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Immune cell therapies and immune cell engineering
Original research
Distinct host preconditioning regimens differentially impact the antitumor potency of adoptively transferred Th17 cells
  1. Megen C Wittling1,2,
  2. Hannah M Knochelmann1,3,
  3. Megan M Wyatt1,
  4. Guillermo O Rangel Rivera1,4,
  5. Anna C Cole1,
  6. Gregory B Lesinski5 and
  7. Chrystal M Paulos1
  1. 1Surgery/Oncology & Microbiology/Immunology, Emory University, Atlanta, Georgia, USA
  2. 2School of Medicine, Emory University, Atlanta, Georgia, USA
  3. 3Medicine, Stanford University School of Medicine, Stanford, California, USA
  4. 4Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA
  5. 5Hematology and Oncology, Emory University, Atlanta, Georgia, USA
  1. Correspondence to Megen C Wittling; megen.wittling{at}emory.edu; Dr. Gregory B Lesinski; gregory.b.lesinski{at}emory.edu; Dr. Chrystal M Paulos; chrystal.mary.paulos{at}emory.edu

  • GBL and CMP are joint senior authors.

Abstract

Background How distinct methods of host preconditioning impact the efficacy of adoptively transferred antitumor T helper cells is unknown.

Methods CD4+ T cells with a transgenic T-cell receptor that recognize tyrosinase-related peptide (TRP)-1 melanoma antigen were polarized to the T helper 17 (Th17) phenotype and then transferred into melanoma-bearing mice preconditioned with either total body irradiation or chemotherapy.

Results We found that preconditioning mice with a non-myeloablative dose of total body irradiation (TBI of 5 Gy) was more effective than using an equivalently dosed non-myeloablative chemotherapy (cyclophosphamide (CTX) of 200 mg/kg) at augmenting therapeutic activity of antitumor TRP-1 Th17 cells. Antitumor Th17 cells engrafted better following preconditioning with TBI and regressed large established melanoma in all animals. Conversely, only half of mice survived long-term when preconditioned with CTX and infused with anti-melanoma Th17 cells. Interleukin (IL)-17 and interferon-γ, produced by the infused Th17 cells, were detected in animals given either TBI or CTX preconditioning. Interestingly, inflammatory cytokines (granulocyte colony stimulating factor, IL-6, monocyte chemoattractant protein-1, IL-5, and keratinocyte chemoattractant) were significantly elevated in the serum of mice preconditioned with TBI versus CTX after Th17 therapy. The addition of fludarabine (FLU, 200 mg/kg) to CTX (200 mg/kg) improved the antitumor response to the same degree mediated by TBI, whereas FLU alone with Th17 therapy was ineffective.

Conclusions Our results indicate, for the first time, that the antitumor response, persistence, and cytokine profiles resulting from Th17 therapy are impacted by the specific regimen of host preconditioning. This work is important for understanding mechanisms that promote long-lived responses by adoptive cellular therapy, particularly as CD4+ based T-cell therapies are now emerging in the clinic.

  • Chemotherapy
  • Radioimmunotherapy
  • Skin Cancer
  • Adoptive cell therapy - ACT
  • T cell

Data availability statement

Data are available upon reasonable request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-008715

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Adoptive cellular therapy (ACT) has been a breakthrough in terms of treatment for patients with hematologic malignancies yet has room for improvement for the treatment of solid tumors. One major way in which the efficacy of this therapy is impacted is via lymphodepletion prior to cell transfer, as multiple mechanisms then assist the transferred cell’s ability to engraft and persist. The two major methods for preconditioning include total body irradiation and chemotherapy, and yet the difference between these two approaches has not been previously explored on its impact on CD4+ ACT.

