Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells’ effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using in vitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12– and IL-27–induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27–induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12–mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

The transmembrane ectonucleotidase, CD39, is recognized as an exhaustion marker for CD8+ T cells (1, 2). Given that CD39 facilitates the production of immunosuppressive adenosine, it has been theorized that CD39 expression by CD8+ tumor-infiltrating lymphocytes (TILs) may contribute to their exhaustion and could negatively impact their antitumor immune response (2). Immunosuppressive immune cells, such as tumor-associated macrophages, use ectonucleotidases to produce adenosine, which binds A2A and A2B receptors on conventional T cells; upregulating intracellular cAMP and inhibiting T cell activation, proliferation, and cytokine production (3).

Recent studies have identified CD39 as a marker for tumor-specific CD8+ TILs in both humans and mice (4–9). Although CD39 is a putative exhaustion marker, there are studies demonstrating that CD39+CD8+ TILs can exhibit meaningful antitumor activity (5, 10–13). For example, chimeric Ag receptor T cells and personalized tumor-reactive CD8+ T cells expressing CD39 exhibited superior cytotoxic functions, which was diminished on CD39 knockdown (12). Furthermore, several clinical studies have shown that CD39+CD8+ TILs positively correlate with response to treatment with immune checkpoint inhibitors and improved survival in various cancer types (13–17). Due to the significance of tumor-specific T cells in antitumor immunity and response to immunotherapy, better understanding of the factors that contribute to CD39 expression and their consequential effects on CD8+ T cell function is required.

Proinflammatory cytokines IL-12 and IL-27 have established roles in driving CD8+ T cell differentiation and promoting effector functions (18). These cytokines are produced by macrophages, dendritic cells, and to a lesser extent, B cells, which themselves facilitate T cell activation (18–20). There is considerable evidence demonstrating that IL-12, and more recently IL-27, can stimulate CD8+ T cell–mediated antitumor immunity, both directly and indirectly (21–33). Because IL-12 and IL-27 contribute to CD8+ T cell effector functions and tumor-specific T cells express CD39, these cytokines could play a role in regulating CD39 expression. This notion is supported by research demonstrating that IL-12 and IL-27 can induce CD39 expression on regulatory T cells (34, 35).

In this study, we investigated the ability of IL-12 and IL-27 to induce CD39 expression on CD8+ T cells isolated from tumor Ag–naive or –experienced mice. In this study, we show that IL-12 and IL-27 differentially induced CD39 expression on CD8+ T cells, and these cells exhibit different immunophenotypes marked by differential inhibitory receptor expression. We report, for the first time to our knowledge, that IL-27–induced CD39+CD8+ T cells appeared less exhausted than IL-12–induced CD39+CD8+ T cells. IL-12–induced CD39+CD8+ T cells displayed increased inhibitory receptor expression and diminished cytotoxic capabilities. IL-12 neutralization in vivo validated that IL-12 plays a role in the induction of CD39+CD8+ T cells within an immunogenic neuro-2a tumor model. These data establish a distinct role for IL-12 and IL-27 in modulating CD39 expression on CD8+ T cells and illustrate that extrinsic factors (i.e., the cytokine milieu) play an integral role in the phenotype and functionality of CD39+CD8+ T cells, which could provide an explanation for the controversial reports about a positive or negative role of CD39 expression on CD8+ T cells.

The neuro-2a cell line was obtained from the American Type Culture Collection. Mismatch repair (MMR) deficiency was induced in neuro-2a cells through CRISPR/Cas9 knockout of the MLH1 gene, as previously described (36). Neuro-2a cells, in which the knockout of MLH1 was successful, are termed idMMR (induced MMR–deficient) neuro-2a cells. Cells in which MLHI knockout was not successful are termed MMR-proficient (pMMR). For all experiments, pMMR and idMMR neuro-2a cells cultured for 8–9 wk after transfection were used. Cells were maintained in RPMI 1640 medium (Wisent Bio Products) supplemented with 10% heat-inactivated FBS (Life Technologies) and incubated at 37°C and 5% CO2.

For whole-cell lysate preparation, pMMR or idMMR neuro-2a cells were suspended at 5 × 107 cells/ml in PBS and subjected to five freeze-thaw cycles. Aliquots were stored at −80°C until further use.

Female A/J mice were purchased from the Jackson Laboratory. Mice were 6–10 wk of age and housed under specific pathogen-free conditions in the Victoria Research Laboratories Vivarium. All studies were approved by the Animal Care Committee at Western University (London, ON, Canada), Animal Use Protocol 2021-102. Littermates were randomly assigned to experimental groups.

For tumor model experiments, mice were s.c. injected with 100 μl of 5 × 105 pMMR or idMMR neuro-2a cells (day 0). Tumors were measured every 2–3 d with calipers; volumes were calculated with the following formula: 0.5(length × width2). All mice were euthanized via CO2 asphyxiation once the first tumor volumes reached ∼1500 mm3.

