Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets. Successful immunotherapy surprisingly resulted in an expansion of CD4+Foxp3+ regulatory T (Treg) cells concurrent with a reduction of conventional CD4+ T (Tconv) cells, despite the marked antitumor responses. Following immunotherapy, we observed differential up-regulation of PD-1 on the surface of CD4+Foxp3+ Treg cells and CD4+Foxp3 Tconv cells. Interestingly, it was the ligand for PD-1, B7-H1 (PDL-1), that correlated with Tconv cell loss after treatment. Furthermore, IFN-γ knockout (IFN-γ−/−) and IFN-γ receptor knockout (IFN-γR−/−) animals lost up-regulation of surface B7-H1 even though PD-1 expression of Tconv cells was not changed, and this correlated with CD4+ Tconv cell increases. These results suggest that subset-specific expansion may contribute to marked shifts in the composition of the T cell compartment, potentially influencing the effectiveness of some immunotherapeutic approaches that rely on IFN-γ.

Immunotherapeutic use of an agonist CD40 mAb in combination with IL-2 has been shown to have synergistic antitumor effects in mouse models of advanced renal cell carcinoma and lung carcinoma (1). More recently, we reported that treatment of mice after immunization combined with this and other immunotherapeutic regimens can lead to an IFN-γ-dependent loss of CD4+ T cells and subsequent inability to mount an effective memory response after a delayed live tumor challenge despite successful initial antitumor responses (2). Additionally, other investigators using a viral Ag-challenge model have shown similar effects after administration of anti-CD40 alone. Administration of anti-CD40 to lymphocytic choriomeningitis virus (LCMV)4-infected mice was associated with loss of virus-specific CD8+ T cells upon secondary challenge in vitro. Although a loss of CD4+ T cells was also observed, the dominant effector outcome was due to the loss of CD8+ T cells and was mediated by the Fas-FasL pathway (3). Most tumor models focus on CD8+ T cell effector pathways. However, in addition to helping generate tumor Ag-specific CD8+ T cell memory, recent studies suggest a more direct role for CD4+ T cells in some antitumor responses (4). Although we previously reported a loss in CD4+ T cell numbers after anti-CD40 and IL-2 immunotherapy despite increases in CD8+ T cells, the mechanism underlying this lack of CD4+ T cell expansion was not clear.

CD4+ T cells are a very diverse lymphocyte population with respect to the cytokines they can produce, and understanding their polarization toward stimulatory or inhibitory activity is important for understanding how they can affect treatment in a disease setting (5). Regulatory CD4+ T cells expressing the hallmark forkhead transcription factor 3 (Foxp3) are of particular interest with respect to cancer immunotherapy due to their potent immunosuppressive effects. It has therefore been suggested that their presence should be evaluated with all immunotherapeutic regimens because increases in regulatory T (Treg) cells can be counterproductive to the desired outcome (6). We therefore examined the effects of CD40-based immunotherapeutic regimens on CD4+ T cell subsets and key markers correlating with their expansion or loss. Our current observations presented herein report a differential expression pattern of the cell-surface marker programmed death-1 (PD-1, CD279) in response to anti-CD40 and IL-2 immunotherapy on the surface of conventional CD4+ T (Tconv) cells and Treg cells. PD-1 is found on most cells of hemopoietic origin, and its surface expression has been associated with programmed cell death of thymocytes after TCR ligation (7, 8). PD-1 up-regulation after T cell activation has been implicated as being important for the peripheral tolerance of CD8+ T cells to tissue Ags, as well as self Ags early in their development (9, 10, 11).

We observed markedly increased expression of PD-1 on the surface of CD4+ Tconv cells, but not on Treg cells, after treatment with anti-CD40 and IL-2. Additionally, B7-H1 was up-regulated in an IFN-γ-dependent fashion, consistent with previous reports (12), and we found this up-regulation of B7-H1 to correlate with the observed loss of CD4+ T cells. These findings caused us to look more closely at CD4+ T cell subsets in the context of immunotherapy-induced alterations of CD4+ T cell subsets and overall changes in the composition of the T cell compartment. The results reported herein led us to hypothesize that IFN-γ-dependent up-regulation of B7-H1 after immunotherapy is met with a differential expression of PD-1 on CD4+ Tconv cells vs Treg cells. From these results, we suggest that the differential expression pattern of the regulatory marker PD-1 following immunotherapy contributes to the loss of Tconv cells while simultaneously allowing Treg cells to expand. This may have ramifications in the length and extent of antitumor effects after immunotherapy.

Female C57BL/6 and BALB/c mice were purchased from the Animal Production Area of the National Cancer Institute (Frederick, MD). B6.129S7-Ifngtm1Ts (IFN-γ−/−) and B6.129S7-IfngR (IFN-γR−/−) as well as some aged-matched control wild-type C57BL/6 mice were purchased from The Jackson Laboratory. Mice were between 8 and 12 wk of age at the start of experiments, and they were housed in microisolator cages or, in the case of genetically engineered and aged matched control mice, on a Hepa-filtered vent rack. Under all settings, mice were housed under specific pathogen-free conditions. All experiments were in accordance with Institutional Animal Care and Use Committee guidelines.

Agonist rat anti-mouse CD40 (FGK115B3) was purified by ammonium sulfate precipitation from ascites. The endotoxin level of the anti-mouse CD40 Ab was <1 endotoxin unit/mg Ab as determined by quantitative chromogenic Limulus amebocyte lysate kit (Biowhittaker QCL-1000, Cambrex). Recombinant human IL-2 (TECIN (Teceleukin)) was provided by the National Cancer Institute. Recombinant human IL-15 was purchased from PeproTech. Purified rat IgG was purchased from Jackson ImmunoResearch Laboratories.

