Abstract

Introduction

Although CD8⁺ T cells have been shown to be potent mediators of antitumor immunity, the role of CD4⁺ T cells is largely undefined (1, 2). In the absence of CD4⁺ T cell help, CD8⁺ T cells against viral or foreign antigens can become lethargic(3), deleted(4), or lose the capacity to become and remain memory CD8⁺ T cells upon rechallenge(5-7). Therefore, the use of self-reactive CD8⁺ T cells in the adoptive immunotherapy of cancer may face similar fates, as T cells must remove tumor-antigen in the context of persisting self-antigen. One theoretical means of improving immunotherapy to self may involve the provision of CD4⁺ T cell help, since helper cells facilitate CD8⁺ T cell activation, function and survival(1, 6, 8). Nonetheless, naturally occurring CD4⁺ T cells represent a double-edged immunological sword: in addition to their helper functions, one T cell subset, naturally occurring CD4⁺CD25⁺ T regulatory cells (hereafter referred to as T_reg), suppresses T cells and controls immunological tolerance to self-antigens (9-12).

In recent years, naturally occurring CD4⁺CD25⁺ T regulatory (T_reg) cells have emerged as the dominant T cell population governing peripheral self-tolerance (13-15). CD4⁺CD25⁺ T cells develop in the thymus and represent 5-10% of the peripheral CD4⁺ T cell compartment. They constitutively express the high affinity IL-2 receptor or CD25 (IL-2Rα), glucocorticoid-induced TNFR (GITR), CTLA-4, and the transcription factor Foxp3 (forkhead box P3)(13). The mechanism of suppression is through cell-cell contact but how T_reg cells induce and maintain self-tolerance in vivo is still unknown.

In mice, autoimmune destruction of a variety of tissues can be triggered by the removal of T_reg cells(12, 16). Organ-specific destruction of tissue expressing a self-antigen can be further enhanced by self-antigen vaccination or through the provision of inflammatory signals when functional T_reg cells are absent(17). When lymphopenic mice are reconstituted with a normal repertoire of CD4⁺CD25^- T cells (hereafter referred to as T_helper), autoimmunity is observed in multiple tissues and co-transfer of T_reg cells abrogates these effects(18). Furthermore, depletion of T_reg cells can enhance tumor protection to tumor-associated antigens that are expressed as self-antigens(19, 20).

Spontaneous autoimmunity is also seen in IL-2^-/-, IL-2 receptor (IL-2R) α^-/-, IL-2 receptor (IL-2R) β^-/-, JAK-3^-/-, STAT-5^-/-, and Foxp3 deficient mice(15, 21, 22). The CD4⁺ T cell subset that remains in each of these knockouts is devoid of functional T_reg cells. Together, these observations suggest a role for IL-2 and T_reg cells in controlling effector T lymphocytes specific for self-antigens in vivo.

Because T_reg cells are able to control autoimmunity to naturally expressed self-antigens, they may play a role in T cell tolerance to self-antigens naturally or over-expressed by tumors. We hypothesized that by removing the T_reg cell subset we could use the autoimmune potential present within the normal repertoire of CD4⁺ T cells to help self-reactive CD8⁺ T cells mediate antitumor immunity against tumors expressing self-antigens.

Previously we have shown that adoptive transfer of CD8⁺ T cells from pmel-1 transgenic mice which recognize the melanocyte differentiation antigen, gp100, could cause autoimmunity and the regression of established tumors in tumor-bearing syngeneic animals. This therapy was successful after vaccination with a virus encoding a tumor-antigen, and exogenous administration of a γ_c-signaling cytokine, either IL-2 or IL-15(23, 24). In the present report, we transferred different CD4⁺ T cell subsets along with pmel-1 CD8⁺ T cells, and vaccination into CD4⁺ T cell-deficient tumor-bearing animals. Although help against foreign and viral antigens has been described numerous times (reviewed in(2)), we sought to understand the effect of different CD4⁺ T cell subpopulations on adoptive immunotherapy of established tumors as well as the mechanisms that cause and break tolerance to tumors in an environment of persisting self-antigen. We conclude that naturally occurring T_helper cells can help self-reactive CD8⁺ T cells break tolerance to self through an IL-2-dependent mechanism, but require the absence of naturally occurring T_reg cells to be effective.

Material and Methods

Mice and tumor cells. Pmel-1 TCR transgenic mice have been described previously (23). Pmel-1 TCR transgenic and pmel-1 Thy1.1⁺ mice were bred and kept at the NIH animal facilities. C57BL/6, C57BL/6 CD45.1, CD4^-/-, CD8^-/-, RAG-1^-/-, IL-2^+/- and Thy1.1⁺ mice were obtained from Jackson laboratories (Bar Harbor, ME) and bred at the NIH animal facility. IL-2^-/- mice were obtained by crossing IL-2^+/- mice together and verified by PCR (Jackson Laboratories). B16.F10 (H-2^b), hereafter called B16, is a gp100⁺ spontaneous murine melanoma obtained from the National Cancer Institute tumor repository and was maintained in culture media (CM) as previously described (23, 62).

Peptides and recombinant fowlpox and vaccinia viruses. All synthetic peptides were synthesized using regular F-MOC chemistry. The synthetic H-2D^b-restricted peptide, hgp100_25-33, KVPRNQDWL, was synthesized by Peptide Technologies (Washington, D.C.) to a purity > 99% by HPLC and amino acid analysis. All recombinant viruses encoding hgp100 have been described before and were kindly provided by Therion Biologics (Cambridge, MA) (23).

In vitro activation of pmel-1 T cells and cytokine release assay. Splenocytes from mice were depleted of erythrocytes by hypotonic lysis, cultured in CM with 30 IU/ml rhIL-2 in the presence of 1 μM hgp100_25-33 peptide and used on day 6 after start of the culture. For cytokine release assays, sorted Thy1.1⁺ pmel-1 T cells (5.0 × 10⁴) were cocultured in CM with 10⁵ irradiated (3000 rads) splenocytes pulsed with 1μM hgp100_25-33 peptide. Supernatants were collected after 24 hrs and tested using a mIFN-γ ELISA kit (Endogen, Cambridge, MA) according to manufacturer's protocol.

