Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4+CD25hi regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti–Gr-1 antibody was immunologically and clinically more efficacious than anti–Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti–Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved. Cancer Res; 72(8); 2089–99. ©2012 AACR.
Among known factors associated with cancer development, advancing age remains the leading risk (1). Immune therapy for cancer is a scientifically sound approach, but clinical effects have generally been modest. Much of our understanding of tumor immunity comes from studies in young hosts. Few studies have examined the effects of age on response to immune therapy and on tumor-associated immune dysfunction.
Although antitumor immunity declines with age, as do other immune effector functions (2, 3), T-cell functions in aged hosts can sometimes be improved (4). Thus, to the extent that underlying tumor-associated immune dysfunction can be reversed in aged hosts with cancer, clinically relevant antitumor immune responses could potentially be achieved.
Regulatory T cells (Treg) are key mediators of tumor immune dysfunction, and reducing Treg function is a rational cancer immunotherapy strategy (5–8). Treg contributions to age-related decline in immune responses is contradictory, with some studies showing increases in Treg prevalence and/or function with age in humans and mice (9–12), whereas others show no changes or reduced Treg contributions (13, 14). Myeloid-derived suppressor cells (MDSC) are regulatory cells that also increase in tumors (15–18) and suppress antitumor immunity (19). They are a heterogeneous population usually identified as CD11b+Gr-1+ cells in mice (17).
Although Treg depletion is an effective approach to improving antitumor immunity and responses to immunotherapy (6, 8), conflicting studies report the effect of Treg depletion as cancer immunotherapy in aged hosts (20, 21). MDSC depletion is another potentially effective approach to reverse cancer-associated immune dysfunction. Whereas MDSCs contribute to immunopathology in aged hosts including in cancer (22, 23), the subject remains little studied, as does potential interactions between MDSCs and Tregs.
We examined immune dysregulatory mechanisms in young versus aged hosts with cancer and assessed age-specific responses to Treg depletion, MDSC modulation, or both. We showed that aged and young hosts with cancer responded differently to those immune therapies in a tumor-dependent fashion, but that, more importantly, clinically significant antitumor immune responses can be achieved even in aged tumor-bearing mice using age-specific immune therapies. These data suggest means to improve the efficacy of tumor immunotherapy in the population most at risk for cancers and show that age and tumor interactions with the aged host are significant considerations in preclinical and clinical testing of immunotherapeutic strategies.
Materials and Methods
Four- to 8-week old (National Cancer Institute repository) and 17- to 19-month old (NIA repository) C57BL/6 (BL6) mice were purchased. Young FoxP3-IRES-red fluorescent protein (FIR) mice (24) provided by Richard Flavell and Foxp3DTR mice (25) from Alexander Rudensky were aged in our facility. Young mice are 2 to 6 months old. Aged mice are 22 to 26 months old. All animal studies were approved by the The University of Texas Health Science Center at San Antonio (UTHSCSA) Institutional Animal Care and Use Committee.
B16F10 melanoma and MC-38 colon carcinoma were purchased from the American Type Culture Collection. We engineered OVA-expressing B16F10 cells (26) and MC-38 cells (27), herein referred to as B16 and MC-38, respectively, for simplicity. Tumor challenge was injection of 250,000 B16 or 1 × 106 MC-38 tumor cells subcutaneously as described (26). Tumor growth was measured with Vernier calipers and volume calculated as (length × width2)/2.
Starting 2 days after tumor challenge, BL6 mice were injected i.p. every other day with 5 μg denileukin diftitox (DT; Eisai) or PBS control to deplete Tregs, and/or with 200 μg of anti–Gr-1 monoclonal antibody (mAb; clone RB6.8C5–18) or isotype control to deplete Gr-1+ cells. Foxp3DTR mice were injected with 15 μg diphtheria toxin per kilogram or PBS once, and sacrificed 7 to 9 days later as indicated. B16-bearing mice were treated with 25 or 50 mg 5-fluorouracil per kilogram or PBS once, 1 week after tumor challenge, and sacrificed 7 days later.