For stimulation assays using neuro-2a Ag–experienced mice (i.e., primed mice), the mice were vaccinated by i.p. injection of 100 μl of pMMR or idMMR whole neuro-2a cell lysate twice: 10 and 3 d before the start of the assay.

For IL-12 neutralization experiments, tumors were palpable ∼12 d after tumor inoculation. At such time, mice were injected i.p. with 0.5 mg of InVivoMAb anti-mouse IL-12 p75 (anti–IL-12, clone R2-9A5; BioXCell) or Ultra-LEAF Purified Rat IgG2b, κ Isotype Ctrl Ab (Ab, isotype control, clone RTK4530; BioLegend) in PBS every 3–4 d until end point.

Spleens and/or tumors were harvested from mice and mechanically disrupted. Tumors were processed using the Mouse Tumor Dissociation Kit and GentleMACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. RBCs were removed from spleen and tumor samples using ammonium-chloride-potassium lysis buffer.

For CD8+ T cell stimulation assays, CD8+ T cells were isolated from spleens of naive or neuro-2a cell lysate-primed mice with the MojoSort Mouse CD8 T-cell Negative Selection Isolation Kit (BioLegend) according to the manufacturer’s instructions. Cells were washed and resuspended in lymphocyte medium (RPMI 1640 with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 2 mM l-glutamine, and 50 μM 2-ME) supplemented with recombinant mouse IL-2 (30 ng/ml) alone (control condition) or with IL-12 (10 ng/ml) and/or IL-27 (50 ng/ml) (all recombinant cytokines were obtained from BioLegend) (28, 37–39). Cells were activated at 5 × 104 T cells/well in a 96-well round-bottom plate with an equivalent number of washed anti–CD3/CD28 Dynabeads (ThermoFisher). After 24 h, beads were magnetically removed, and cells were cultured for an additional 24 h in cytokine-supplemented lymphocyte media. After the incubation period, cells were stained for flow cytometric analysis.

For functional stimulation assays, bulk splenocytes were isolated from spleens of neuro-2a cell lysate-primed mice. Cells were suspended at 1 × 106 cells/ml in cytokine-supplemented lymphocyte medium and activated with plate-bound anti-CD3 (8 μg) and anti-CD28 (8 μg) Abs. After 48 h, cells from each group were divided into three conditions: experimental (anti-CD3/CD28 activation was continued), positive control (PMA/ionomycin was added according to manufacturer’s instructions; BioLegend), and negative control (no activation reagents included). For all conditions, cells were incubated for 5 h with brefeldin A (5 μg/ml; BioLegend), anti–CD16/32, and anti-mouse CD107a-Brilliant Violet 510 Ab (1/200 dilution; BioLegend); cells were stained for CD107a to detect T cell degranulation. After the incubation period, cells were stained for flow cytometric analysis.

For all experiments, 1 × 106 cells were incubated with Zombie viability dye (1/1000; BioLegend) (Supplemental Table I), followed by a brief incubation with anti-CD16/32 (1/50; BioLegend) to block Fc receptors. Cells were then stained with a mixture of extracellular Abs (all BioLegend; Supplemental Table I). For the surface marker staining, cells were fixed with 2% paraformaldehyde.

For intracellular cytokine staining, the Cyto-Fast Fix/Perm Buffer Set was used according to the manufacturer’s instructions after staining for extracellular markers. Cells were then stained with a mixture of intracellular Abs (all BioLegend; Supplemental Table I).

Data were acquired with the BD LSRII flow cytometer (BD Biosciences). Data analysis and calculation of median fluorescence intensity (MFI) values were conducted using FlowJo v10.8 software (BD Biosciences).

Data are represented as mean and SEM. Statistical analyses were performed with GraphPad Prism v9 software using the unpaired t test or one-way or two-way ANOVA with Tukey’s multiple comparison test. For all tests, p values ≤0.05 were considered significant, as indicated by an asterisk (*) (“ns” indicates not significant).

IL-12 and IL-27 can regulate CD8+ T cell differentiation, effector function, and, consequently, the antitumor potential of tumor-specific CD8+ T cells, suggesting that these cytokines may contribute to the induction of specific proteins, such as CD39, on these cells (21–33). To understand the effects of IL-12 and IL-27 on CD8+ T cell expression of CD39, irrespective of tumor Ags, we examined CD39 expression via flow cytometry after acute activation of CD8+ T cells isolated from naive mice. As shown in Fig. 1A and 1B, elevated frequencies of CD39highCD8+ T cells were detected after stimulation with IL-12 and, to a lesser extent, IL-27. In addition, when either IL-12 or IL-27 was present, CD8+ T cells expressed higher levels of CD39, measured by the MFI (Fig. 1C, 1D). These results indicate that IL-12 and IL-27 can upregulate CD39 expression on CD8+ T cells, irrespective of previous Ag exposure.

FIGURE 1.