Flow cytometry and CFSE staining. From fresh splenocytes, erythrocytes were removed by hypotonic lysis and cells were stained with the indicated mAbs: CD8α-Allophycocyanin (53-6.7), CD4-Allophycocyanin (H129.19), Vb13-FITC (MR12-3), CD25-PE (PC61), Thy1.1-PE (OX-7). For IFN-γ intracellular staining, splenocytes were activated with lymphocyte activating cocktail (BD Biosciences) for 6 hours, and then fixed and stained using cytofix and cytoperm intracellularing staining protocol (BD Biosciencs). All antibodies were purchased from BD Biosciences. Propidium iodide staining cells were excluded from analysis. Samples were analyzed using a FACScalibur™ flow cytometer and CELLquest™ software. For CFSE staining, pmel-1 T cells were activated and cultured for 1 week before being sorted on CD8⁺ enrichment columns (R&D systems) and labeled with 2uM CFSE dye (CFDA SE Cell Tracer Kit; Molecular Probes). Pmel-1 T cells were prepared by resuspending in prewarmed PBS (37°C) containing the appropriate concentration of CFSE dye for 15 minutes. Cells were then washed and incubated with fresh PBS for an additional 30 minutes to allow complete modification of the probe before adoptive cell transfer. 4 days later, pmel-1 T cells were isolated from mouse splenocytes and analyzed by FACS.

Purification of CD4⁺ T cell subsets and pmel-1 T cells. Unfractionated CD4⁺ T cells were purified from single cell suspensions of IL-2^-/-, C57BL/6, or C57BL/6 CD45.1 spleens, using a CD4⁺ enrichment column (R&D Systems). T_reg cells were subsequently purified using MACS CD4⁺CD25⁺ Isolation Kits (Miltenyi Biotech) to a purity >95%. T_helper cells were purified on a LS⁺ selection column twice (Miltenyi Biotech) to obtain >98% depletion of CD4⁺CD25⁺ T cells. Cells were either cultured overnight in CM or transferred immediately. Cells were also used for suppression assays to confirm their function. Pmel-1 Thy1.1⁺ T cells were purified from splenocytes of RAG-1^-/- mice by labeling with Thy1.1-PE (15 μg/ 1.0 × 10⁸ cells/mL) for 10 minutes. Cells were subsequently washed and sorted from whole splenocytes with anti-PE microbeads using LS⁺ selection columns (Miltenyi Biotech).

In vitro suppression assays. CD4⁺CD25⁺ and CD4⁺CD25^- T cells were isolated from peripheral lymph nodes of C57BL/6 mice by FACS sorting as previously described (28). Subsequently, they were activated with irradiated TΔS (T-depleted splenocytes; 1:1 ratio), soluble anti-CD3 (0.5 μg/mL), and human IL-2 (5 ng/mL, 100 U/mL) for 72 h and then were split and maintained in IL-2 medium for 7-14 days (28). Pmel-1 transgenics were activated with 1 μM of hgp100_25-33 peptide, which was pulsed onto γ-irradiated (3000 rads) TΔS for 30 minutes and washed twice before co-culture. In vitro suppression assays were performed by stimulating pmel-1 CD8⁺ cells (5.0 × 10⁴) alone or in the presence of titrated numbers of either freshly isolated or activated CD4⁺CD25⁺ or CD4⁺CD25^- T cells. Cultures were stimulated with either soluble anti-CD3 (0.5ug/ml) or in the presence of peptide-pulsed TΔS for 72hrs, as previously described (28). Supernatants were taken on day 3 of co-culture for IFN-γ release and 1 μCi [³H]-TdR was added for the last 8 hours. All data represent the average cpm of triplicate determinations. IFN-γ was measured using an ELISA kit (R&D systems).

Adoptive cell transfer. Mice were injected subcutaneously with 1.0-5.0 × 10⁵ B16 melanoma cells as depicted. The standard treatment regimen consisted of the intravenous administration of 1.0 × 10⁶ pmel-1 T cells activated for 1 week in vitro with 1 μM hgp100 peptide and subsequently purified using CD8⁺ enrichment kits (R&D Systems) to a purity > 98%. CD4⁺CD25⁺ (1.0 × 10⁵), CD4⁺CD25^- (1.0 × 10⁶), unfractionated CD4⁺ T cells (1.0 ×10⁶), IL-2^-/- CD4⁺CD25^- T cells (1.0 × 10⁶), or a mixture of T_reg to T_helper (1:10) were co-injected with pmel-1 T cells as indicated. One day before adoptive cell transfer of T cells, C57BL/6 mice underwent sub-lethal whole-body irradiation (500 cGy) (24). Mice were vaccinated by intravenous injection with 2.0 × 10⁷ PFU of a recombinant fowlpox virus encoding human gp100 (rFPVhgp100) on the same day of transfer. IL-2 (Chiron) was administered for 4 days directly following vaccination by daily intraperitoneal injections of 600,000 IU rhIL-2 in PBS. Tumors were measured in a blinded fashion using calipers and the products of perpendicular diameters were recorded.

Masked uveitis score. Eyes were enucleated from mice and placed in 4% gluteraldehyde for 30 minutes. Subsequently, eyes were transferred in 10% formalin for 48 hours, and then embedded in methylarcylate. Four to five micron sections were taken along pupillary-optical axis. Sections were evaluated by a masked ophthalmic pathologist using the score as follows: minimal = 0.5, mild = 1, moderate = 2, severe = 3. Scores where given for iridiocyclitis, choroiditis, vitritis, and retinal involvement. The grading was then combined for a final masked uveitis score.

Statistics. Tumor graphs were compared using Wilcoxon rank sum test. Factorial ANOVA was used to compare autoimmunity in the eye. The t-test for means was used to analyze IFN-γ ELISA results.

Results

Naturally occurring CD4⁺ T cells prevent immunotherapy to established tumors.