We isolated and stained cells and carried out cell sorts as previously described (28), using LSR II and FACSAria flow cytometers and FACSDiva software (BD Bioscience). Anti-CD11b (M1/70), anti-interferon (IFN)-γ (XMG1.2), anti-CD25 (PC61), anti-CD69 (H1.2F3), anti-CD4 (GK1.5), anti-CD3 (500A2), anti-Gr-1 (RB6-8C5), Ly-6C (AL-21), and matched isotype control antibodies (Ab) were from BD Pharmingen. Anti-CD62L (MEL14), anti-CD44 (1M7), anti-interleukin (IL)-17A (17B7), and anti-Foxp3 (FJK-16a) were purchased from eBioscience; anti-CD8α (5H10) from Caltag Laboratories; Ly6-G (1A8) and anti-CD44 (1M7) mAbs from Biolegend. CD8+ OVA-specific T cells were detected with OVA-specific pentamers (ProImmune).
Treg suppression assays were done as previously described (26). Briefly, Tregs (CD4+CD25hi from BL6 mice or CD4+ Foxp3+CD25hi or CD4+ Foxp3+CD25lo from FIR mice) were sorted from spleens after CD4+ T-cell enrichment (StemCell). For MDSC suppression assays, CD11b+Gr-1hi cells were sorted from spleens. Carboxyfluorescein succinimidyl ester-labeled CD4+ responder T cells from naïve mice were incubated at 3 × 104 cell per well in 96-well plates with Tregs or MDSCs, and CD3/CD28 T Cell Expander beads (Invitrogen). After 4 days incubation at 37°C, responder cell proliferation was measured by carboxyfluorescein succinimidyl ester dilution by fluorescence-activated cell sorting (FACS).
Bone marrow cultures
At sacrifice, femurs were flushed, washed in PBS, and bone marrow (BM) cells were cultured in 10% fetal calf serum/RPMI at 5 × 105 cells per well in 4 ng/mL GM-CSF (PeProTech Inc.) at 37°C in 5% CO2. Cells were recovered for counting and FACS analysis on day 4.
Serum cytokines were assessed by a custom multiplex Bio-Plex Pro assay kit (Bio-Rad) according to the manufacturer's protocol. Cytokines levels were measured with a Luminex 200 analyzer (Luminex Corp.).
Statistical analyses were conducted with Prizm software (GraphPad Inc.). For tumor growth measurements, we used a one-way ANOVA comparing treatment arms. For all other single measurement assays, we used a 2-tailed Mann–Whitney test. P < 0.05 was considered significant.
Treg depletion is clinically efficacious in young but not in aged B16-bearing mice
As CD4+CD25+Foxp3+ Tregs contribute to immune dysfunction in cancer (6, 8), we first showed that naïve young and aged mice had comparable numbers of CD4+ Foxp3+CD25hi spleen phenotypic Tregs (P = 0.1, Fig. 1A). B16-challenged aged mice had a higher prevalence of spleen CD4+Foxp3+CD25hi T cells compared with young mice (Fig. 1B) but absolute numbers were similar (P = 0.5, Fig. 1A). Following challenge, B16 tumor grew similarly in young and aged mice in the absence of specific treatment (Fig. 1C). DT treatment, which depletes young mouse Treg efficiently (29), significantly reduced B16 growth in young mice as expected (P = 0.0001, young DT vs. PBS), but strikingly had no effect in aged mice (P = 0.4, Fig. 1A). DT reduced numbers (Fig. 1A) and prevalence (Fig. 1B) of CD4+Foxp3+CD25hi T cells in spleens of young mice as expected and was even more effective in depleting Tregs in aged mice (Fig. 1A and B). Spleen CD4+CD25hi Tregs at baseline and after tumor challenge from young and aged mice were comparably suppressive in vitro, with no significant change after DT treatment (Fig. 1D).