    WHAT THIS STUDY ADDS

    • This study explored how total body irradiation versus chemotherapy affected the antitumor activity of transferred CD4+ T helper 17 cells. Interestingly, these preconditioning methods differentially impacted the success of this therapy, highlighting that more optimal ways of priming the host before cell transfer are needed. Additionally, as cytokine profiles and T-cell persistence were different between these conditioning methods, it suggests that total body irradiation has additional unexplored benefits that aid CD4+ ACT.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • This research has major implications as while total body irradiation (TBI) versus cyclophosphamide (CTX) did not appear to differentially impact responses in CD8+ ACT, it does differentially impact the antitumor activity of CD4+ ACT. Our results show that preconditioning enhances CD4+ ACT and that TBI is more effective than single-agent chemotherapies, perhaps by inducing unique cytokine factors that could be used in the future to further bolster preconditioning methods more broadly used in the clinic. Combining fludarabine with CTX, a regimen often received by patients prior to ACT, improved survival, engraftment, and antitumor activity to levels comparable with TBI.

    Background

    Adoptive T-cell transfer (ACT) therapy involves the infusion of tumor-specific T cells into patients and ranges from tumor-infiltrating lymphocytes (TIL) to lymphocytes engineered with antigen receptors (chimeric antigen receptor or T-cell receptor (TCR)).1–6 Optimizing ACT to treat patients with aggressive malignancies is an important undertaking, given that this treatment shows promise in the clinic.7–10 In particular, lymphodepleting preconditioning regimens prior to ACT are standard modalities to enhance the potency of ACT and do so via multiple mechanisms.11–14 Yet, it remains unknown what type of host preconditioning is most ideal for CD4+ T helper cell therapies, a lymphocyte population that is emerging as promising to treat patients.15 16 To date, how different forms of non-myeloablative preconditioning regimens impact the efficacy of transferred antitumor T helper cells has not been explored in a systematic manner.

    Previous research has shown that lymphodepletion via total body irradiation (TBI) or cyclophosphamide is an effective regimen to enhance the antitumor activity of transferred anti-melanoma CD8+ T cells.17 Lymphodepletion transiently ablates immunosuppressive host cells, such as regulatory T cells and myeloid-derived suppressor cells, which actively blunt CD8+ T-cell functionality.18–20 Both forms of lymphodepletion create “space” via depleting host natural killer (NK) cells and lymphocytes, in turn inducing homeostatic cytokines that can be used to instead support the transferred T cells.21 22 Additionally, TBI mediates the systemic translocation of microbial ligands from the gut, known to activate antigen-presenting cells, in turn enhancing the function of transferred CD8+ TIL.14 Radiation can also activate dendritic cells (DCs) by mediating the systemic release of damage-associated molecular patterns and high-mobility group box 1 protein, resulting in improved adaptive responses to tumors.23 Cyclophosphamide has specific benefits as well—increasing interferon (IFN)-α production and aiding T helper 17 (Th17) differentiation24 25 as does fludarabine, which can lead to cytotoxicity through its effects on DNA synthesis.26 These two chemotherapy agents are also often combined for lymphodepletion prior to ACT.27 Most recently, both Th17 cells and hybrid Th1/17 cells (able to co-secrete interleukin (IL)-17A and IFN-γ) were enriched in patients with breast cancer responsive to neoadjuvant chemotherapy.28 However, despite these known benefits of lymphodepletion, the effects of these regimens on antitumor Th17 therapy are unknown.

    Non-myeloablative TBI is a common method to mediate host lymphodepletion in mice prior to ACT therapy.17 29–33 Aligned with this preclinical approach, a series of clinical trials at the National Cancer Institute (NCI) were conducted using TIL in patients and giving them various regimens of total body irradiation.9 Yet, most individuals given ACT are preconditioned with non-myeloablative chemotherapies, including cyclophosphamide (CTX) and/or fludarabine (FLU), as TBI at a dose of 1,200 cGy in humans has been associated with toxicities including profound neutropenia and thrombotic microangiopathy.34 In mice, it was reported that escalating the intensity of lymphodepletion by increasing doses of fractionated TBI stepwise elevated features of toxicity in animals, based on heightened cytokine storm signatures and translocation of gut microbes.35 While this approach improved outcomes in the preclinical setting, it was not without undue side effects. This work points to focusing on using non-myeloablative preparative regimens, including CTX and/or FLU (commonly used in the clinic) to augment T helper therapy. However, the mechanistic understanding of how these various preconditioning regimens impact infused cells and their therapeutic efficacy is unclear. Further, we posit that these approaches of lymphodepletion are not interchangeable and harbor distinct mechanistic differences that may impact subsequent ACT in vivo. A greater appreciation for these differences may inform the field for optimizing preclinical studies of CD4+ T-cell-based ACT in murine tumor models.