We have previously demonstrated that adoptive cell therapy using either 10⁷ naïve or activated pmel-1 T cells, recombinant fowl-pox virus encoding human gp100 (rFPVhgp100) vaccination, and exogenous IL-2 could effectively cure established B16 melanoma in wild-type (WT) syngeneic mice(23). This therapy was not dependent on host T- or B-lymphocytes for its effectiveness. However, because the transfer of a large precursor frequency of CD8⁺ transgenic T cells can be independent of the effects of CD4⁺ T cells(25), we evaluated whether host lymphocytes could have a positive or negative effect by transferring a smaller dose of pmel-1 T cells (1.0 × 10⁶/mouse).

To evaluate the impact of host lymphocytes on a CD8⁺ T cell-mediated adoptive cell therapy, we tested the relative efficacy of treatment in mice with either a selected loss of lymphocyte subsets through genetic knockouts or through whole body irradiation. Recombination-activating gene-1 deficient mice (RAG)-1^-/-, CD4^-/- mice, CD8^-/-, or C57BL/6 mice were inoculated with the highly aggressive, poorly immunogenic B16 melanoma and treated intravenously 14 d later with the tripartite treatment regimen comprised of activated pmel-1 T cells (CD25⁺, CD44^high, CD62L^low, CD69^high), rFPVhgp100, and IL-2(23). Treatment of B16 melanoma in mice either sub-lethally irradiated (500 cGy) or on a RAG-1^-/- background was markedly enhanced when compared to non-irradiated WT C57BL/6 mice (Fig. 1a). Importantly, B16 melanoma grew at the same rate in the no treatment controls indicating that the absence of lymphocytes did not alter the growth kinetics of the B16 tumor.

Open in a separate window

Figure 1

Naturally occurring CD4⁺ T cells inhibit effective immunotherapy to established tumors. (a-d) Mice were inoculated with 1.0 × 10⁵ cells of B16 melanoma on day -14 before adoptive cell transfer with 1.0 × 10⁶ pmel-1 T cells (P), 2.0 × 10⁷ PFU rFPVhgp100 (F), and 600,000 IU of exogenous IL-2 (I), which was given daily for 3-4 days. (a) Tumor regression in C57BL/6 mice (□) is compared with RAG-1^-/- mice (•) and C57BL/6 mice receiving 500 (cGy) whole body irradiation on day -1 of treatment (□). (b) Tumor regression in C57BL/6 mice (□) is compared with RAG-1^-/- mice (•) and CD4^-/- mice (□). (c) Tumor regression in C57BL/6 mice (□) is compared with RAG-1^-/- mice (•) and CD8^-/- mice () in the same experiment. Data are represented as mean tumor size ± S.E.M. Experiments were independently repeated twice. (d-e) Tumor regression is IL-2 dependent. (d) Transfer of pmel-1 T cells alone (Δ) or pmel-1 T cells and rFPVhgp100 vaccine (•) into tumor-bearing RAG-1^-/- hosts is similar to no treatment (). Addition of exogenous IL-2 with cells and vaccine is required for tumor regression (□). (e) CFSE profile of adoptively transferred pmel-1 T cells into RAG-1^-/- hosts. Pmel-1 CD8⁺ T cells were labeled with CFSE and adoptively transferred into tumor-bearing RAG-1^-/- hosts alone (P), with vaccination (PF), or with vaccination and exogenous IL-2 (PFI). 4 days later, splenocytes from treated mice were analyzed by flow cytometry. Gated on CD8⁺ T cells and displayed as Vβ13-PE vs. CFSE.

Next, we continued with a genetic dissection of the adaptive immune system by using selective knockout mice for different T cell subsets. In the same experiment, tumor regression was augmented in CD4^-/- mice, similar to that seen in RAG-1^-/- mice (Fig. 1b). Because both RAG-1^-/- and CD4^-/- do not develop CD4⁺ T cells, we employed our adoptive cell transfer regimen in CD8^-/- mice, whose immune system contains CD4⁺ T cells. As shown in figure 1c, there was no augmentation of tumor treatment in CD8^-/- mice when compared with WT C57BL/6 controls. Tumor regression in MHC class II^-/-, athymic nude, and severe combined immunodeficient (SCID) mice was also similar to RAG-1^-/- mice and CD4^-/- mice in the same experiment (data not shown). Therefore, the endogenous CD4⁺ T cell repertoire is capable of suppressing antitumor immunity to established tumors as demonstrated 20 years earlier(26).

T_reg cells suppress self-reactive CD8⁺ T cells in vitro.

Because T_reg cells can suppress CD4⁺ and CD8⁺ T cells(28), we evaluated whether T_reg cells could suppress transgenic pmel-1 CD8⁺ T cells. Therefore, naturally occurring CD4⁺ T cells were purified from lymph nodes of C57BL/6 mice and fractionated into T_reg and T_helper cells. Sorted T_reg and T_helper cell subsets were separately activated with anti-CD3 and IL-2 as previously described(28) and then cocultured with either anti-CD3 activated (0.5 μg/mL) or peptide stimulated (1 μM hgp100) pmel-1 T cells. From this assay, we observed a dose-dependent suppression of proliferation (Fig. 2a) and IFN-γ production (Fig. 2b) when pmel-1 T cells were mixed with T_reg cells. By contrast, coculture with CD4⁺CD25^- T_helper cells did not suppress proliferation or IFN-γ production. As reported in the literature, similar results were found when T_reg cells were cocultured with T_helper cells (data not shown and (28)). T_reg cells cultured alone proliferated minimally (data not shown). These results indicated that T_reg cells could profoundly suppress tumor/self-antigen-specific pmel-1 T cells and naturally occurring CD4⁺CD25^- T_helper cells in vitro.