Treg depletion improves tumor antigen–specific immunity in young but not aged hosts with B16
Untreated, aged B16-bearing mice have a higher prevalence of IL-17+ (Fig. 2C, Supplementary Fig. S1) and activated T cells (Supplementary Fig. S2) compared with young mice, consistent with greater inflammation in age (27, 30). DT increased tumor-antigen–specific CD8+ T cells (Fig. 2A) in spleens of young but not aged B16-bearing mice. DT increased the prevalence and numbers of IFN-γ- and IL-17–producing CD4+ and CD8+ spleen T cells in young B16-bearing mice while reducing these cells in aged mice (Fig. 2B and C; Supplementary Fig. S1), suggesting differential age-related regulatory control of these cytokines producing T cells in B16-bearing mice. Finally, DT increased the prevalence of CD44−CD62L+ effector memory CD8+ T cells in young but not aged mice and their activation as measured by CD69 expression (Supplementary Fig. S2A and S2B). DT increased the prevalence and activation of naïve CD62L+CD4+ T cells in young mice but reduced CD69 expression in aged mice (Supplementary Fig. S2B and S2D). Together, these data are consistent with better immune improvement in DT-treated young versus aged hosts with B16 melanoma.
Lack of efficacy of DT in aged mice could result from: (i) a defect in aged Treg in hampering antitumor immunity, (ii) a defect in aged effector cells to produce significant antitumor immunity following Treg depletion, or (iii) additional age-dependent regulatory mechanisms preventing effective antitumor immunity after Treg depletion.
Functional Foxp3+CD25lo Tregs increase with age
To test for potential additional Treg populations, we used FIR mice in which Tregs are identified as red fluorescent protein+Foxp3+ cells (24). Foxp3+CD4+ T-cell prevalence increased with age (Fig. 3A and B) and, in particular, Foxp3+CD4+ T cells expressing low levels of CD25 (CD4+FoxP3+CD25lo; Fig. 3C), previously described as being equally suppressive as conventional CD4+Foxp3+CD25hi Tregs in young naïve mice (31). CD4+Foxp3+CD25lo Tregs from aged naïve mice were functional but less suppressive than conventional CD4+Foxp3+CD25hi Tregs in vitro (Fig. 3D).
DT depletes both CD25hi and CD25lo Foxp3+ Tregs in tumor-bearing aged mice
Because DT targets IL-2 receptor–expressing cells and not Tregs specifically (6), it was possible that DT failed to deplete this Foxp3+CD25lo Treg subpopulation prominent in aged tumor-bearing mice. Aged FIR mice were challenged with B16 and treated with DT. Tumor growth in aged FIR mice was comparable with that in wild-type (WT) mice, and DT treatment was again clinically ineffective (Fig. 4A). Two days after the last DT injection, both CD25hi and CD25loFoxp3+CD4+ spleen T cells were reduced, although conventional Foxp3+CD25hi Tregs were preferentially depleted (Fig. 4B). The proportion of CD25hi Tregs among total Foxp3+ cells was significantly reduced by DT treatment (P = 0.01, Fig. 4C).
CD25hi and CD25lo Foxp3+CD4+ T cells are equally suppressive in aged B16-bearing mice
We tested the suppressive function of CD25hi and CD25lo Foxp3+CD4+ populations and found that both Treg subsets were equally suppressive in aged B16-bearing mice (Fig. 4D) in contrast to their differential suppression capacity in aged naïve mice (Fig. 3D), suggesting that tumors can alter the function of a specific Treg subset, in this case making it more suppressive. Nonetheless, DT treatment did not affect the suppressive function of remaining Tregs in aged mice (Fig. 4D). As in WT mice, DT treatment did not significantly increased tumor specific or IFN-γ+CD8+ T cells in spleens of DT-treated B16-bearing FIR mice (Fig. 4E). Thus, lack of clinical efficacy of DT treatment in aged B16-bearing mice is not due to lack of depletion of a specific Treg subpopulation.
Treg depletion increases MDSC numbers in B16-bearing aged mice
As differential Treg depletion seemed unlikely to explain differential treatment effects, we tested DT treatment effects on MDSCs as they are reportedly increased in aged mice and contribute to immune dysfunction in cancer (32), including in aged hosts (22). Consistent with prior reports (22, 33), aged mice had a higher prevalence (Fig. 5A) and numbers (Fig. 5B) of spleen CD11b+Gr-1hi MDSCs at baseline and after tumor challenge. DT treatment slightly reduced the numbers and prevalence of CD11b+Gr-1hi MDSC in young tumor-bearing mice (Fig. 5A and B). By striking contrast, DT significantly increased the prevalence and numbers of CD11b+Gr-1hi MDSCs in aged B16-bearing mice (Fig. 5A and B). MDSCs were also significantly more suppressive in aged versus young mice, both at baseline and after B16 challenge, which was not affected by DT treatment (Fig. 5C).