IL-12 and IL-27 can induce CD39 expression on CD8+ T cells. Splenic CD8+ T cells from naive mice were stimulated with anti-CD3/CD28 Dynabeads and IL-2, in the presence or absence of IL-12 or IL-27. After 48 h, samples were stained for flow cytometric analysis of CD39 expression. (A) Representative dot plots depicting CD39high expression, gated on CD8+CD3+ cells. (B) The frequency of CD39highCD8+ T cells. (C) Quantification of MFI of CD39-AF647 on CD39+CD8+ T cells. (D) Representative histogram of CD39 expression, in which the vertical black line separates CD39 (left) and CD39+ (right) CD8+ T cells. Error bars indicate mean and SEM (B). Bars display median values (C). Statistical analyses were conducted by one-way ANOVA (n = 6; data are from two independent experiments). *p ≤ 0.05.

FIGURE 1.

IL-12 and IL-27 can induce CD39 expression on CD8+ T cells. Splenic CD8+ T cells from naive mice were stimulated with anti-CD3/CD28 Dynabeads and IL-2, in the presence or absence of IL-12 or IL-27. After 48 h, samples were stained for flow cytometric analysis of CD39 expression. (A) Representative dot plots depicting CD39high expression, gated on CD8+CD3+ cells. (B) The frequency of CD39highCD8+ T cells. (C) Quantification of MFI of CD39-AF647 on CD39+CD8+ T cells. (D) Representative histogram of CD39 expression, in which the vertical black line separates CD39 (left) and CD39+ (right) CD8+ T cells. Error bars indicate mean and SEM (B). Bars display median values (C). Statistical analyses were conducted by one-way ANOVA (n = 6; data are from two independent experiments). *p ≤ 0.05.

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Within the tumor microenvironment (TME), tumor-specific T cells can be exposed to their cognate Ags. Vaccination using whole-tumor lysates can be used to prime and activate T cells. In previous work, we have demonstrated that immune-cold murine neuroblastoma tumors could be turned immunologically hot by inducing MMR deficiency (36). As such, we used idMMR and pMMR neuro-2a cells to generate immunogenic and poorly immunogenic neuro-2a tumor lysates, respectively. We therefore determined whether IL-12 and IL-27 could modulate CD39 expression on CD8+ T cells after vaccination of animals with immunogenic or poorly immunogenic tumor lysates.

CD8+ T cells isolated from the spleen of immunogenic idMMR neuro-2a–primed mice showed a significantly greater frequency of CD39highCD8+ T cells when activated with IL-12 or IL-27. However, this was not observed for CD8+ T cells isolated from the poorly immunogenic pMMR neuro-2a–primed mice (Fig. 2A, 2B). The combination of IL-12 and IL-27 resulted in the greatest frequency of CD39highCD8+ T cells, regardless of the CD8+ T cell source (Fig. 2A, 2B). Similarly, expression levels of CD39 on CD39+CD8+ T cells were highest when both IL-12 and IL-27 were present, as measured by MFI (Fig. 2C, 2D). Compared with the control conditions, CD8+ T cells from the idMMR-vaccinated group that were stimulated with either IL-12 or IL-27 showed high CD39 expression. (Fig. 2C, 2D). Collectively, these data suggest that IL-12 and IL-27 can individually induce the expression of CD39 on CD8+ T cells that have encountered immunogenic Ags, whereas those from a poorly immunogenic model require stimulation with both cytokines in combination.

FIGURE 2.

IL-12 and IL-27 in combination induce high levels of CD39 expression on CD8+ T cells. Splenic CD8+ T cells from neuro-2a–primed mice were stimulated with anti-CD3/CD28 Dynabeads and IL-2 (control conditions), in the absence or presence of IL-12 and/or IL-27. After 48 h, samples were stained for flow cytometric analysis of CD39 expression. (A) Representative dot plots depicting CD39high expression, gated on CD8+CD3+ cells, from mice primed with the pMMR (left) or idMMR (right) neuro-2a lysates. (B) Frequency of CD8+ T cells expressing high levels of CD39 from pMMR (left) or idMMR (right) neuro-2a–primed mice. (C) Quantification of MFI of CD39-AF647 on CD39+CD8+ T cells from mice primed with pMMR (left) or idMMR (right) neuro-2a lysates. (D) Representative histograms of CD39 expression, in which the vertical black line separates CD39 and CD39+CD8+ T cells from mice primed with pMMR (left) or idMMR (right) neuro-2a lysates. Error bars indicate mean and SEM (B). Bars display median values (C). Statistical analyses were conducted by two-way ANOVA (n ≥ 6; data are from at least two independent experiments). *p ≤ 0.05.

FIGURE 2.