Open in a separate window

Figure 2

CD4⁺CD25⁺ T_reg cells suppress pmel-1 CD8⁺ T cell proliferation and function in vitro. (a-b) Highly purified T_reg cells from C57BL/6 lymph nodes suppress (a) proliferation and (b) IFN-γ production of pmel-1 T cells in vitro. T_reg or T_helper cells were activated with irradiated APC (1:1 ratio) and soluble anti-CD3 antibody (0.5 μg/mL) for 3 days in the presence of 100 IU human IL-2. Cells were then split and maintained for an additional 7-14 days in 100 IU human IL-2. (a) Pmel-1 T cells were stimulated with soluble anti-CD3 or 1 μM hgp100_25-33 peptide-pulsed T-depleted spleen cells in the presence of activated T_reg (■) or T_helper (□) cells for 3 days in the absence of IL-2 at the indicated ratios. Maximum suppression of pmel-1 T cell proliferation is seen at a 1:1 suppressor to effector ratio. (b) Pmel-1 T cells were stimulated with anti-CD3 or 1 μM hgp100_25-33 peptide-pulsed TΔS cells in the presence of activated T_reg (■) or T_helper (□) cells for 3 days in the absence of IL-2 at the indicated ratios. Maximal suppression of IFN-γ production by pmel-1 T cells for both modes of stimulation is seen at a 1:1 suppressor to effector ratio. Experiments were independently repeated twice.

T_helper cells are required to maintain effector self-reactive CD8⁺ T cells in vivo

We observed that transfer of pmel-1 T cells with or without rFPVhgp100 vaccination into tumor-bearing RAG-1^-/- hosts could not induce regression of B16 melanoma, despite the fact that RAG-1^-/- hosts lack T_reg cells (Fig. 1d). As seen in figure 1d, the addition of exogenous IL-2 was necessary for full therapeutic effectiveness. Therefore, we hypothesized that exogenous IL-2, in this setting, was substituting for a T helper cell. Because absence of T cell help can hinder the in vivo maintenance of CD8⁺ T cells and development of memory T cells(4, 7, 29, 30), we surmised that effector CD8⁺ T cells also needed help to induce the regression of established tumors, in addition to removal of T_reg cells.

To test if the transfer of CD4⁺ T cells might replace the requirement for exogenous IL-2 and help pmel-1 T cells eradicate tumors, we transferred unfractionated CD4⁺ T cells and sorted CD4⁺CD25^- T cells with pmel-1 T cells and rFPVhgp100 vaccine into tumor-bearing RAG-1^-/- hosts. The combination of pmel-1 T cells, vaccine, and CD4⁺CD25^- T cells induced tumor regression and long-term survival without exogenous IL-2 (Fig. 3a and 3b), whereas no or minimal therapeutic effect was seen with unfractionated CD4⁺ T cells (Fig. 3a). Furthermore, adoptive transfer of CD4⁺CD25^- T cells alone or in combination with pmel-1 cells without vaccine did not induce durable and stable tumor regression, showing the requirement for vaccination in this treatment model (data not shown).

Open in a separate window

Figure 3

T_helper cells maintain self-reactive effector CD8⁺ T cells in vivo. (a-d) RAG-1^-/-mice were inoculated with 3.0 × 10⁵ cells of B16 melanoma between day -7 and -14 before adoptive cell transfer. (a) T_helper cells help CD8⁺ T cell mediated antitumor immunity to established B16 melanoma. Mice receiving 1.0 × 10⁶ pmel-1 T cells (P), 2.0 × 10⁷ PFU fowlpox virus encoding human gp100 (F), and 1.0 × 10⁶ T_helper cells maintained long-term, durable regression of established B16 melanoma (□). Data are represented as mean tumor size ± S.E.M. Data represent 6 independent experiments. (b) Survival of mice in (a) treated with pmel-1 T cells (P), rFPVhgp100 vaccine (F), and T_helper cells was maintained up to 80 days post treatment (□). (c) Adoptive cell transfer of sorted 1.0 × 10⁵ T_reg cells (•) or a mixture of 1.0 × 10⁵ T_reg and 1.0 × 10⁶ T_helper cells (1:10 ratio; CD4⁺ T cells; □) with pmel-1 T cells and vaccination does not maintain tumor regression of B16 melanoma. Removal of the T_reg subset from the unfractionated CD4⁺ T cell pool allows the helper function of the remaining T_helper cells to become apparent (□). Data represent 5 independent experiments. Experiment in (c) was stopped at 35 days post treatment to allow for analysis of adoptively transferred pmel-1 T cells. (d) T_helper cells maintain maximal pmel-1 T cell numbers (solid black bars) and function (cross-hatched bars) in the absence of IL-2. †, P <0.037. Proliferation bar graphs represent fold increase in the absolute number of pmel-1 T cells (% CD8⁺ Thy1.1⁺ Vβ13⁺ T cells × splenocyte count) taken from pooled spleens 5 wks after transfer (n=2). Fold increase is defined as: (absolute #pmel-T cells in a group divided by absolute #pmel-1 T cells from the group which received no CD4⁺ T cells (PF)). For all functional assays, pmel-1 Thy1.1⁺ CD8⁺ T cells were purified from spleens of treated mice 5 wks after transfer and stimulated with 1μM hgp100_25-33 peptide-pulsed γ-irradiated spleen cells for 24 hours (n = 2). All groups were also tested against non-peptide-pulsed targets, which resulted in no production of IFN-g (data not shown). Data are represented as IFN-γ (pg/mL) ± S.E.M. Experiments were repeated twice. The symbol, *, represents an undetectable value.

Although in figure 3a there was no tumor regression observed when unfractionated CD4⁺ T cells were used, we found in repeated experiments that the transfer of unfractionated CD4⁺ T cells had a variable effect on tumor regression; ranging from negligible (Fig. 3a) to a modest suppression of tumor growth (Fig. 3c). We found in sorted CD4⁺ T cell preparations that the CD4⁺CD25⁺ T cell population varied from 2-13% (data not shown). Therefore, we hypothesized that the variability of the antitumor responses was due to the relative percentages of regulatory and helper T cell subsets. To solve the variability between these subsets, we prepared T_reg and T_helper cells from a common pool of CD4⁺ T cells or from different congenic strains (CD4⁺CD25⁺CD45.1⁺ or CD4⁺CD25^-CD45.2⁺ T cells) and fixed the ratio at 1:10 (T_reg: T_helper) for subsequent experiments, which is the accepted physiological ratio in vivo(13). This ratio was verified in vivo by flow cytometry (data not shown).