MDSC prevalence was inversely correlated with Treg prevalence only in aged B16-bearing mice (Fig. 5D) suggesting that Tregs could regulate MDSC numbers in aged B16-bearing mice and that Treg depletion in aged mice leads to an undesired increase in MDSCs, thus negating the beneficial antitumor effects of DT-mediated Treg depletion observed in young mice. Increased spleen MDSCs following DT treatment and their highly suppressive character was confirmed in aged B16-bearing FIR mice (Supplementary Fig. S3A–S3C). Because MDSC subsets could have differential function, we then showed that DT had similar effects on CD11b+Gr-1med MDSC (Supplementary Fig. S3D).
MDSC depletion is clinically efficacious in aged but not in young B16-bearing mice
We tested effects of MDSC depletion given their deleterious effects. Anti–Gr-1 antibody depleted CD11b+Gr1hi MDSC in spleens of young and aged B16-bearing mice (Fig. 6A and B). Similar to DT treatment, anti–Gr-1 antibody did not significantly affect the suppressive function of remaining MDSCs in young or aged mice (Fig. 6C). However, by contrast to DT-mediated Treg depletion, anti–Gr1-mediated depletion of MDSC resulted in significantly slower tumor growth in aged but not young B16-bearing mice (P = 0.007 for aged, Fig. 6D). MDSC depletion yielded significantly more IFN-γ+CD8+ T cells in aged versus young mice (Fig. 6E) suggesting improved antitumor immunity as a mechanism for treatment efficacy, without significant effect on the prevalence of Foxp3+CD25hi Tregs (Fig. 6F) or Treg function (Supplementary Fig. S4A) in both young and aged mice. Surprisingly, anti–Gr-1 antibody increased CD11b+Gr1med MDSC in young but not aged hosts (Supplementary Fig. S4B).
Combining Treg depletion with MDSC depletion increases clinical response in aged B16-bearing mice
As MDSC seem to contribute more to cancer-related immune dysfunction in aged versus young mice with B16, and because DT-mediated Treg depletion increased these dysfunctional MDSC in aged B16-bearing mice, we hypothesized that combining both Treg and MDSC depletion would significantly boost antitumor immunity and clinical efficacy in aged mice. Thus, we challenged aged BL6 mice with B16 and treated with anti–Gr-1 antibody alone or combined with DT. Combination therapy significantly reduced tumor growth and weight in aged mice better than anti–Gr-1 treatment alone (Fig. 7A). Reduced tumor growth was associated with increased IFN-γ+CD4+ and IFN-γ+CD8+ T-cell prevalence (Fig. 7B), and increased tumor-specific CD8+ T-cell prevalence in spleen and tumor draining lymph nodes (DLN; Fig. 7C). Anti–Gr-1 treatment again reduced MDSCs in spleen and tumor (Fig. 7D). Combination therapy reduced CD25hiCD4+ phenotypic Tregs similar to DT treatment alone (Fig. 7E). In confirmation, combination therapy significantly reduced tumor growth in aged FIR mice over anti–Gr-1 antibody alone (Supplementary Fig. S4C and S4D).
Tregs restrain MDSC numbers in young and aged naïve and B16-bearing hosts
DT clearly depletes Tregs, but is not Treg specific (6). To test specific Tregs contributions to MDSC effects, we challenged syngeneic Foxp3DTR mice with diphtheria toxin, which depletes only Tregs (34). Following a single injection of diphtheria toxin, young and aged B16-bearing mice experienced similar MDSC increases 9 days later (Fig. 5E) confirming Treg-mediated MDSC control in B16 tumor, but not just in aged hosts. To test whether effects depended on tumor, we depleted Tregs from young and aged naïve Foxp3DTR mice and found similar MDSC increases (Fig. 5F). Thus, DT has an age- and tumor-specific effect on MDSC control, but this effect might not be exclusively from Treg depletion.