IL-12 and IL-27 in combination induce high levels of CD39 expression on CD8+ T cells. Splenic CD8+ T cells from neuro-2a–primed mice were stimulated with anti-CD3/CD28 Dynabeads and IL-2 (control conditions), in the absence or presence of IL-12 and/or IL-27. After 48 h, samples were stained for flow cytometric analysis of CD39 expression. (A) Representative dot plots depicting CD39high expression, gated on CD8+CD3+ cells, from mice primed with the pMMR (left) or idMMR (right) neuro-2a lysates. (B) Frequency of CD8+ T cells expressing high levels of CD39 from pMMR (left) or idMMR (right) neuro-2a–primed mice. (C) Quantification of MFI of CD39-AF647 on CD39+CD8+ T cells from mice primed with pMMR (left) or idMMR (right) neuro-2a lysates. (D) Representative histograms of CD39 expression, in which the vertical black line separates CD39 and CD39+CD8+ T cells from mice primed with pMMR (left) or idMMR (right) neuro-2a lysates. Error bars indicate mean and SEM (B). Bars display median values (C). Statistical analyses were conducted by two-way ANOVA (n ≥ 6; data are from at least two independent experiments). *p ≤ 0.05.

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To determine the importance of anti–CD3/CD28 in the IL-12– and IL-27–mediated induction of CD39, we stimulated bulk splenocytes with cytokines, but without anti-CD3/CD28. We found IL-12 and IL-27 were able to induce CD39 expression on CD8+ T cells relative to the control (Supplemental Fig. 1). Moreover, the greatest frequency of CD39+CD8+ T cells was observed after stimulation with both IL-12 and IL-27. These data suggest that these cytokines are capable of modulating CD39 expression on CD8+ T cells independent of anti-CD3/CD28 activation signaling.

CD39 is considered an exhaustion marker and is commonly found to be coexpressed with inhibitory receptors on CD8+ T cells (1). In accordance with the literature, we found that more CD39+CD8+ T cells expressed inhibitory receptors programmed cell death-1 (PD-1), lymphocyte-activation gene 3 (LAG-3), and T cell Ig and mucin-domain containing-3 (TIM-3), compared with CD39CD8+ T cells (Fig. 3A–C). Given that CD39 is considered an exhaustion marker, we hypothesized that IL-12– and IL-27–mediated upregulation of CD39 on T cells can be correlated with coexpression of other inhibitory receptors on CD8+ T cells. Using the in vitro activation assay with CD8+ T cells from immunogenic idMMR neuro-2a Ag–experienced mice, we found that IL-12–induced CD39+CD8+ T cells showed a higher level of PD-1, LAG-3, and TIM-3 coexpression (Fig. 3D–F). Although the combination of IL-12 and IL-27 produced a greater effect on CD39 induction of CD8+ T cells (Fig. 2B, 2C), these cells, stimulated with both cytokines, had reduced levels of PD-1 and TIM-3 compared with IL-12 individually (Fig. 3D, 3F).

FIGURE 3.

IL-12 promotes inhibitory receptor expression to a greater extent than IL-27. Splenic CD8+ T cells from idMMR neuro-2a–primed mice were stimulated with anti-CD3/CD28 Dynabeads and IL-2 (control conditions). After 48 h, samples were stained for flow cytometric analysis of CD39, PD-1, LAG-3, and TIM-3 expression. (AC) Frequency of PD-1+ (A), LAG-3+ (B), and TIM-3+ (C) CD39 and CD39+CD8+ T cells. Splenic CD8+ T cells from idMMR neuro-2a–primed mice were stimulated similar to (A)–(C), in the presence or absence of IL-12 and/or IL-27. (DF) Quantification of PD-1+ (D), LAG-3+ (E), and TIM-3+ (F) CD39+CD8+ T cells. (G) Quantification of CD39+CD8+ T cells coexpressing TIM-3, PD-1, and LAG-3. Error bars indicate mean and SEM. Statistical analyses were conducted by unpaired t tests (A–C) or one-way ANOVA (D–G) (n = 6; data are from two independent experiments). *p ≤ 0.05.

FIGURE 3.

IL-12 promotes inhibitory receptor expression to a greater extent than IL-27. Splenic CD8+ T cells from idMMR neuro-2a–primed mice were stimulated with anti-CD3/CD28 Dynabeads and IL-2 (control conditions). After 48 h, samples were stained for flow cytometric analysis of CD39, PD-1, LAG-3, and TIM-3 expression. (AC) Frequency of PD-1+ (A), LAG-3+ (B), and TIM-3+ (C) CD39 and CD39+CD8+ T cells. Splenic CD8+ T cells from idMMR neuro-2a–primed mice were stimulated similar to (A)–(C), in the presence or absence of IL-12 and/or IL-27. (DF) Quantification of PD-1+ (D), LAG-3+ (E), and TIM-3+ (F) CD39+CD8+ T cells. (G) Quantification of CD39+CD8+ T cells coexpressing TIM-3, PD-1, and LAG-3. Error bars indicate mean and SEM. Statistical analyses were conducted by unpaired t tests (A–C) or one-way ANOVA (D–G) (n = 6; data are from two independent experiments). *p ≤ 0.05.