Adoptive transfer of a mixture of T_reg and T_helper cells at a 1:10 ratio resulted in tumor growth that was similar to transfer of unfractionated CD4⁺ T cells that had a comparable ratio (Fig. 3c and data not shown). As seen in figure 3a, the transfer of sorted T_helper cells (1.0 × 10⁶) with pmel-1 T cells and rFPVhgp100 vaccine resulted in durable and stable tumor regression (Fig. 3c). However, transfer of sorted T_reg cells (1.0 × 10⁵) with pmel-1 T cells and vaccine resulted in minimal tumor regression that was similar to pmel-1 T cells and vaccination alone (Fig. 3c).

To understand the differences between the treatment groups, we analyzed the impact of these CD4⁺ T cell subpopulations on the persistence and function of adoptively transferred pmel-1 T cells. By using a congenic marker system (Thy1.1⁺/Thy1.2⁺), we were able to measure the persistence of Thy1.1⁺ pmel-1 T cells 5 wks after vaccination. We found that the absolute numbers of pmel-1 T cells was enhanced by cotransfer with T_helper cells, but not with a mixture of T_reg: T_helper (1:10 ratio) (Fig. 3d). Function of sorted Thy1.1⁺ pmel-1 T cells as measured by IFN-γ enzyme-linked immunosorbent assay (ELISA) after peptide stimulation (1 μM hgp100) ex vivo was significantly enhanced with the addition of T_helper cells (Fig. 3d; †; P = 0.037).

Next, pmel-1 T cells were taken from mice treated with T_reg cells and analyzed ex vivo for T cell numbers and IFN-γ secretion. We found that pmel-1 T cell numbers were decreased approximately 4-fold when compared with T_helper cells and function was similar to the transfer of no CD4⁺ T cells as measured by IFN-γ ELISA (Fig. 3d). Suppression could only be observed when T_reg cells were cotransferred with the T_helper population, which contained approximately 90% CD4⁺CD25^- T cells (Fig. 3d; †; P = 0.037). Thus, T_reg cells suppressed adoptive immunotherapy of an established tumor, but T_helper cells improved tumor immunotherapy by maintaining the functionality of tumor/self-reactive CD8⁺ T cells.

T_helper cells enhance exogenous IL-2 therapy

In the current report, we noticed that lower dosages of pmel-1 T cells (10⁶/mouse) could induce stable tumor regression when given in combination with vaccination and T_helper cells but not with exogenous IL-2 (Fig. 4a). IL-2 treatment induced transient tumor regression that lasted for approximately 20 d after adoptive transfer. However, in the presence of T_helper cells, IL-2 therapy was enhanced and induced stable tumor regression beyond 20 d (Fig. 4a).

Open in a separate window

Figure 4

T_helper cells can replace exogenous IL-2 and maintain function of tumor-reactive CD8⁺ T cells but exogenous IL-2 therapy fails in the presence of T_reg cells. (a) The combination of 1.0 × 10⁶ pmel-1 T cells, 2.0 × 10⁷ PFU rFPVhgp100 vaccination, and exogenous IL-2 (600,000 IU) given daily for 3 days in RAG-1^-/- hosts enhances but does not maintain tumor regression (□). Only cotransfer of 1.0 × 10⁶ T_helper cells with pmel-1 T cells and vaccination into RAG-1^-/- hosts helped maintain tumor regression with (Δ) or without (□) exogenous IL-2. Data represent 3 independent experiments. (b) Exogenous IL-2 does not maintain function (crosshatched bars) of pmel-1 T cells unless given in combination with T_helper cells (PFI CD4⁺CD25^- vs PFI, ‡, P = 0.001). Pmel-1 T cell absolute numbers were increased approximately 2-fold in the presence of T_helper cells (solid black bars). Fold increase is defined as: (absolute #pmel-T cells in a group divided by absolute #pmel-1 T cells from the group which received no CD4⁺ T cells (PF)). The symbol, *, represents an undetectable value. (c) Function of adoptively transferred pmel-1 T cells 2 weeks (solid black bars) and 4 weeks (crosshatched bars) after transfer. Function (IFN-g (pg/mL)) declines with time unless T_helper cells are also transferred: PFI vs. PF CD25^- or PFI CD25^- (d) Activated pmel-1 CD8⁺ T cells (CD25⁺, CD44^high, CD62L^low, CD69^high; 1.0 × 10⁶) were transferred with 1.0 × 10⁵ sorted T_reg cells, rFPVhgp100 vaccination (2.0 × 10⁷ pfu), and exogenous IL-2 on day 7 after tumor inoculation. CD8⁺ T cells required vaccine and IL-2 for tumor treatment (□). T_reg cells inhibited tumor treatment by effector CD8⁺ T cells in the presence of exogenous IL-2 (Ο) and treatment was similar to groups receiving no exogenous IL-2 (■). Experiments repeated independently three times.

Next, to understand how IL-2 and T_helper cells affected the adoptive immunotherapy differently, we analyzed the function of pmel-1 CD8⁺ T cells co-transferred with vaccination, exogenous IL-2 and/or T_helper cells ex vivo beyond 20 days after transfer (week 5). We found that the co-transfer of T_helper cells resulted in a significant increase in pmel-1 T cell function that was maintained in vivo, as measured by IFN-γ ELISA (PFI CD25^-; ‡, P < 0.001; Fig. 4b). Surprisingly, the provision of pmel-1 T cells, vaccine, and exogenous IL-2 in the absence of T_helper cells was insufficient to maintain long-term CD8⁺ T cell function (5 wks after transfer) (PFI; Fig. 4b). A more detailed analysis revealed that T cell function goes down with time in the absence of T_helper cells (Fig. 4c). Thus, adoptive cell transfer of T_helper cells could replace and/or enhance exogenous IL-2 therapy of established tumors by maintaining the effector function and numbers of adoptively transferred CD8⁺ T cells.