High-order Treg depletion improves immune and clinical responses to B16 in aged hosts
Treg depletion is essentially complete in Foxp3DTR mice treated with diphtheria toxin (34; and data not shown), contrasting to lesser Treg depletion with DT (Fig. 1A and B). This high-order Treg depletion in aged Foxp3DTR mice improved clinical tumor control (Fig. 2D), IFN-γ production, and antitumor immunity (Fig. 2E). Thus, with sufficient Treg reduction alone, aged hosts can mount clinically effective antitumor immunity (Fig. 2D and E).
Treatment effects are tumor dependent
Because DT treatment effects could be tumor- as well as age dependent, we assessed treatment effects in MC-38 colon carcinoma. Unlike results in B16, DT-mediated Treg depletion was beneficial to tumor growth (Fig. 1E and F) and antitumor immunity (Supplementary Fig. S5A–S5F) in young and aged mice. Anti–Gr-1 antibody further boosted clinical effects in young but not aged hosts (Fig. 1E and F) although DT increased CD11b+Gr-1hi MDSC in both (Fig. 5G). A significant increase in CD11b+Gr-1med was also observed only in young MC-38–bearing mice treated with DT (Supplementary Fig. S3E). Tumor weights (Fig. 1F) were similar in aged B16-bearing mice treated with PBS and anti–Gr-1–treated mice despite volumetric differences.
DT and Treg depletion affect myelopoiesis in vitro
To test whether Treg depletion effects on MDSCs related to altered myelopoiesis, we cultured BM from B16-bearing Foxp3DTR mice and MC-38-bearing WT mice with 4 ng/mL GM-CSF for 4 days. High-order Treg depletion or DT treatment skewed BM differentiation toward the Ly-6ChiLy-6G− subset in B16-bearing Foxp3DTR mice (Supplementary Fig. S6A), and MC-38–bearing C57/BL6 mice (Supplementary Fig. S7A and S7B). These data establish that Tregs and DT treatment can alter myelopoiesis in tumor-bearing mice, but additional work is required to establish the in vivo significance. GM-CSF, VEGF, M-CSF, IL-6 (among others) can drive MDSC generation in vivo (32), but we found similar serum levels of these cytokines in B16 (Supplementary Fig. S6B) or MC-38 (Supplementary Fig. S7C) bearing young or aged mice following high-order Treg depletion or DT treatment, and equivalent increases in GM-CSF+ T lymphocytes in B16-bearing (Supplementary Fig. S6C and S6D) and MC-38–bearing mice (Supplementary Fig. S7D). Furthermore, DT did not alter GM-CSF, VEGF, M-CSF (not shown), or IL-6 in young or aged B16-bearing mice (Supplementary Fig. S8). These data establish that Tregs control factors related to MDSC generation in tumor-bearing mice.
5-fluorouracil does not treat B16 melanoma effectively
Finally, we tested an agent contemplated for MDSC depletion in humans, 5-fluorouracil. In striking contrast to a published report (35), 5-fluorouracil did not deplete MDSC in B16 melanoma and worsened clinical tumor response (Fig. 6H) with no effect on non-Treg T cells, B cells, or non-MDSC myeloid cell subsets (not shown), and no alteration of MDSC or Treg function in vitro (Supplementary Fig. S9).
Although increasing age is the biggest risk factor for cancer, surprisingly few studies have addressed the specific consequences of age on antitumor immunity, and fewer still have examined age effects on tumor immunotherapy. Age-dependent immune alterations including the generally decreased performance of effector T cells could reduce antitumor immunity. Naive T cells from older individuals show functional defects such as impaired ability to proliferate, produce relevant cytokines, and reduced differentiation into effector T cells (12–13). Thus, many immunotherapies effective in young hosts are less so in aged hosts. For example, tumor rejection mediated through OX40 signals decreases with age, as does effector T-cell differentiation (36). However, effective cancer immunotherapy for aged individuals is a realistic goal, as some age-associated immune defects are reversible. As examples, tumor immunity in aged mice can be rescued with sufficient cosignaling (37), and reduced T-cell priming boosted by the immune cosignaling CD137 (41BB) pathway (38).