Spleen and lymph node cells were prepared by gentle dissociation and were filtered to remove excess debris followed by washing twice in Dulbecco’s PBS containing 5% FBS (HyClone) and 1% penicillin/streptomycin (Mediatech). Cell counts were determined by a lyse/no wash procedure a with known concentration of fluorescent beads or on a Coulter Z1 particle counter (Coulter Electronics). Blood was collected in tubes containing EDTA; RBC were lysed in blood samples with FACSLyse (BD Biosciences).

Cell suspensions from lymph node or spleen or whole blood were incubated with Abs labeled with FITC and R-PE, PE-cyanine 5, and/or PE-cyanine 7 and PE-Texas Red followed by wash and resuspension in PBS + 5% FBS (HyClone) + 1% penicillin/streptomycin (Mediatech). Intracellular Foxp3 labeling was completed using the Ready-SET-Go! Foxp3 labeling kit (eBiosciences), and all samples were resuspended in 1% formaldehyde (Sigma-Aldrich) in 1× Dulbecco’s PBS (Mediatech). Abs were purchased from either eBioscience or BD Biosciences. Listmode data files were collected on a three-color FACScan flow cytometer using CellQuest software (BD Biosciences), on a four-color Beckman Coulter XL/MCL using system II software, or on a five-color FC 500 MPL (Beckman Coulter). All data sets were analyzed using FlowJo software (TreeStar).

Agonist rat anti-mouse CD40 (FGK115B3) was administered i.p. at 65 μg (BALB/c and in experiments using IFN-γ−/−or IFN-γR−/−) or 80 μg per dose for 5 consecutive days. Recombinant human IL-2 (0.5–1.0 × 106 IU/dose) was administered i.p. four times per week in two sets of two injections, with the second injection in a set being 8–20 h from the previous one. In experiments where IL-15 was used in combination with anti-CD40, 2.5 μg of recombinant human IL-15 was administered i.p. twice daily in place of IL-2 injections.

Statistical analysis was performed using Prism software (GraphPad Software). Flow cytometry data were analyzed using Student’s t test; a Welch’s correction was applied to data sets with significant differences in variance. Survival data were analyzed using a log-rank test. A minimum of 3 mice/group was used in all experiments. Experiments using C57BL/6 mice were repeated at least three times. BALB/c experiments were performed once with 3 mice/group to support the observations made using C57BL/6 mice. Data were tested for normality and variance, and p < 0.05 was considered significant.

Evaluation of splenic CD4+ T cell percentages 11 days after the start of immunotherapy showed a marked lack of expansion of CD4+ T cells compared with CD8+ T cells (Fig. 1,A). Despite reported initial antitumor effects (1), this result was found to be due to cell death, as CD4+ T cells were shown to be entering into the cell cycle after immunotherapy even though no expansion was taking place. Similar to our previous observations in multiple immunotherapeutic models (2), evaluation of CD4+ and CD8+ T cells shortly after immunotherapy administration resulted in an alteration of the normal ratio between CD4+ and CD8+ T cells (Fig. 1,B). We next examined the effect of immunotherapy on the different CD4+ T cell subsets. Surprisingly, while CD4+ T cells did not expand as a whole population, the regulatory subset of CD4+ T cells defined by the expression of Foxp3 significantly (p < 0.05) expanded following administration of immunotherapy (Fig. 1,C). In addition to total cell number, Treg cell expansion concurrent with the lack of Tconv cell expansion resulted in Treg cells making up a larger percentage of the CD4+ T cell compartment (Fig. 1,D). Because IL-2, and not IL-15, is reported to be a strong promoter of Treg cells in vivo, anti-CD40 was combined with IL-15 to determine whether this combination would also result in a significant expansion of Treg cells. In addition to anti-CD40 and IL-2, anti-CD40 and IL-15 combined immunotherapy resulted in similar preferential expansion of Treg cells and not Tconv cells (Fig. 1, E and F). This appears to be due to a dominant effect of CD40, as IL-15 alone did not promote Treg cell expansion (data not shown). These results suggest that administration of immunotherapy results in an early loss of Tconv cells and simultaneous expansion of Treg cells, despite the occurrence of marked antitumor effects.

FIGURE 1.

Αnti-CD40 and IL-2 combination immunotherapy results in skewing of normal lymphocyte ratios. Spleens from C57BL/6 mice harvested on day 11 of treatment with anti-CD40 and IL-2 showed expansion of CD4+ Foxp3+ Treg cells. A, Animals that had received immunotherapy showed a significant (p < 0.05) increase in the number of splenic CD8+ T cells, but not in CD4+ T cells, as determined by flow cytometry. B, After treatment with anti-CD40 and IL-2, animals showed a significant (p < 0.0001) decrease in the ratio of CD4+ to CD8+ T cells in the spleen. C and D, Despite the lack of expansion of splenic CD4+ T cells after immunotherapy, we observed a significant (p < 0.05) increase in Treg cells in animals that had been treated with anti-CD40 and IL-2. This increase in Treg cells was determined to be in total CD4+Foxp3+ cell numbers (C) and as a percentage (D) of the total CD4+ T cell population. E and F, IL-15 was used in combination with anti-CD40 in place of IL-2, and Treg cells were analyzed by flow cytometry for expansion (E) in cell numbers and (F) as a percentage of all CD4+ T cells. Data in AD were repeated at least three times with similar results; the data in E and F were repeated two times. Analysis for all parts of Fig. 1 were analyzed using an unpaired Student’s t test; a Welch’s correction was applied for any set of data with significantly different variances.

FIGURE 1.