T cell help is IL-2 dependent, not programmed, and lost in the presence of T_reg cells

We showed earlier that treatment was either IL-2 dependent or required T_helper cells to be effective. Therefore we hypothesized that tumor regression observed following adoptive transfer of T_helper cells was the result of IL-2 production by the T_helper cell population. To be able to test this hypothesis we derived T helper cells from IL-2^-/- mice. However, because precursor T_reg cells may be resident in IL-2^-/- mice(31) (Fig. 5a), we transferred sorted T_helper cells from IL-2^-/- mice together with pmel-1 T cells and vaccine into tumor-bearing RAG-1^-/- hosts. In more than 5 independent experiments, we did not observe stable tumor regression using T_helper cells derived from IL-2^-/- mice, whereas sorted T_helper cells from IL-2^+/+ mice effectively enhanced tumor regression (Fig. 5b).

Open in a separate window

Figure 5

T cell help is IL-2 dependent and lost in the presence of T_reg cells. (a) Flow cytometry analysis of mouse splenocytes shows that IL-2^-/- mice do not develop T_reg cells (n=3). (b-c) RAG-1^-/- mice were inoculated with 3.0 × 10⁵ cells of B16 melanoma between day -7 before adoptive cell transfer with 1.0 × 10⁶ pmel-1 T cells (P), 2.0 × 10⁷PFU rFPVhgp100 (F), plus T_helper cells from IL-2^-/- mice or naturally occurring T_helper cells plus/minus exogenous IL-2 (I). (b) Transfer of T_helper cells from IL-2^-/- mice with pmel-1 T cells and vaccination into tumor-bearing RAG-1^-/- hosts does not help treatment of established B16 melanoma (). (c) Addition of exogenous IL-2 does not restore the helper function of T_helper cells from IL-2^-/- mice (■). *, P = 0.021. Data are derived from a single experiment that was independently repeated 3 times. (d) T_helper cells do not program tumor-reactive CD8⁺ T cells. Depletion of T_helper cells 4 days after transfer with 500 mg of GK1.5 CD4 depleting mAb (Δ). Data represents 3 independent experiments with similar results. Isotype control antibody had no effect on CD4⁺ T cells and depletion of CD4⁺ T cells was confirmed by flow cytometry. (e) T_helper cells utilize IL-2 in vivo. CD25 expression on adoptively transferred T_helper cells alone, T_helper cells with T_reg(CD4⁺unfractionated), and T_helper cells derived from IL-2^-/- mice, 35 d after treatment. (f) Spleens were taken from tumor-bearing RAG-1^-/- mice and analyzed by flow cytometry for the congenic marker Thy1.1 and CD8, which represents the transferred pmel-1 T cells 3 weeks after treatment with the indicated regimen. Two mice were used per group. Data is indicative of 3 independent experiments. (g) Absolute number of pmel-1 T cells 3 weeks after transfer from the same experiment in (f). (h) Intracellular IFN-γ 3 wks after adoptive cell therapy. Cells were activated with lymphocyte activating kit and analyzed by flow cytometry 6 hours later. Two mice were analyzed per group. Data is indicative of 3 independent experiments.

In an attempt to understand the kinetics of IL-2 dependency in this system, we gave exogenous IL-2 for 4 days after treatment together with T_helper cells derived from IL-2^-/- mice. Initially, we observed tumor regression, but this regression was not maintained (Fig. 5c; * P = 0.021).

To assess whether T_helper cells could program CD8⁺ T cells to treat established tumors, we transferred T_helper cells for 4 days and then depleted with injection of 500 μg of CD4⁺ T cell depleting mAb (GK1.5; Fig. 5d), which was confirmed by flow cytometry (data not shown). Tumor treatment in mice receiving depleting mAb was similar to mice that had received no T_helper cells or exogenous IL-2 (Fig. 5d) or T_helper cells derived from IL-2^-/- mice (Fig. 5b). Isotype control antibody had no effect on adoptively transferred T_helper cells in vivo (data not shown).

Next, the expression of CD25 on transferred CD4⁺ T cells was determined. IL-2 upregulates it's own receptor expression(32). Therefore, to determine whether the major source of IL-2 was from the transferred cells or from the host, CD4⁺ T cells were analyzed by flow cytometry 35 days after transfer into tumor-bearing RAG-1^-/- hosts. CD25 expression was between 5-10% as expected for whole CD4⁺ T cells (Fig. 5e). Sorted T_helper cells had 0.23% CD25 expression and T_helper cells from IL-2^-/- mice had 0.8% CD25 expression (Fig. 5e). After 35 days in vivo, whole CD4⁺ T cells and sorted T_helper cells upregulated their receptor (Fig. 5e). T_helper cells from IL-2^-/- mice also upregulated their receptor, but at a much lower level (Fig. 5e). Thus, even though whole CD4⁺ cells, which contain T_reg cells, upregulated their CD25 expression to comparable levels as T_helper cells; tumor regression was minimal. Together, these results indicated that IL-2 mainly comes from transferred activated T_helper cells.

Next we looked at the persistence of tumor-reactive CD8⁺ T cells after adoptive transfer with different CD4⁺ T cell subsets by flow cytometry 3 wks after treatment. As shown in Figure 5f, Thy1.1⁺ pmel-1 T cells required the presence of T_helper cells in order to persist. A 10-fold reduction in Thy1.1⁺ pmel-1 T cell frequency was seen in groups that received T_reg cells and T_helper cells at a 1:10 ratio when compared to groups receiving pmel-1 T cells and T_helper cells alone. The same reduction in Thy1.1⁺ pmel-1 T cell frequency was seen in groups that received T_helper cells from IL-2^-/- mice or no T_helper cells. Persistence of Thy1.1⁺ pmel-1 T cells was even more depressed in groups receiving only T_reg cells, approximately a 30-fold reduction when compared to the T_helper cell group. As a comparison, absolute number of pmel-1 CD8⁺ T cells was also calculated for the same experiment (Fig. 5g). Function, as measured by intracellular IFN-γ, of adoptively transferred pmel-1 T cells with T_helper cells was also assessed and shown to be suppressed when T_reg cells were cotransferred at 1:10 ratio, or when T_helper cells were derived from IL-2^-/- mice (Fig. 5h). Thus, these results highlight that IL-2 from T_helper cells was essential for the induction of antitumor immunity to a self-antigen, and this effect was lost in the presence of T_reg cells.