A complementary approach to boosting effector arms of immunity in cancer immunotherapy is to reduce tumor-associated immune dysfunction (5, 6). As we and others have previously demonstrated the utility of depleting Tregs as a means to reverse tumor-associated immune dysfunction to treat cancer in (young) mouse models (6), we tested the concept in aged hosts.
In our B16 melanoma model, Treg depletion with DT was immunologically and clinically effective in young mice as expected. Disappointingly, though, depleting Tregs with DT in aged hosts did not improve antitumor immunity or immune-mediated tumor rejection. For humane reasons, we studied tumor growth, not survival, but tumor growth and weights are good surrogates for survival in B16 melanoma. We showed that Treg numbers and in vitro function of young and aged Tregs were comparable, and that DT equally depleted young and aged Tregs. Thus, poor clinical efficacy of Treg depletion in aged hosts could be attributed to a defect in aged antitumor effector cells, other immunosuppressive mechanisms, or to tumor-specific factors (21). To assess for additional regulatory populations, we found an age-associated increase in CD25loFoxp3+ Tregs as reported, and showed that they were more suppressive in the tumor environment than in naïve mice. It is likely that Tregs contribute differentially to immunopathology in a tumor-dependent fashion as seen in both young and aged hosts (6, 20, 21), but effects of distinct Treg subsets remain to be defined. We further found an age-associated increase in MDSC that were more suppressive in vitro compared with young counterparts as reported (22). Surprisingly, DT-mediated Treg depletion increased MDSC numbers in aged but not young hosts with B16 melanoma, suggesting a mechanism for DT treatment failure, and suggesting that MDSC depletion combined with Treg depletion could be useful cancer immunotherapy in aged hosts in this model.
In support, anti–Gr-1 antibody depleted MDSC effectively in young and aged hosts, but improved antitumor immunity and was clinically effective only in aged hosts with B16. Consistent with increased MDSC reducing DT efficacy is aged hosts, addition of anti–Gr-1 antibody improved Treg depletion efficacy in aged, but not young hosts in this model. These data confirm that aged effector cells remain competent and can mediate important effector functions provided that the impeding immune dysfunction is reduced. As anti–Gr-1 antibody is not specific for MDSC, dysfunctional contributions from other Gr-1+ cells, such as plasmacytoid dendritic cells, are not entirely excluded and require further investigation.
As DT only incompletely depletes Tregs, and targets IL-2 receptor–expressing cells, not Tregs specifically, we used Foxp3DTR mice in which specific, high-order Treg depletion is possible. High-order Treg depletion in young and aged naïve or B16-bearing hosts equally boosted MDSCs. These data confirm that Treg constrain tumor MDSC, but show that DT-mediated Treg depletion differs from high-order Treg depletion in MDSC effects, which could be due to better Treg depletion in Foxp3DTR mice, lack of depletion of a specific Treg subset by DT, DT effects on another IL-2 receptor–expressing cell, or reduced Treg function from the greater inflammation in Foxp3DTR versus DT-treated mice. Understanding DT-mediated effects on MDSC restraint helps define mechanisms of treatment responses, important to clinical translation, as DT is U.S. Food and Drug Administration (FDA)-approved for some cancers, and depletes Tregs in humans (6). Understanding mechanisms of MDSC restraint by Tregs in cancer furthers basic knowledge of cancer immunopathology. Both approaches are important to developing optimal, translatable cancer immunotherapies.
MDSCs are myeloid cells generated through cooperation of numerous factors (32). We found that DT and high-order Treg depletion had similar effects on in vitro myelopoiesis, including skewing to potentially more immunosuppressive Ly-6C+ MDSC, and increasing in vivo T-cell GM-CSF production but without clear effects on global GM-CSF, VEGF, M-CSF, or IL-6. Thus, Tregs and DT each can affect myelopoiesis, which might affect immunotherapy outcomes. DT effects on MDSCs deserve additional attention to help translate concepts, as selective high-order Treg depletion in humans is currently unachievable and could pose autoimmune risks in any regard.