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Because it is the coexpression of multiple inhibitory receptors or CD39 that has been associated with terminal exhaustion of CD8+ T cells (1, 40, 41), we analyzed the frequency of CD39+CD8+ T cells that were triple positive (TP; i.e., TIM-3+PD-1+LAG-3+). Our results showed that the highest frequency of TP CD39+CD8+ T cells was found on stimulation with IL-12 (Fig. 3G). Notably, CD8+ T cells from control and IL-27–stimulated conditions showed comparable levels of TP CD39+CD8+ T cells. Overall, our data indicate that IL-27 can attenuate the induction of PD-1 and TIM-3 expression by IL-12 on CD39+CD8+ T cells.

Given the discrepancies in the literature regarding the antitumor activity of CD39+CD8+ T cells, we sought to investigate the effector capabilities of CD39+CD8+ T cells using our in vitro assay. CD39+CD8+ T cells activated with anti-CD3, anti-CD28, and IL-2 demonstrated increased cytotoxic potential, measured by CD107a expression, compared with CD39CD8+ T cells (Fig. 4A). CD39+CD8+ T cells also produced higher levels of IFN-γ, TNF-α, and IL-2 than CD39CD8+ T cells (Fig. 4B–D). The frequencies of TNF-α and IL-2 were relatively low (measuring <5%) under control conditions; as such, we used PMA/ionomycin to hyperstimulate cells for validation purposes. Consistent with previous data, CD39 expression was positively correlated with TNF-α+CD8+ T cells (Supplemental Fig. 2A). However, CD39+CD8+ T cells showed impairment in IL-2 production under hyperstimulated conditions (Supplemental Fig. 2B). Overall, our data suggest that CD39+CD8+ T cells have increased effector and cytotoxic potential outside the conditions experienced within the TME.

FIGURE 4.

CD39+CD8+ T cells exhibit elevated and malleable functionality. (AD) Bulk splenocytes from idMMR neuro-2a–primed mice were stimulated with anti-CD3 and anti-CD28 Abs and IL-2. Frequency of CD107a+ (A), IFN-γ+ (B), TNF-α+ (C), and IL-2+ (D) CD39 and CD39+CD8+ T cells. (EH) Bulk splenocytes from idMMR neuro-2a–primed mice were stimulated similar to (A)–(D), in the presence or absence of IL-12 and/or IL-27. Error bars indicate mean and SEM. Statistical analyses were conducted by unpaired t tests (A–D) or one-way ANOVA (n ≥ 6; data are from at least two independent experiments). *p ≤ 0.05.

FIGURE 4.

CD39+CD8+ T cells exhibit elevated and malleable functionality. (AD) Bulk splenocytes from idMMR neuro-2a–primed mice were stimulated with anti-CD3 and anti-CD28 Abs and IL-2. Frequency of CD107a+ (A), IFN-γ+ (B), TNF-α+ (C), and IL-2+ (D) CD39 and CD39+CD8+ T cells. (EH) Bulk splenocytes from idMMR neuro-2a–primed mice were stimulated similar to (A)–(D), in the presence or absence of IL-12 and/or IL-27. Error bars indicate mean and SEM. Statistical analyses were conducted by unpaired t tests (A–D) or one-way ANOVA (n ≥ 6; data are from at least two independent experiments). *p ≤ 0.05.

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To further examine the functionality of CD39+CD8+ T cells after IL-12 and IL-27 induction, we analyzed effector cytokine and IL-2 production and CD107a expression in IL-12– and IL-27–stimulated cells. Our data revealed that IL-27–induced CD39+CD8+ T cells displayed the greatest frequency of CD107a expression, but this effect was lost when IL-12 was added in combination (Fig. 4E). Interestingly, IL-12–induced CD39+CD8+ T cells showed the highest frequency of IFN-γ+ cells, whereas those stimulated with IL-27 had the lowest frequency (Fig. 4F). We found no difference in the expression of TNF-α across conditions (Fig. 4G). The highest level of IL-2+ cells was observed in the IL-27–induced CD39+CD8+ T cell groups, whether individual or in combination with IL-12 (Fig. 4H). Altogether, these results show that the functionality of CD39+CD8+ T cells is differentially affected when stimulated by IL-12 or IL-27.

CD8+ T cells from idMMR neuro-2a–primed mice showed a higher frequency of CD39highCD8+ T cells when stimulated with IL-12 than IL-27 (Fig. 2B). In addition, IL-12–stimulated CD8+ T cells showed greater frequencies of TP CD39+CD8+ T cells. In an effort to examine the relevance of IL-12 on the induction of CD39 expression on CD8+ T cells in vivo, we neutralized the biological activity of IL-12 in pMMR and idMMR neuro-2a tumor-bearing mice (Fig. 5A).

FIGURE 5.