Αnti-CD40 and IL-2 combination immunotherapy results in skewing of normal lymphocyte ratios. Spleens from C57BL/6 mice harvested on day 11 of treatment with anti-CD40 and IL-2 showed expansion of CD4+ Foxp3+ Treg cells. A, Animals that had received immunotherapy showed a significant (p < 0.05) increase in the number of splenic CD8+ T cells, but not in CD4+ T cells, as determined by flow cytometry. B, After treatment with anti-CD40 and IL-2, animals showed a significant (p < 0.0001) decrease in the ratio of CD4+ to CD8+ T cells in the spleen. C and D, Despite the lack of expansion of splenic CD4+ T cells after immunotherapy, we observed a significant (p < 0.05) increase in Treg cells in animals that had been treated with anti-CD40 and IL-2. This increase in Treg cells was determined to be in total CD4+Foxp3+ cell numbers (C) and as a percentage (D) of the total CD4+ T cell population. E and F, IL-15 was used in combination with anti-CD40 in place of IL-2, and Treg cells were analyzed by flow cytometry for expansion (E) in cell numbers and (F) as a percentage of all CD4+ T cells. Data in AD were repeated at least three times with similar results; the data in E and F were repeated two times. Analysis for all parts of Fig. 1 were analyzed using an unpaired Student’s t test; a Welch’s correction was applied for any set of data with significantly different variances.

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PD-1/B7-H1 ligation has been shown to have inhibitory and even proapoptotic effects on CD8+ T cells (7, 13). However, the effect of immunotherapy on this pathway with regard to CD4+ T cells has not previously been investigated. Therefore, we assessed surface PD-1 expression on CD4+ Tconv cells and CD8+ T cells as well as CD4+ Treg cells by flow cytometry immediately following administration of an anti-CD40 and IL-2 regimen. Following immunotherapy, we observed a significant increase of the percentage of Tconv cells expressing PD-1 on the cell surface (p < 0.001), which was not observed in the Treg cell subset (p > 0.05). We also observed a significant increase in the percentage of CD8+ T cells that expressed surface PD-1 after treatment with anti-CD40 and IL-2 (Fig. 2, A and B); however, the fold increase in the percentage of CD8+ T cells expressing surface PD-1 was significantly (p < 0.01) lower than that for CD4+ Tconv cells (Fig. 2,C). The percentage of CD8+ T cells from control-treated animals did not significantly differ from naive animals (Fig. 2,D). Although we observed a higher baseline percentage of CD8+ T cells that were also PD-1+, CD8+ T cells had a consistently lower (p < 0.0001) level of receptor expression than did CD4+ T cells as determined by median fluorescence intensity (data not shown). Further studies performed in both C57BL/6 mice and BALB/c mice showed that administration of anti-CD40 and IL-2 induced similar expression patterns of PD-1 on the surface of CD4+ T cells in both strains (Fig. 2,E). In addition to PD-1, we also examined the expression of the PD-1 ligand, B7-H1. B7-H1 is widely expressed on most hemopoietic cell types (12), and therefore we evaluated its expression by flow cytometric analysis on all CD45+ splenocytes. Following anti-CD40 and IL-2 administration, we observed a significant (p < 0.0001) increase in the median fluorescence intensity of surface B7-H1 on CD45+ splenocytes (Fig. 3, A and B). B7-H1 expression was also significantly (p < 0.05) higher on the surface of the CD11c+ population of leukocytes (data not shown); however, the expression was not limited to myeloid or lymphoid cells, and we therefore evaluated surface B7-H1 expression on all hemopoietic (CD45+) cells. We did observe some variation in the baseline level of B7-H1 in our control-treated animals between experiments, but a comparison between naive and control-treated animals did not show an effect of the rat Ig and PBS treatment in the relative levels of B7-H1 on CD45+ cells (Fig. 3 C). These data show that anti-CD40 and IL-2 results in the up-regulation of B7-H1 on CD45+ cells while simultaneously increasing surface PD-1 on conventional CD4+ T cells but not on Treg cells. These changes correlate directly with the observed loss in CD4+ T cell numbers, suggesting that changes in the expression of B7-H1 and PD-1 may contribute to the decrease in conventional CD4+ T cells in the absence of similar effects on CD4+ Treg cells.

FIGURE 2.

Treg cells fail to up-regulate PD-1 as a result of anti-CD40 and IL-2 immunotherapy. Relative levels of PD-1 on the surface of Foxp3+CD4+ Treg cells were compared with conventional Foxp3CD4+ T cells 11 day after initiation of immunotherapy by flow cytometry. A, Representative histograms of PD-1 labeling show unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). B, The relative percentage of Treg cells expressing surface PD-1 was not significantly (p > 0.05) greater in animals that had received anti-CD40 and IL-2 immunotherapy. However, surface PD-1 expression was significantly (p < 0.01) higher on conventional CD4+ T cells and CD8+ T cells from animals that had received immunotherapy. C, The increase in the percentage of cells expressing surface PD-1 as a fold increase in animals treated with anti-CD40 and IL-2 over control treated animals. D, The percentage of CD8+ T cells expressing surface PD-1 is not significantly (p > 0.05) increased when animals are treated with the control therapy. E, Representative histograms show similar up-regulation of surface PD-1 on CD4+ T cells from C57BL/6 mice as well as BALB/c mice: unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). The data in A and B were repeated at least three times with similar results; the data in C were collected once. Student’s t test, with Welch’s correction for any data sets with significant differences in variance, was used in B; a one-way ANOVA was used in C and D.

FIGURE 2.

