell therapy efficiency in mouse models, the results from clinical trials in treatment of melanomas and renal cell carcinomas were very disappointed, showing no improvement in T-cell therapeutic efficiency^36,37. In addition, the systemic administration of IL-2 also induced severe side-effects such as the vascular leak syndrome³⁸. An alternative is the use of CD4⁺ Th cells providing IL-2 help to CD8⁺ Tc cells¹³. However, the molecular mechanism by which the CD4⁺ Th cell's IL-2 is specifically delivered to CD8⁺ Tc cells in vivo is unclear. T cell death or apoptosis formation can be derived from lymphokine withdrawal²⁹. In this study, we demonstrated that CD4⁺ Th1 cells can reduce apoptosis formation of active CD8⁺ Tc1 cells by 3-folds in the culture medium without IL-2 stimulation, indicating that this help effect may be mediated via its secreted helper cytokine IL-2. In addition, we also demonstrated that (i) the CD4⁺ Th1 cells greatly augmented the in vivo CD8⁺ Tc1 cell-therapy in treatment of established tumors, and (ii) the Th1's help effect was substantially lost in mice with administration of CD4⁺ Th1(IL-2^–/–) cells, indicating that the in vivo Th1's help effect is dependent upon its IL-2.

T cell-to-T cell (T-T) Ag presentation, dependent upon CD4⁺ T cells first acquiring MHC and costimulatory molecules from APCs, and then becoming Th-APCs with the capacity in stimulation of other T cells, is increasingly attracting attention^39,40. Recently, Brandes et al. have shown that active human T cells expressed a similar phenotype as APC and can act as Th-APCs in stimulation of CTL responses after directly pulsed with soluble Ag⁴¹. Although these active CD4⁺ T cells pulsed with Ag can act as T-APCs in stimulation of CTL responses, they are different from the concept of CD4⁺ Th-APCs as recently described²². We have recently demonstrated that in vitro DC-activated CD4⁺ T cells with acquired DC membrane molecules by DC activation can act as Th-APCs in stimulation of CTL responses and antitumor immunity²² and CD4⁺ Th(pMHC I^–/–) cells without acquired pMHC I complexes lost its antitumor protection (data not shown). In addition, we also found that in vivo DC-activated CD4⁺ T cells can also acquire APC membrane molecules by DC activation, and induce antitumor immunity (data not shown), indicating the physiological significance of APC membrane molecule acquisition of CD4⁺ T cells in vivo. Kennedy et al. have also demonstrated that CD4⁺ T cells can acquire APC membrane molecules in vivo, and induce memory CTL responses⁴² further confirming the physiological significance of the above concept. In this study, in addition to the acquired costimulatory molecules²² we further demonstrated that CD4⁺ T cells acquired pMHC I complexes by using a specific antibody recognizing pMHC I complexes. More importantly, for the first time, we elucidated the molecular mechanism on targeted delivery of CD4⁺ T cell help to active CD8⁺ Tc cells in vivo by using the recently established control CD4⁺ Th1(pMHC I^–/–) cells. We showed that it is the acquired pMHC I complex on active CD4⁺ Th1 cells that mediate the specific delivery of the CD4⁺ Th cytokine IL-2 to CD8⁺ Tc1 cells in vivo, leading to an enhanced Tc1-cell immunotherapy of established EG7 solid tumors. In addition, we also found that the DC_OVA-activated CD4⁺ Th1 cells with acquired pMHC I complexes were able to stimulate naive CD8⁺ T cell proliferation in vitro and in vivo, and induce antitumor immunity²² whereas CD4⁺ Th1(pMHC I^–/–) cells with the same cytokine profiles as CD4⁺ Th1 cells, but without acquired pMHC I complexes completely lost its stimulatory effect (data not shown), thus confirming the importance of acquired pMHC I complexes in targeting CD4⁺ Th1's helper effects to both naïve or active CD8⁺ Tc cells in vivo.

The importance of successful Tc-cell immunotherapy of Tc-cell tumor infiltration is increasingly being recognized^2,26,43. The systemic administration of IL-2 has been shown to enhance CD8⁺ T cells tumor infiltration¹⁰. However, the severe side-effects³⁸ derived from the systemic administration of IL-2 limit its use in clinic trials. It has also been shown that the transferred CD8⁺ T cells infiltrated into tumors only in presence CD4⁺ T cell help by immunohistochemical analysis¹⁶. In this study, we kinetically and quantitatively examined CD8⁺ Tc1 cell tumor infiltration by flow cytometric analysis. Our data showed that a 3-fold more of the transferred OVA-specific CD8⁺ Tc1 cells can be detected in EG7 tumors of mice with treatment of both CD4⁺ Th1 and CD8⁺ Tc1 cells, indicating that CD4⁺ Th1 cells can significantly promote CD8⁺ Tc1 cell tumor infiltration. Interestingly, we also clearly demonstrated that the in vivo CD4⁺ Th1 cell's helper effect in promoting CD8⁺ Tc1 cell tumor infiltration is mediated by its IL-2. More importantly, we further demonstrated that the critical role of acquired pMHC I complexes on CD4⁺ Th1 cells in targeting its helper effect in promoting CD8⁺ Tc1 cell tumor infiltration. In addition, some other factors may also be involved in enhanced CD8⁺ Tc1 cell tumor infiltration, including (i) an augmented CD8⁺ Tc1 cell pool in the mice, thereby allowing for enhanced CD8⁺ T cell infiltration of tumors, and (ii) a prolonged survival of tumor infiltrating CD8⁺ Tc1 cells in tumors. In addition, some other mechanisms may also be involved. For example, it has been reported that the costimulatory signals^41,42,44 and MHC class II molecules⁴⁵ were involved in survival of the responding cells such as T cells. Therefore, the active Th1 cells with acquired CD80 and pMHC II may also provide its helper effects to active Tc1 cells in vivo through these mechanisms.

Taken together, our results provide clear evidence that CD4⁺ Th1 cells are capable of enhancing adoptive CD8⁺ Tc1-cell immunotherapy by prolonging CD8⁺ Tc1 cell survival in vitro and promoting CD8⁺ Tc1 cell tumor localization and memory responses in vivo. The CD4⁺ Th1’s effect is mainly mediated by its own helper cytokine IL-2 specifically targeted to CD8⁺ Tc1 cells in vivo via acquired pMHC I complexes. Therefore, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of established solid tumors.

Abbreviations

APC

antigen-presenting cells

CTL

cytotoxic T lymphocyte

DC

dendritic cell

PCR

polymerase chain reaction

pMHC

major histocompatibility complex/peptide

TCR

T cell receptor

Tc

CD8⁺ cytotoxic T

Th

CD4⁺ helper T