IL-12 neutralization results in reduced frequency of CD39+ and exhausted CD8+ TILs. (A) Mice were inoculated s.c. with 5 × 105 idMMR neuro-2a cells. Isotype control (IgG2b, k) or anti–IL-12 treatment began on day ∼12. At the end point, tumors and spleens were processed and stained for flow cytometric analysis of CD8+ T cell populations. (B) Frequency of CD8+ TILs with naive (Tnaive = CD44CD62L+Fas) or effector (TEFF = CD44+CD62L) phenotype. (C) Frequency of CD3+ TILs expressing CD8. (D) Frequency of CD8+ TILs expressing CD39, CD38, PD-1 and CD39, TIM-3 and CD39, LAG-3 and CD39, PD-1 and TIM-3, and PD-1 and CD38, from left to right, respectively. (E) Frequency of splenic CD8+ T cells expressing CD39. Error bars indicate mean and SD (B) or mean and SEM (C–E). Statistical analyses were conducted by two-way ANOVA (B) and single (C and E) or multiple (D) unpaired t tests (C and E) (n ≥ 4; data are from two independent experiments). *p ≤ 0.05.

FIGURE 5.

IL-12 neutralization results in reduced frequency of CD39+ and exhausted CD8+ TILs. (A) Mice were inoculated s.c. with 5 × 105 idMMR neuro-2a cells. Isotype control (IgG2b, k) or anti–IL-12 treatment began on day ∼12. At the end point, tumors and spleens were processed and stained for flow cytometric analysis of CD8+ T cell populations. (B) Frequency of CD8+ TILs with naive (Tnaive = CD44CD62L+Fas) or effector (TEFF = CD44+CD62L) phenotype. (C) Frequency of CD3+ TILs expressing CD8. (D) Frequency of CD8+ TILs expressing CD39, CD38, PD-1 and CD39, TIM-3 and CD39, LAG-3 and CD39, PD-1 and TIM-3, and PD-1 and CD38, from left to right, respectively. (E) Frequency of splenic CD8+ T cells expressing CD39. Error bars indicate mean and SD (B) or mean and SEM (C–E). Statistical analyses were conducted by two-way ANOVA (B) and single (C and E) or multiple (D) unpaired t tests (C and E) (n ≥ 4; data are from two independent experiments). *p ≤ 0.05.

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To confirm the anti–IL-12 Ab was indeed neutralizing IL-12 in vivo, we analyzed differentiation states of CD8+ TILs, because IL-12 plays a key role in effector T cell differentiation and activation (42, 43). As expected, the ratio of naive and effector TILs was altered in idMMR mice treated with anti–IL-12 versus isotype control (Fig. 5B). Moreover, we found fewer effector CD8+ TILs present in anti–IL-12–treated idMMR neuro-2a tumors, providing evidence that IL-12 neutralization was effective. No conclusions could be made for the pMMR group because of the low frequency of CD8+ TILs as these tumors are poorly immunogenic. However, fewer activated (CD38+) CD8+ TILs were found in anti–IL-12–treated pMMR neuro-2a tumors (Supplemental Fig. 3B), providing evidence for successful IL-12 neutralization in these tumors.

For idMMR neuro-2a tumors, treatment with anti–IL-12 resulted in a reduced frequency of CD39+CD8+ TILs relative to the control group, whereas the frequency of CD8+ TILs remained the same (Fig. 5C, 5D). Alternatively, we found IL-12 neutralization did not affect the frequency of CD39+CD8+ TILs from the poorly immunogenic pMMR neuro-2a tumors (Supplemental Fig. 3C). Within idMMR neuro-2a tumors, we observed less activated (CD38+), exhausted (PD-1+TIM-3+, PD-1+CD39+, TIM-3+CD39+, and LAG-3+CD39+), and dysfunctional (PD-1+CD38+) CD8+ TILs with anti–IL-12 treatment (Fig. 5D). These results indicate that IL-12 is functionally relevant to the induction of CD39+CD8+ TILs within an immunogenic tumor model and suggest that IL-12 contributes to the exhausted phenotype of CD8+ TILs in the immunogenic idMMR neuro-2a tumors. In addition, there was no difference in the frequency of splenic CD39+CD8+ T cells between anti–IL-12–treated and control groups (Fig. 5E), highlighting the importance of IL-12 for the induction of CD39 on CD8+ TILs specifically.

In this study, we showed that CD8+ T cells from immunogenic, idMMR neuro-2a Ag–experienced mice upregulated CD39 expression when activated in the presence of IL-12 or IL-27 (Fig. 2). These results are in agreement with previous studies demonstrating that regulatory T cells upregulate CD39 in response to stimulation with IL-12 or IL-27 (34, 35). Our results showed that stimulation with IL-12 was capable of inducing CD39 on CD8+ T cells isolated from the idMMR neuro-2a–primed mice, and coordinately, neutralization in idMMR neuro-2a tumor-bearing mice resulted in fewer CD39+CD8+ TILs (Figs. 2, 5D). This was not the case for the poorly immunogenic pMMR neuro-2a model, because IL-12 neutralization had no effect on the frequency of CD39+CD8+ TILs (Supplemental Fig. 3C). This demonstrates the requirement for immunogenic neoantigens for IL-12 to exert meaningful effects on CD39 expression by CD8+ TILs. In accordance with the aforementioned data, we found in vitro stimulation with IL-12 individually was insufficient to induce CD39 expression on CD8+ T cells from the poorly immunogenic, pMMR neuro-2a–primed mice (Fig. 2). Taken together, these data suggest that immunogenic neoantigens are critical in allowing IL-12 to induce CD39 expression upon restimulation. An important limitation of this study was the inability to assess the effects of IL-27 neutralization in vivo and confirm the importance of IL-27 on the induction of CD39+CD8+ T cells in vivo. There are currently no commercially available Abs that specifically neutralize the murine heterodimeric IL-27 cytokine without affecting other cytokines such as IL-30 and IL-12. However, we speculate that blocking IL-27 in vivo would result in a reduced frequency of CD39+CD8+ TILs, similar to the results observed from blocking IL-12.