Treg cells fail to up-regulate PD-1 as a result of anti-CD40 and IL-2 immunotherapy. Relative levels of PD-1 on the surface of Foxp3+CD4+ Treg cells were compared with conventional Foxp3CD4+ T cells 11 day after initiation of immunotherapy by flow cytometry. A, Representative histograms of PD-1 labeling show unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). B, The relative percentage of Treg cells expressing surface PD-1 was not significantly (p > 0.05) greater in animals that had received anti-CD40 and IL-2 immunotherapy. However, surface PD-1 expression was significantly (p < 0.01) higher on conventional CD4+ T cells and CD8+ T cells from animals that had received immunotherapy. C, The increase in the percentage of cells expressing surface PD-1 as a fold increase in animals treated with anti-CD40 and IL-2 over control treated animals. D, The percentage of CD8+ T cells expressing surface PD-1 is not significantly (p > 0.05) increased when animals are treated with the control therapy. E, Representative histograms show similar up-regulation of surface PD-1 on CD4+ T cells from C57BL/6 mice as well as BALB/c mice: unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). The data in A and B were repeated at least three times with similar results; the data in C were collected once. Student’s t test, with Welch’s correction for any data sets with significant differences in variance, was used in B; a one-way ANOVA was used in C and D.

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FIGURE 3.

Anti-CD40 and IL-2 immunotherapy results in an increased surface expression of B7-H1 on all hemopoietic cells. Mice were treated with anti-CD40 and IL-2 according to the standard regimen listed in Materials and Methods. Eleven days after treatment initiation, spleens were harvested and analyzed by flow cytometry for surface B7-H1 expression. A, B7-H1 (PDL-1) expression was determined by median fluorescence intensity analysis and is significantly (p < 0.0001 by unpaired Student’s t test) higher on splenocytes from animals that received anti-CD40 and IL-2. B, Representative histograms show a similar pattern in both C57BL/6 mice as well as BALB/c mice: unlabeled control (gray line) and labeling with anti-B7-H1 Ab (clone MIH5) on CD45+ splenocytes from control-treated animals (dashed black line) and animals treated with anti-CD40 and IL-2 (solid black line). C, A comparison of the median fluorescence intensity of B7-H1 on CD45+ cells in control-treated animals vs anti-CD40 and IL-2-treated animals (a one-way ANOVA was used for statistical analysis). Each experiment was made up of three mice per group. The data shown in B and C were collected on two different flow cytometers (Beckman Coulter XL and FacScan, respectively), and the y-axes are representative of the mean fluorescence intensity as reported by the two instruments. Experiments shown in A were completed at least three times with similar results; data in B and C were completed once (n = 3 for each).

FIGURE 3.

Anti-CD40 and IL-2 immunotherapy results in an increased surface expression of B7-H1 on all hemopoietic cells. Mice were treated with anti-CD40 and IL-2 according to the standard regimen listed in Materials and Methods. Eleven days after treatment initiation, spleens were harvested and analyzed by flow cytometry for surface B7-H1 expression. A, B7-H1 (PDL-1) expression was determined by median fluorescence intensity analysis and is significantly (p < 0.0001 by unpaired Student’s t test) higher on splenocytes from animals that received anti-CD40 and IL-2. B, Representative histograms show a similar pattern in both C57BL/6 mice as well as BALB/c mice: unlabeled control (gray line) and labeling with anti-B7-H1 Ab (clone MIH5) on CD45+ splenocytes from control-treated animals (dashed black line) and animals treated with anti-CD40 and IL-2 (solid black line). C, A comparison of the median fluorescence intensity of B7-H1 on CD45+ cells in control-treated animals vs anti-CD40 and IL-2-treated animals (a one-way ANOVA was used for statistical analysis). Each experiment was made up of three mice per group. The data shown in B and C were collected on two different flow cytometers (Beckman Coulter XL and FacScan, respectively), and the y-axes are representative of the mean fluorescence intensity as reported by the two instruments. Experiments shown in A were completed at least three times with similar results; data in B and C were completed once (n = 3 for each).

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Previous data from our laboratory indicated that the selective loss of CD4+ T cells following anti-CD40 and IL- 2 was dependent on IFN-γ (2). Therefore, we evaluated the relative levels of PD-1 on the surface of CD4+ T cells from wild-type mice vs mice lacking either IFN-γ (IFN-γ−/−) or the IFN-γ receptor (IFN-γR−/−). After treatment with anti-CD40 and IL-2, we found surface expression of PD-1 on CD4+ T cells was still significantly (p < 0.001) up-regulated in IFN-γR−/− mice (Fig. 4, A and B) and IFN-γ−/− mice (Fig. 4, C and D). Surface up-regulation of PD-1 on CD4+ T cells occurred in the absence of IFN-γ signaling despite increases in CD4+ T cell numbers following treatment (2). These data suggest that IFN-γ is not influencing the observed reduction in CD4+ T cells through direct alteration of the surface expression of PD-1 on CD4+ T cells.

FIGURE 4.

Surface expression of PD-1 on CD4+ T cells is not affected by IFN-γ. Surface PD-1 expression on CD4+ T cells was evaluated by flow cytometry 11 days after immunotherapy initiation in animals lacking either IFN-γ (IFN-γ−/−) or the IFN-γ receptor (IFN-γR−/−). A, Representative histograms of PD-1 in IFN-γR−/− mice show unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). B, Cells isolated from the spleens of IFN-γR−/− and wild-type C57BL/6 animals showed a significantly (p < 0.001) higher percentage of CD4+ T cells in the spleen expressing surface PD-1 from animals that had been administered immunotherapy. C, Histograms of PD-1 labeling in IFN-γ−/− mice show unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). D, Evaluation of splenocytes from IFN-γ−/− mice 11 days after the start of immunotherapy showed a higher (p < 0.001) percentage of CD4+ T cells expressing surface PD-1 in animals that had been administered anti-CD40 and IL-2. Experiments using IFN-γR−/− animals were completed one time with three animals per group and were supported by IFN-γ−/− data, which were completed three times with three animals per group. An unpaired Student’s t test was used to determine significant differences between animals that had received immunotherapy and the control immunotherapy; a Welch’s correction was used for data sets with significant differences in variance.