Breakdown of tolerance to the gp100 self-antigen is IL-2 dependent.

We notice mice treated with pmel-1 T cells, vaccine, and IL-2 or T_helper cells developed profound autoimmune vitiligo following 5 weeks after adoptive cell transfer. This vitiligo usually started periorbitally and spread in a random fashion as shown in figure 6a (n = 26). Conversely, limited or no autoimmune vitiligo was seen in mice that didn't receive exogenous IL-2 (n = 25) or received T_helper cells derived from IL-2^-/- mice (n = 25).

Open in a separate window

Figure 6

Breakdown of self-tolerance to the gp100 antigen requires IL-2. (a) Mice receiving pmel-1 T cells, rFPVhgp100 vaccination, and T_helper cells develop autoimmune vitiligo that spreads in an unpredictable fashion 5 wks after adoptive cell transfer. Mice receiving pmel-1 T cells, rFPVhgp100 vaccination, and exogenous IL-2 also develop vitiligo (as shown in (23)). Two representative mice receiving pmel-1 T cells, rFPVhgp100, and T_helper cells are shown (n = 25). (b) Uveitis during immunotherapy of B16 melanoma is only present with the administration of pmel-1 T cells, rFPVhgp100 vaccine, and exogenous IL-2, *, P < 0.05, or with cotransfer of T_helper cells without exogenous IL-2, §, P < 0.05. Uveitis is not observed in mice receiving pmel-1 T cells, rFPVhgp100 vaccine, and T_helper cells derived from IL-2^-/- mice. Uveitis in the eyes of treated mice was scored as follows: (0 = none, 1 = mild, 2 = moderate, 3 = severe). Data represent 2 independent experiments.

Since the eyes of C57BL/6 mice also express gp100 tumor/self-antigen(33), we evaluated the requirement for IL-2 production in the destruction of normal eye tissue. We looked for the induction of autoimmunity in the eye, as evidenced by uveitis. We found in repeated experiments that exogenous IL-2 caused significant uveitis (10 fold increase) when compare to no exogenous IL-2 treatment (PFI vs. PF; *, P < 0.05; Fig. 6b and manuscript in preparation; Palmer, D. et al.). We also found that the addition of T_helper cells induced uveitis that was similar to groups receiving IL-2 (PF CD25^- IL-2^+/+ vs PFI; **, P > 0.05; Fig. 6b). Importantly, as seen with autoimmune vitiligo, no uveitis was observed when T_helper cells were derived from IL-2^-/- mice (PF CD25^- IL-2^-/-; Fig. 6b; §, P < 0.05). Together, these results indicated that naturally occurring T_helper cells facilitated the induction of tumor regression and autoimmunity against a tumor/self-antigen through an IL-2-dependent mechanism.

Discussion

A fundamental question unanswered in immunology is how to raise T cell help against a persisting self-antigen, which subsequently results in the breakdown of self-tolerance(2). We describe here the requirements for the initiation of autoimmunity and thus the induction of anti-tumor immunity to established tumors expressing the gp100 melanocyte differentiation antigen, an antigen also expressed in the skin and eyes of C57BL/6 mice(33).

Recently depletion of T_reg cells has been shown to augment reactivity to tumor/self-antigens in tumor prevention models(19, 20, 34, 35), but we show for the first time that T_reg cells can inhibit help of self-reactive CD4⁺ T cells and prevent effector CD8⁺ T cells from initiating autoimmunity. T_reg cells control peripheral self-tolerance through yet unknown mechanisms, but we believe that progressively growing tumors shed or secrete self-antigens that subsequently activate naturally occurring T_reg cells(9-11, 36). Although depletion of T_reg cells enhances tumor protection in many models using artificial self-antigens, we show that complete absence of T_reg cells is not enough for treatment of established tumors against a self-antigen when using adoptive immunotherapy(37). Even in the absence of T_reg cells, treatment still required CD8⁺ T cells, vaccination, and some type of help either provided through exogenous cytokines or through T_helper cells. Therefore we suspect as with acute infections(6) that ongoing tumor regression will need continuous T cell help to eradicate established tumors, in addition to removing T_reg cells, because T_helper cells were unable to program self-reactive CD8⁺ T cells.

Since self-antigens can activate T regulatory cells(38), their role may have been over looked in artificial systems modeling self-antigens(36). The modeling of self-reactivity is likely to be important in the development of new immunotherapies that target tumor/self-antigens. Many of the currently available tumor models show the complete destruction of established tumors targeting ″foreign″ antigens and represent tumors given for only a short period of time(8, 39-41). These models have shed valuable light on basic immunologic principles, but it is unclear to what extent the results obtained from these models reflects immune responses against true self/tumor antigens(42). The present study was designed to elucidate the requirements for raising help to a tumor-antigen that was also expressed in normal tissues, a situation that models the clinical scenario in patients with cancer.

We found that CD8⁺ T cell-mediated immunotherapy and vaccination was ineffective in CD4⁺ T cell-deficient hosts unless given in combination with IL-2, and this effect was dramatically diminished in the presence of T_reg cells. Additionally, we showed that T_helper cells were superior to exogenous IL-2 therapy but that cotransfer of T_reg cells also inhibited this effect. Importantly, T_helper cells derived from IL-2^-/- mice contributed to neither antitumor immunity nor autoimmunity, even in mice lacking T_reg cells. All together these findings show the importance of T_helper cell derived IL-2 in the help of CD8⁺ T cells in vivo.