Importantly, we showed that the highest level of CD39 expression was found in CD8+ T cell samples stimulated with both IL-12 and IL-27, regardless of the immunogenicity of the neuro-2a lysate (Fig. 2). To the best of our knowledge, this is the first study to identify that these cytokines produce an additive effect in stimulating CD39 expression on CD8+ T cells from tumor Ag–experienced mice (Fig. 2). The effects of IL-12 and IL-27 on CD39 induction may be attributed to the different signaling pathways these cytokines stimulate; IL-12 signaling predominantly activates STAT4, whereas IL-27 activates STAT1 and STAT3. The STAT transcription factors have both shared unique target genes. For example, STAT3 and STAT4 augment PD-1 expression, whereas STAT1 hinders PD-1 expression (44). As such, the overlaps and differences in signaling cascades produced by IL-12 and IL-27 may extend to the regulation of CD39.

Although CD39 can largely identify tumor-specific CD8+ T cells, CD39 is also a putative exhaustion marker (1, 2). Given that CD39 is considered an exhaustion marker, we hypothesized that these cytokines could induce other exhaustion markers, such as inhibitory receptors. Our data revealed the highest frequencies of PD-1+, LAG-3+, TIM-3+, and TP (TIM-3+PD-1+LAG-3+) CD39+CD8+ T cells were observed after stimulation with IL-12 (Fig. 3D–G). These results agree with previous studies, which showed that IL-12 upregulates these inhibitory receptors (44–46). The combination of immune checkpoint inhibitors, such as PD-1 blockade, with tumor-targeted IL-12 therapies in preclinical models demonstrated the ability to promote CD8+ T cell activity and persistence, while restricting suppressive signals that augment exhaustion (47, 48). These data indicate that combination therapies can harness the antitumor effects of IL-12 for improved CD8+ T cell functions. From our findings, it appears that IL-27 attenuates IL-12–induced expression of these inhibitory receptors on CD39+CD8+ T cells. Recent studies have highlighted that IL-27 can enhance the survival and function of tumor-specific CD8+ T cells (29, 30, 49). Based on our results, it is possible that IL-27 can act to limit inhibitory receptor expression, thereby mitigating the suppressive signals that contribute to CD8+ T cell exhaustion and dysfunction. These findings should be taken into consideration when developing cytokine-based therapies using IL-12 and/or IL-27, to support the antitumor potential of CD8+ T cells without driving the exhaustive phenotype commonly displayed by tumor-specific CD8+ TILs.

The current literature contains conflicting evidence as to the antitumor potential of CD39+CD8+ T cells (1, 2, 5, 10–13). We found, using control conditions, that CD39+CD8+ T cells exhibited superior effector functions relative to their CD39 counterparts (Fig. 4A–C, Supplemental Fig. 2A). Previous studies have demonstrated CD8+ T cell exhaustion is, in part, characterized by the progressive loss of effector function. The ability to produce IL-2 and TNF-α is lost during the initial phases, while cells can still produce IFN-γ and degranulate (marked by CD107a) (50). Overall, our data indicate that CD39+CD8+ T cells are more capable of IFN-γ production and cytotoxicity than their CD39 counterparts (10–13). Although CD39+CD8+ T cells did display low-level characteristics of exhaustion, they were less affected than that of CD39CD8+ T cells (Fig. 4A–C, Supplemental Fig. 2A), suggesting that the use of CD39 as a putative marker of terminal CD8+ T cell exhaustion may not be appropriate. Nevertheless, the expression of CD39 on tumor-specific CD8+ T cells given the immunosuppressive function of CD39 is puzzling, especially considering that outside of the immunosuppressive TME, CD39+CD8+ T cells display enhanced functional potential (Fig. 4A–D). This may parallel with recent discoveries of the protective effects of the widely recognized immunosuppressive cytokine, IL-10. Such studies demonstrated that IL-10 could prevent activation-induced exhaustion of CD8+ T cells through chromatin remodeling and restore the function of exhausted CD8+ T cells via metabolic reprogramming (51, 52). Given that IL-12 and IL-27 themselves promote antitumor immunity, it is possible that IL-12– and IL-27–induced CD39 expression yields different levels of protective qualities for tumor-specific CD8+ TILs. For example, limiting ATP signaling through CD39-mediated ATP hydrolysis could prevent desensitization of P2 receptors by tumor-specific CD8+ TILs (53, 54). Further investigation is warranted to support this claim, and future studies should focus on uncovering the physiological effects of CD39 expression by tumor-specific CD8+ TILs.