FIGURE 4.

Surface expression of PD-1 on CD4+ T cells is not affected by IFN-γ. Surface PD-1 expression on CD4+ T cells was evaluated by flow cytometry 11 days after immunotherapy initiation in animals lacking either IFN-γ (IFN-γ−/−) or the IFN-γ receptor (IFN-γR−/−). A, Representative histograms of PD-1 in IFN-γR−/− mice show unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). B, Cells isolated from the spleens of IFN-γR−/− and wild-type C57BL/6 animals showed a significantly (p < 0.001) higher percentage of CD4+ T cells in the spleen expressing surface PD-1 from animals that had been administered immunotherapy. C, Histograms of PD-1 labeling in IFN-γ−/− mice show unlabeled control (gray line), rat Ig/PBS-treated animals (dashed black line), and anti-CD40 and IL-2-treated animals (solid black line). D, Evaluation of splenocytes from IFN-γ−/− mice 11 days after the start of immunotherapy showed a higher (p < 0.001) percentage of CD4+ T cells expressing surface PD-1 in animals that had been administered anti-CD40 and IL-2. Experiments using IFN-γR−/− animals were completed one time with three animals per group and were supported by IFN-γ−/− data, which were completed three times with three animals per group. An unpaired Student’s t test was used to determine significant differences between animals that had received immunotherapy and the control immunotherapy; a Welch’s correction was used for data sets with significant differences in variance.

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Because surface expression of PD-1 on CD4+ T cells in IFN-γR−/− and IFN-γ−/− mice did not directly correlate with IFN-γ-dependent loss of CD4+ T cells despite restoration of CD4+ T cell expansion, we evaluated the relative levels of B7-H1 expression following immunotherapy. Surface expression of B7-H1, and not PD-1, is reported to be dependent on IFN-γ (12). To examine a possible correlation between B7-H1 expression and immunotherapy-induced CD4+ T cell loss, flow cytometric analysis was used to determine the relative levels of B7-H1 on CD45+ cells from IFN-γR−/− mice and IFN-γ−/− mice after administration of anti-CD40 and IL-2. As expected, CD45+ splenocytes from wild-type mice showed a significant (p < 0.001) up-regulation of surface B7-H1 expression after treatment with anti-CD40 and IL-2. In contrast, CD45+ splenocytes from both IFN-γR−/− mice (Fig. 5, A and B) and IFN-γ−/− mice (Fig. 5, C and D) did not show elevated cell-surface B7-H1 expression after treatment with anti-CD40 and IL-2. These data suggest that the direct effects of IFN-γ on B7-H1 expression patterns correlated with the observed loss of CD4+ T cells following anti-CD40 and IL-2. As selective immunotherapy induced expression of PD-1 on CD4+ Tconv cells and not Treg cells, this allowed for Treg cells to avoid the inhibitory effects of anti-CD40 and IL-2 via up-regulation of B7-H1. This selective up-regulation results in a marked alteration of the CD4+ Tconv/CD4+ Treg/CD8+ T cell ratio, which may contribute to the loss of secondary responses at a later time, after immunotherapy.

FIGURE 5.

B7-H1 expression correlates with IFN-γ. B7-H1 expression on all CD45+ leukocytes was evaluated in wild-type animals and animals deficient in either IFN-γ (IFN-γ−/−) or the IFN-γ receptor (IFN-γR−/−). Unlike wild-type mice where a significantly (p < 0.001) higher level of B7-H1 was observed on cells from animals that had received immunotherapy, B7-H1 was not higher after immunotherapy in either IFN-γR−/− (A and B) or IFN-γ−/− (C and D) mice. An unpaired Student’s t test was used to determine significant differences between animals that had received immunotherapy and the control immunotherapy. A and C, Representative histograms of IFN-γR−/− mice and IFN-γ−/− mice, respectively, show control labeling (gray line), labeling of splenocytes from animals that received rat Ig and PBS (black dashed line) or anti-CD40 and IL-2 (black solid line).

FIGURE 5.

B7-H1 expression correlates with IFN-γ. B7-H1 expression on all CD45+ leukocytes was evaluated in wild-type animals and animals deficient in either IFN-γ (IFN-γ−/−) or the IFN-γ receptor (IFN-γR−/−). Unlike wild-type mice where a significantly (p < 0.001) higher level of B7-H1 was observed on cells from animals that had received immunotherapy, B7-H1 was not higher after immunotherapy in either IFN-γR−/− (A and B) or IFN-γ−/− (C and D) mice. An unpaired Student’s t test was used to determine significant differences between animals that had received immunotherapy and the control immunotherapy. A and C, Representative histograms of IFN-γR−/− mice and IFN-γ−/− mice, respectively, show control labeling (gray line), labeling of splenocytes from animals that received rat Ig and PBS (black dashed line) or anti-CD40 and IL-2 (black solid line).

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In this manuscript, we show that tumor immunotherapy regimens that can lead to successful initial antitumor responses paradoxically result in a lack of CD4+ Tconv cell expansion concurrent with CD4+ Treg cell expansion, and this correlates with PD-1/B7-H1 expression patterns following immunotherapy. Specifically, we present evidence that up-regulation of the inhibitory molecule PD-1 on Tconv cells following immunotherapy is a likely mechanism that contributes substantively to an imbalance between potentially beneficial Tconv cells and deleterious Treg cells. Treg cells are important mediators of the inflammatory immune response through their inhibitory actions on CD4+ and CD8+ T cells as well as NK cells (14, 15). Their presence has been shown to hinder the promotion of an effective immune-mediated antitumor response (6). The selective expansion of Treg cells after immunotherapy described here may present a mechanism by which the immune system attempts to down-regulate itself after being exposed to such a powerful stimulus such as anti-CD40 and IL-2. Therefore, these cells may be a critical determining factor in the outcome of at least some immunotherapeutic approaches to cancer treatment.