High-order Treg depletion alone also improved immune and clinical responses to B16 in aged hosts, further showing the underlying immune competence of aged antitumor effector cells, despite the difficulty in translating this approach clinically. In contrast to DT effects, though, high-order Treg depletion was clinically and immunologically efficacious despite increases in MDSCs. This result could be due to Treg-specific effects in Foxp3DTR mice, to alterations in the inflammatory environment from high-order Treg depletion, or differential MDSC functions after DT versus high-order Treg depletion, among other factors. If clinical effects are specifically from high-order Treg depletion, as opposed to from effects on some IL-2–expressing cell population, that suggests that IL-2 receptor targeting agents for human Treg depletion such as DT or daclizumab might be ultimately inferior to agents that specifically target human Tregs (which do not yet exist). Understanding this point could greatly improve Treg management strategies.
Strikingly, in another tumor, MC-38 colon carcinoma, MDSC depletion was effective in young hosts and further boosted utility of DT-mediated Treg depletion. By contrast, and distinct from B16 results, DT alone was useful in aged hosts but not further augmented by MDSC depletion. Reminiscent of Foxp3DTR results, DT improved immunity in young and aged hosts, despite increasing MDSC in both. Increased antitumor immunity correlated with clinical outcomes supporting the concept that immune dysfunction was improved with the distinct approaches. These data suggest that immune dysfunction is not only age-dependent but also tumor-dependent, affecting both Tregs and MDSC. Our preliminary ex vivo studies did not disclose a noticeable difference in myelopoiesis in BM of mice bearing MC-38 or B16, or in cytokines affecting MDSC generation following Treg depletion in each tumor. Additional work is required to understand specifics of MDSC generation and immunopathogenesis in individual tumors as a function of age and as a consequence of distinct treatment modalities.
Because MDSC depletion alone and in combination with Treg depletion was effective in distinct tumors and hosts, we assessed a translational approach. 5-fluorouracil, is FDA approved and thus relatively easily tested as human cancer immunotherapy. Our surprising finding that 5-fluorouracil does not deplete MDSC in B16 and worsens the clinical course suggests that MDSC depletion agents will also have variable effects depending on the tumor and host. Thus, unlike DT, which depletes Tregs in humans analogous to animal models, MDSC depletion studies in preclinical models might not predict human clinical effects as well.
Together, our data show that the function of aged antitumor effector T cells can be improved by reducing age-related immune dysfunction, with positive clinical benefits. Strategies to reduce immune dysfunction must be tailored to account for age-related and tumor-related immune dysfunctions for optimal utility. Thus, improved and efficacious cancer immune therapy for aged hosts, who are at the greatest risk for cancer, is a realistic goal that can be met with a better understanding of the specific effects of age on basal and tumor-related immune dysfunction. Strategies we propose are clinically translatable as FDA-approved agents such as DT, cyclophosphamide, and daclizumab deplete Tregs (6), and FDA-approved agents such as 5-fluorouracil kill MDSCs in some models (35).
Disclosure of Potential Conflicts of Interest
T.J. Curiel is a consultant on the advisory board of Eisai.
Conception and design: V. Hurez, B.J. Daniel, S.R. Thibodeaux, T. Shin, T.J. Curiel
Development of methodology: V. Hurez, B.J. Daniel, L. Sun, S.R. Thibodeaux, C. Livi, T. Shin, B. Zhang, T.J. Curiel
Acquisition of data: V. Hurez, B.J. Daniel, L. Sun, A.-J. Liu, S. Ludwig, M. Kious, S.R. Thibodeaux, S. Pandeswara, C. Livi, S. Wall, M.J. Brumlik, T. Shin
Analysis and interpretation of data: V. Hurez, B.J. Daniel, L. Sun, S.R. Thibodeaux, C. Livi, S. Wall, M.J. Brumlik, T. Shin, T.J. Curiel
Writing, review, and/or revision of the manuscript: V. Hurez, B.J. Daniel, L. Sun, S.R. Thibodeaux, T. Shin, B. Zhang, T.J. Curiel
Administrative, technical, or material support: V. Hurez, S. Pandeswara, K. Murthy, S. Wall, T.J. Curiel
Study supervision: V. Hurez, T. Shin, T.J. Curiel
The authors thank Kristina M. Church, Xiuhua Sun, and Pei Yi Lin for technical help.
This work was supported by the Voelcker Foundation, The Holly Beach Public Library Association, 1RC2 AG036613, 5P30CA54174, Texas STARS, and the Owens Foundation.
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.