We considered that IL-12 and IL-27 might have distinct effects on the cytokine production and cytotoxic capacity of CD39+CD8+ T cells, because they differentially affected inhibitory receptor expression (Fig. 3D–G). We found that CD39+CD8+ T cells stimulated with IL-27 had the greatest cytotoxic abilities, as shown by the highest frequency of CD107a+ cells (Fig. 4E). When analyzing inhibitory receptor expression, stimulation with IL-27 resulted in the lowest TP CD39+CD8+ T cells (Fig. 3G). Coupled with the higher expression of CD107a, it appears that cells stimulated with IL-27 are less exhausted than those stimulated with IL-12 (55). We also found that activation in the presence of IL-27 resulted in the highest level of IL-2–producing CD39+CD8+ T cells (Fig. 4H). Effector CD8+ T cells that intrinsically produce IL-2 are found to preferentially survive and develop memory traits (56). Collectively, these suggest that IL-27 may contribute to the expansion and persistence of CD39+CD8+ T cells. Further investigation is warranted to support this claim; however, the idea is also supported by studies showing IL-27 promotes memory formation and enhances the survival and self-renewal of tumor-specific CD8+ T cells (29, 30, 49).

In this study, it is important to recognize that although IL-12 induced the highest expression of PD-1+, LAG-3+, TIM-3+, and TP CD39+CD8+ T cells (Fig. 3D–G), IL-12–stimulated cells were functional upon in vitro stimulation (Fig. 4). This was specifically true for the production of IFN-γ, because the frequency of IFN-γ+ cells was highest when stimulated in the presence of IL-12 (Fig. 4F). IL-27–induced CD39+CD8+ T cells showed the lowest proportion of IFN-γ+ cells, which differs from other studies that found IL-27 induces IFN-γ production by CD8+ T cells (26–30); perhaps the difference is specific to CD39+ cells. Our results did show that CD39+CD8+ T cells stimulated with both IL-12 and IL-27 showed the same frequency of IFN-γ+ cells as those stimulated with IL-12 individually. Similarly, one study showed that although IL-27 and TCR stimulation alone induced minimal IFN-γ+CD8+ T cells, IL-27 synergized with IL-12 to produce high levels of IFN-γ (31).

Proinflammatory cytokine-based immunotherapies, such as IL-12, have strong potential; however, their use has been restricted because of systemic toxicities (57). Current efforts focus on methods to localize the cytokines to the TME, and the adoptive transfer of chimeric Ag receptor T cells or tumor-specific or CD8+ T cells engineered to express cytokines represents a promising avenue in cancer immunotherapy (57, 58). In preclinical models, cell-surface–tethered IL-12– or IL-27–secreting adoptive CD8+ T cells show enhanced antitumor activity (30, 59). Our results indicate that the combination of IL-12 and IL-27 would promote the antitumor activity and limit the exhaustive phenotype of CD39+CD8+ T cells (Figs. 3, 4). Two studies have demonstrated the synergistic antitumor effects of IL-12– and IL-27–based cytokine therapies in preclinical models using intratumoral delivery of IL-12 and IL-27 mRNA via lipid nanoparticles or sequential IL-12 and IL-27 gene therapy through i.m. electroporation (60, 61). These studies support the combinational use of these cytokines for cancer therapy.

Overall, results from this study expand our knowledge of the differential effects of IL-12 and IL-27 on CD39+CD8+ T cell development and function, providing a rationale for the combination of both cytokines as a therapeutic strategy for T cell–based immunotherapies. Furthermore, our results highlight the significance of the source of CD39 induction on CD8+ T cells, which may explain the controversial observations about these cells in the literature.

S.M.V. is a member of the Board of Directors of IMV Inc. The rest of the authors have no financial conflicts of interest.

We thank Dr. Kristin Chadwick at the London Regional Flow Cytometry Facility for extending expertise. We also thank the Victoria Research Laboratories Vivarium staff for adept animal husbandry. Schematics were created with BioRender.com (2023).

This work was supported by the Canadian Institutes of Health Research grant (MOP 389137 to S.M.V.) and the London Regional Cancer Program’s Catalyst Grant Program, Keith Samitt Translational Research Grant (to S.M.V.).

The online version of this article contains supplemental material.

idMMR

induced mismatch repair–deficient

LAG-3

lymphocyte-activation gene 3

MFI

median fluorescence intensity

MMR

mismatch repair

PD-1

programmed cell death-1

pMMR

mismatch repair proficient

TIL

tumor-infiltrating lymphocyte

TIM-3

T cell Ig and mucin-domain containing-3

TME

tumor microenvironment

TP

triple positive (TIM-3+PD-1+LAG-3+)

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