In a previous publication, we reported substantial effects of anti-CD40 and IL-2 on the ratio of CD4+ to CD8+ T cells. In tumor-bearing mice, we showed long-term effects, which changes in this ratio can have on memory CD8+ T cell responses (2). Based on our current observations, we cannot rule out the possibility that PD-1 ligation is also having an effect on CD8+ T cells. However, we observed higher increases in PD-1 expression on CD4+ T cells as measured by median fluorescence intensity and the percentage of PD-1+ cells. Therefore, we think that in our model PD-1 is having a more pronounced effect on CD4+ Tconv cells than on CD8+ T cells. The data presented in this publication extend our previous observations to suggest that when subsets of CD4+ T cells are carefully evaluated, substantial differences in important subsets can be detected. For example, our findings suggest that the ratio of CD4+ Tconv cells to CD8+ T cells may be lower than was originally thought. This is due to a preferential expansion of Treg cells in the CD4+ T cell compartment after treatment with either anti-CD40 and IL-2 or anti-CD40 and IL-15. Although the consequences of such a preferential expansion following immunotherapy have not been previously described, this may contribute to the loss of secondary responses and also may shorten the duration of the initial antitumor response. It is therefore reasonable to suggest that combination immunotherapy in conjunction with Treg cell depletion may further enhance the effectiveness of this approach. One potential way to reduce the induction of Treg cells would be to use IL-15 instead of IL-2 (16), as IL-2, and not IL-15, is known to be a strong promoter of Treg cell expansion (17, 18, 19). However, we did not observe a difference in the expansion of Treg cells between the two immunotherapeutic regimens, and IL-15 alone did not induce Treg cell expansion (data not shown). This suggests that Treg cell expansion may rely more on the administration of anti-CD40 than on the administration of IL-2.

Agonist CD40 mAb administration has been shown to suppress the immune response to LCMV infection, resulting in an increase in viral titers after treatment. In this LCMV model, loss of Ag-specific CD8+ T cells was observed. Interestingly, in this model a significant decrease in CD4+ T cells was also observed after treatment with anti-CD40 alone; however, any potential correlation with PD-1 expression was not discussed (3).

The use of anti-CD40 and IL-2 provides a model for investigating the disadvantages and benefits of potent immune stimulation. This model magnifies differences that may occur in the effectiveness of initial vs long-term immune-mediated tumor responses. In this regard, our recent studies indicate that strong immunotherapeutic regimens such as anti-CD40 and IL-2 combined therapy can hamper long-term responses to Ag through deleterious changes in the CD4+/CD8+ T cell balance (2). Alterations in this balance have been of great interest for some time (20, 21). Most recently there has been a debate about potentially “helpless” CD8+ T cells being incapable of responding to secondary antigenic challenge, which can occur even when primary response capabilities function with complete normalcy and strength (22, 23).

Surface expression patterns of PD-1 on human CD4+ T cells can be used as an indication of disease outcome in various human disease settings such as rheumatoid arthritis, schistosomiasis, and Hodgkin’s lymphoma (24, 25, 26). Similarly, PD-1 expression occurs on the CD8+ T cells of patients infected with HIV who show long-term progressor status (27). However, few reports have discussed the relative expression patterns on CD4+ T cell subsets, and, to our knowledge, no reports have discussed a differential response of CD4+ T cell subsets to immunotherapy dependent on this pathway. Although we observed a differential expression of PD-1 on the surface of Tconv cells and Treg cells after anti-CD40 and IL-2, we did not find a difference in the expression pattern of other immune markers such as Fas or DR5 after treatment (data not shown).

PD-1 has two known ligands, B7-H1 (PDL-1, CD274) and B7-DC (PDL-2, CD273) (12). B7-H1 is found on many cell types, including lymphocytes and myeloid cells as well as cells that are not of hemopoietic origin (8). B7-DC is primarily found on dendritic cells and is not up-regulated in response to IFN-γ; therefore, we have focused on the effects of B7-H1. Ligation of PD-1 by B7-H1 is capable of eliciting either apoptosis or senescence (7, 12). B7-H1 is inducibly up-regulated on tumor cells both in vivo and in vitro, and it is therefore thought to be important in tumor evasion of immune responses (28). PD-1 engagement by B7-H1 has been shown to have potent inhibitory effects on immune stimulation (29, 30) resulting in promotion of CD8+ T cell tolerance to self Ags in the periphery (9). Therefore, the PD-1/B7-H1 pathway is currently under intense investigation because manipulating it has the potential to modulate immune responses in a positive or negative manner (31, 32, 33).

Given our data presented here, further studies in tumor-bearing mice could address the question of whether selective up-regulation of PD-1 on Tconv cells and not Treg cells following immunotherapy might allow the tumor to dampen the effectiveness of tumor-infiltrating lymphocytes while not affecting the immune-inhibiting function of Treg cells. B7-H1 up-regulation may additionally promote immune suppression by supporting cell conversion to suppressive phenotype. In addition to Tconv cells being negatively affected through ligation of PD-1 by B7-H1, Treg cells may benefit from this interaction (34). In Heliobacter pylori infection, T cell anergy at the site of infection has been shown to be dependent on PD-1 ligation by B7-H1. It was shown that the presence of B7-H1 promoted an increase in Treg cell frequency when CD4+ T cells from H. pylori-infected donors were cocultured with H. pylori-infected epithelial cells in vitro. This Treg cell expansion was abrogated when anti-B7-H1 Ab was included (34).