Next we showed that adoptive cell therapy with T_helper cells or exogenous IL-2 failed in the presence of T_reg cells. Since T_reg cells constitutively express the high affinity IL-2R, it is plausible that T_reg cells may be preferentially using IL-2 as shown in vitro(43), as they require IL-2 for their function and maintenance in vivo(44). It is also feasible that T_reg cells suppress CD8⁺ T cells or CD4⁺ T cells by decreasing their access to IL-2 either by suppressing the production of IL-2 by T_helper cells(45, 46), or decreasing the surface expression of the IL-2 receptor(28). Alternatively, T_reg cells may condition the antigen presenting cell towards tolerance(47). Thus, the durable induction of an antitumor (anti-self) response by T_helper cells may be dependent on their continuous production of IL-2, which is lost in the presence of T_reg cells constitutively expressing high affinity IL-2R. Whether T_helper cells that become CD25⁺ T cells are bona fide Foxp3 expressing T_reg cells in unknown in this model, but a recent paper suggests that this may be the case during expansion in a lymphopenic environment(48). Regardless, we still see maintenance of tumor regression and therefore a converted T_helper to an induced T_reg cell may not play a role during treatment of established tumors.

It has been argued that T_helper cells from IL-2^-/- mice have a secondary deficiency that diminishes their helper effect. However, it has been reported in the literature that T cell ontogeny and function in IL-2^-/- mice is not affected(49, 50). Furthermore, mixed bone marrow chimeras of IL-2^-/- and CD25^-/- cells used to reconstitute lethally irradiated hosts resulted in normal T cell homeostasis and engraftment of a stable CD4⁺CD25⁺ T_regpopulation(51). In addition, transfer of T_reg cells into CD25^-/- mice, which have the same phenotype as IL-2^-/- mice, lead to recoverable levels of T_reg cells and suppression of autoimmune disease since CD25^-/- mice still have a cellular source of IL-2(21). However, transfer of T_reg cells into IL-2^-/- mice did not prevent autoimmune disease(44). Taken together, these findings show that the main deficiency in IL-2^-/- mice is the complete absence of functional T_reg cells and not an intrinsic functional T cell defect(44). Lack of T_reg cells, due to absence of IL-2 signaling, leads to uncontrolled CD4⁺ T cell proliferation and activation, which paradoxically is not dependent on IL-2 (15, 21, 31, 51)

However, even though IL-2^-/- mice get autoimmune disease, adoptive transfer of IL-2 deficient T_helper cells were unable to help CD8⁺ T cells treat an established tumor or cause autoimmunity in IL-2^+/+ mice. Autoimmunity in our model is dependent on IL-2 production whereas in IL-2^-/- mice it is independent of IL-2(15). This is an important finding as it shows a disparity between how these two types of autoimmunity can manifest. Most importantly, it shows the risk of using self-antigens to immunize against self-antigen expressing tumors(52, 53).

These findings also point to the deficiencies in the use of high-dose exogenous IL-2 in cancer clinical trials(54). Already known for its toxicity, another danger inherent in the administration of exogenous IL-2 may be the induction of T_reg cell function(21, 44, 46, 55). Thus, depletion of T_reg cells with either ONTAK(56) or another method before adoptive cell transfer may enable unencumbered delivery of IL-2 by T_helper cells to tumor-reactive T cells or to T_helper cells themselves.

A key feature of this immunotherapy regimen is that T_helper cells are derived from wild-type mice, obviating the need for the development of T_helper cells with specificities for tumor-antigens a priori. The identities of the antigens recognized by T_helper cells in this setting remains of considerable interest because isolation of tumor-reactive T_helper cells can lead to more effective class-II-restricted vaccines(57). However, exactly what the requirements of T cell help are in vivo are still being debated(2), but we report here that IL-2 plays an important role in the breakdown of self-tolerance to a persisting antigen. Whether IL-2 is acting on CD8⁺ T cells or T_helper cells or both is unknown(2). It is possible that IL-2 secreted by T_helper cells leads to down-stream events that participate in T cell help, such as release of other cytokines or activation of costimulatory molecules, which license the APC to initiate help of CD8⁺ T cells. Thus, exogenous IL-2 therapy may lose these contributions by T_helper cells when used alone. However, whatever the mechanism of help in vivo, transfer of naturally occurring T_helper cells in combination with tumor-reactive CD8⁺ T cells plus vaccination represents a clinically feasible approach to the immunotherapy of established, progressing tumors in humans, since isolation of tumor-reactive CD4⁺ T cells has been difficult.

One currently used and approved immunotherapeutic approach in humans involves lymphodepletion prior to adoptive transfer(58). The immune-enhancing effects of lymphodepletion can be accomplished through irradiation, chemoablation(27, 58) or through genetic means as demonstrated here. The mechanisms of how lymphodepletion enhances adoptive immunotherapy remain incompletely understood (Klebanoff et al., Trends Immunology, in press), but our data suggests that the removal of T_reg cells is a major contributing factor. But as shown here, removing T_reg cells is not enough to treat established tumors, T cell help must be provided. The mechanisms of lymphodepletion are multifactorial because antitumor immunotherapies in CD4⁺ T cell-deficient mice can be further enhanced with total body γ-irradiation (data not shown). As has been shown in other systems(59), the increased availability of homeostatic γ_c-signaling cytokines such as IL-7, IL-15, or IL-21 could be enhancing T cell function in this model(24, 60, 61).

Nevertheless, we show here that naturally occurring T_helper cells can initiate autoimmunity and tumor regression in an environment of persisting self-antigen through self-reactive CD8⁺ T cells and that naturally occurring T_reg cells represent a formidable barrier to the breakdown of self-tolerance. Therefore, the future of immunotherapy against self-antigens will rely on ways of removing this population and augmenting T cell help of tumor-reactive T cells or tumor-infiltrating lymphocytes(54) isolated from patients. Together, these findings form a new approach for studying T cell help and suppression in vivo against self-antigens and form the basis of a new treatment for many types of cancers expressing self-antigens and chronic persisting infections.

Acknowledgements

Nonstandard abbreviations used:

References