Blockade of PD-1 and/or B7-H1 as well as other inhibitory markers such as CTLA-4 has recently been of interest when attempting to break tolerance (11, 35, 36). Combined PD-1 and B7-H1 blockade, but not B7-DC, is reported not only to enhance CD8+ T cell-mediated antitumor responses, but also to reverse anergy in CD8+ T cells (11). Note, however, that blockade of the PD-1/B7-H1 pathway usually only results in a partial removal of its inhibitory effect. Studies in our model aimed at the blockade of PD-1 or B7-H1 singly with anti-CD40 and IL-2 had no effect in vivo, possibly due to the massive expansion of PD-1+ and B7-H1+ cells, which would require very high levels of blocking Ab to obtain results (data not shown). This demonstrates one of the potential problems when exerting very strong stimulatory signals to amplify immune responses. Studies using PD-1 or B7-H1 knockout mice may be the best way to determine whether antitumor responses after immunotherapy are enhanced by the removal of PD-1 or B7-H1 signaling.

Relieving immune responses from the strict control that is mediated through PD-1/B7-H1 may be beneficial for the development of more effective antitumor immunotherapeutic approaches. Herein we report a previously undescribed preferential expansion of Treg cells that occurs in parallel to the loss of effector cells after administration of anti-CD40 and IL-2. It is of interest that this preferential Treg cell expansion still resulted in marked initial antitumor effects (1, 2). Because of its potent immunomodulating capabilities, the PD-1 and B7-H1 receptor/ligand interactions provide a potentially important component that should be further considered with regard to immune changes and overall responses that can be induced by different forms of immunotherapy. Our observations highlight the strong opposing force that the immune system has to potent stimuli. The different regulatory mechanisms used to protect from overstimulation may hinder efforts toward more effective antitumor immunotherapies.

We thank Myriam Bouchlaka and William Hallett for help with reviewing the manuscript, as well as Weihong Ma and Megan Whitaker for technical help.

W.J.M and B.R.B are members of the Scientific Advisory Board for Seattle Genetics.

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

1

This work was supported by National Institutes of Health Grants R01 A134495, R01 CA72669, R37 HL56067, P01 AI0562991, P20 RR-016464, and R01 CA095572.

2

W.J.M, D.R, L.A.W, B.R.B, and R.W.H contributed research design and experimental oversight as well as data interpretation and help with writing the manuscript. K.L.A, Q.Z., V.B., D.E.C.W, and J.M.W. conducted experiments as well as helped with data analysis and writing the manuscript.

4

Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; PD-1, programmed death-1; Tconv, conventional T; Treg, regulatory T.

1
Murphy, W. J., L. Welniak, T. Back, J. Hixon, J. Subleski, N. Seki, J. M. Wigginton, S. E. Wilson, B. R. Blazar, A. M. Malyguine, et al
2003
. Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses.
J. Immunol.
170
:
2727
-2733.
2
Berner, V., H. Liu, Q. Zhou, K. L. Alderson, K. Sun, J. M. Weiss, T. C. Back, D. L. Longo, B. R. Blazar, R. H. Wiltrout, et al
2007
. IFN-γ mediates CD4+ T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy.
Nat. Med.
13
:
354
-360.
3
Bartholdy, C., S. O. Kauffmann, J. P. Christensen, A. R. Thomsen.
2007
. Agonistic anti-CD40 antibody profoundly suppresses the immune response to infection with lymphocytic choriomeningitis virus.
J. Immunol.
178
:
1662
-1670.
4
Perez-Diez, A., N. T. Joncker, K. Choi, W. F. Chan, C. C. Anderson, O. Lantz, P. Matzinger.
2007
. CD4 cells can be more efficient at tumor rejection than CD8 cells.
Blood
109
:
5346
-5354.
5
Afzali, B., G. Lombardi, R. I. Lechler, G. M. Lord.
2007
. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease.
Clin. Exp. Immunol.
148
:
32
-46.
6
Curiel, T. J..
2007
. Tregs and rethinking cancer immunotherapy.
J. Clin. Invest.
117
:
1167
-1174.
7
Ishida, Y., Y. Agata, K. Shibahara, T. Honjo.
1992
. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death.
EMBO J.
11
:
3887
-3895.
8
Okazaki, T., Y. Iwai, T. Honjo.
2002
. New regulatory co-receptors: inducible co-stimulator and PD-1.
Curr. Opin. Immunol.
14
:
779
-782.
9
Martin-Orozco, N., Y. H. Wang, H. Yagita, C. Dong.
2006
. Cutting edge: Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens.
J. Immunol.
177
:
8291
-8295.
10
Keir, M. E., S. C. Liang, I. Guleria, Y. E. Latchman, A. Qipo, L. A. Albacker, M. Koulmanda, G. J. Freeman, M. H. Sayegh, A. H. Sharpe.
2006
. Tissue expression of PD-L1 mediates peripheral T cell tolerance.
J. Exp. Med.
203
:
883
-895.
11
Goldberg, M. V., C. H. Maris, E. L. Hipkiss, A. S. Flies, L. Zhen, R. M. Tuder, J. F. Grosso, T. J. Harris, D. Getnet, K. A. Whartenby, et al
2007
. Role of PD-1 and its ligand, B7–H1, in early fate decisions of CD8 T cells.
Blood
110
:
186
-192.
12
Flies, D. B., L. Chen.
2007
. The new B7s: playing a pivotal role in tumor immunity.
J. Immunother.
30
:
251
-260.
13
Freeman, G. J., A. J. Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura, L. J. Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, et al
2000
. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.
J. Exp. Med.
192
:
1027
-1034.
14
Picca, C. C., J. Larkin, III, A. Boesteanu, M. A. Lerman, A. L. Rankin, A. J. Caton.
2006
. Role of TCR specificity in CD4+CD25+ regulatory T-cell selection.
Immunol. Rev.
212
:
74
-85.
15
Barao, I., A. M. Hanash, W. Hallett, L. A. Welniak, K. Sun, D. Redelman, B. R. Blazar, R. B. Levy, W. J. Murphy.
2006
. Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells.
Proc. Natl. Acad. Sci. USA
103
:
5460
-5465.
16
Antony, P. A., N. P. Restifo.
2005
. CD4+CD25+ T regulatory cells, immunotherapy of cancer, and interleukin-2.
J. Immunother.
28
:
120
-128.
17
Purton, J. F., J. T. Tan, M. P. Rubinstein, D. M. Kim, J. Sprent, C. D. Surh.
2007
. Antiviral CD4+ memory T cells are IL-15 dependent.
J. Exp. Med.
204
:
951
-961.
18
Burkett, P. R., R. Koka, M. Chien, S. Chai, D. L. Boone, A. Ma.
2004
. Coordinate expression and trans presentation of interleukin (IL)-15Rα and IL-15 supports natural killer cell and memory CD8+ T cell homeostasis.
J. Exp. Med.
200
:
825
-834.
19
Sato, N., H. J. Patel, T. A. Waldmann, Y. Tagaya.
2007
. The IL-15/IL-15Rα on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells.
Proc. Natl. Acad. Sci. USA
104
:
588
-593.
20
Shedlock, D. J., H. Shen.
2003
. Requirement for CD4 T cell help in generating functional CD8 T cell memory.
Science
300
:
337
-339.
21
Sun, J. C., M. J. Bevan.
2003
. Defective CD8 T cell memory following acute infection without CD4 T cell help.
Science
300
:
339
-342.
22
Janssen, E. M., N. M. Droin, E. E. Lemmens, M. J. Pinkoski, S. J. Bensinger, B. D. Ehst, T. S. Griffith, D. R. Green, S. P. Schoenberger.
2005
. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death.
Nature
434
:
88
-93.
23
Badovinac, V. P., K. A. Messingham, T. S. Griffith, J. T. Harty.
2006
. TRAIL deficiency delays, but does not prevent, erosion in the quality of “helpless” memory CD8 T cells.
J. Immunol.
177
:
999
-1006.
24
Chemnitz, J. M., D. Eggle, J. Driesen, S. Classen, J. L. Riley, S. Debey-Pascher, M. Beyer, A. Popov, T. Zander, J. L. Schultze.
2007
. RNA-fingerprints provide direct evidence for the inhibitory role of TGFβ and PD-1 on CD4+ T cells in Hodgkin’s lymphoma.
Blood
110
:
3226
-3233.
25
Colley, D. G., L. E. Sasser, A. M. Reed.
2005
. PD-L2+ dendritic cells and PD-1+CD4+ T cells in schistosomiasis correlate with morbidity.
Parasite Immunol.
27
:
45
-53.
26
Hatachi, S., Y. Iwai, S. Kawano, S. Morinobu, M. Kobayashi, M. Koshiba, R. Saura, M. Kurosaka, T. Honjo, S. Kumagai.
2003
. CD4+PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid.
J. Rheumatol.
30
:
1410
-1419.
27
Zhang, J. Y., Z. Zhang, X. Wang, J. L. Fu, J. Yao, Y. Jiao, L. Chen, H. Zhang, J. Wei, L. Jin, et al
2007
. PD-1 up-regulation is correlated with HIV-specific memory CD8+ T-cell exhaustion in typical progressors but not in long-term nonprogressors.
Blood
109
:
4671
-4678.
28
Dong, H., S. E. Strome, D. R. Salomao, H. Tamura, F. Hirano, D. B. Flies, P. C. Roche, J. Lu, G. Zhu, K. Tamada, et al
2002
. Tumor-associated B7–H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.
Nat. Med.
8
:
793
-800.
29
Keir, M. E., L. M. Francisco, A. H. Sharpe.
2007
. PD-1 and its ligands in T-cell immunity.
Curr. Opin. Immunol.
19
:
309
-314.
30
Grakoui, A., E. John Wherry, H. L. Hanson, C. Walker, R. Ahmed.
2006
. Turning on the off switch: regulation of anti-viral T cell responses in the liver by the PD-1/PD-L1 pathway.
J. Hepatol.
45
:
468
-472.
31
Khoury, S. J., M. H. Sayegh.
2004
. The roles of the new negative T cell costimulatory pathways in regulating autoimmunity.
Immunity
20
:
529
-538.
32
Blazar, B. R., B. M. Carreno, A. Panoskaltsis-Mortari, L. Carter, Y. Iwai, H. Yagita, H. Nishimura, P. A. Taylor.
2003
. Blockade of programmed death-1 engagement accelerates graft-versus-host disease lethality by an IFN-γ-dependent mechanism.
J. Immunol.
171
:
1272
-1277.
33
Hori, J., M. Wang, M. Miyashita, K. Tanemoto, H. Takahashi, T. Takemori, K. Okumura, H. Yagita, M. Azuma.
2006
. B7-H1-induced apoptosis as a mechanism of immune privilege of corneal allografts.
J. Immunol.
177
:
5928
-5935.
34
Beswick, E. J., I. V. Pinchuk, S. Das, D. W. Powell, V. E. Reyes.
2007
. B7-H1 expression on gastric epithelial cells after Helicobacter pylori exposure promotes the development of CD4+CD25+FoxP3+ regulatory T cells.
Infect. Immun.
75
:
4334
-4341.
35
Tsushima, F., S. Yao, T. Shin, A. Flies, S. Flies, H. Xu, K. Tamada, D. M. Pardoll, L. Chen.
2007
. Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy.
Blood
110
:
180
-185.
36
Maker, A. V., P. Attia, S. A. Rosenberg.
2005
. Analysis of the cellular mechanism of antitumor responses and autoimmunity in patients treated with CTLA-4 blockade.
J. Immunol.
175
:
7746
-7754.