    Overarchingly, there are many advantageous aspects of preconditioning that remain to be investigated in the context of CD4+ T-cell therapies. Clinical trial data supports the use of chemotherapy prior to T-cell infusion, with CTX administration leading to both increased T-cell engraftment and persistence in patients.36 37 Given that both CTX and TBI allow for improved engraftment and deplete immunosuppressive host cells, we sought to define how these two distinct methods of lymphodepletion impact the therapeutic efficacy of anti-melanoma Th17 cell therapy, which is emerging as promising in the field. As CTX and FLU are often given in combination to patients prior to T-cell therapy, we also explored how this dual chemotherapy regimen compared with single agent chemotherapy and TBI. Herein we examine how these forms of host preconditioning impact the efficacy of a novel adoptive Th17 cell therapy that our team and others have found are potent against tumors.38–43

    Results

    Th17 cells mediate superior survival protection in mice compared with Th1 cells

    To test the potency of antitumor CD4+ Th1 versus Th17 cells as an adoptive immunotherapy, we used the transgenic tyrosinase-related peptide (TRP)-1 model where CD4+ T cells express a TCR that recognizes TRP on melanoma.40 Mice bearing B16F10 melanoma were preconditioned with 5 Gy TBI and were then treated with infusion of TRP-1 Th1 or Th17-cytokine programmed lymphocytes (figure 1A). As expected, TRP-1 Th1 cells secreted more IFN-γ and nominal levels of IL-17A ex vivo on reactivation (figure 1B,C). Conversely, Th17 cells mainly produced IL-17A but little IFN-γ after programming ex vivo (figure 1B,C). Moreover, the infusion of TRP-1 Th17 cells mediated the most efficacious antitumor responses in mice, as denoted by the ability of 22 of 26 animals (~85%) to survive nearly 2 months post ACT therapy with Th17 versus Th1 cells (figure 1D). These results corroborate those from other laboratories that have also found that Th17 cell therapy is effective against various solid tumors, often mediating long-lived responses in mice.44 ,40–43 45 46

    Figure 1
    Figure 1

    Adoptive transfer of TRP-1 Th17 cells significantly improves survival of mice bearing established melanoma, as compared with transferred Th1 cells. (A) Mouse experimental model. C57BL/6 mice bearing B16F10 melanoma were preconditioned with 5 Gy total body irradiation 1-day prior to adoptive cell transfer of TRP-1 specific Th1 (IL-12, αIL-4, IL-2) or Th17 (IL-6, IL-21, IL-1β, TGF-β, αIFN-γ, αIL-4, αIL-2) cells (1e6). (B) Representative flow plots and (C) Bar graph of IFN-γ and IL-17A production by TRP-1 subsets post reactivation with PMA/ionomycin on day 7 after expansion (n=3). Analysis via t-test. (D) Survival curve from three combined experiments of mice (n=6–10 mice/group) with day 7 established melanoma. Th17 versus Th1 p<0.0001 log-rank (Mantel-Cox) test. ACT, adoptive cellular therapy; α, anti-; IFN, interferon; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; TBI, total body irradiation; TGF-β, transforming growth factor-β; Th17, T helper 17; TRP, tyrosinase-related peptide.

    TBI as lymphodepletion prior to Th17 infusion improves antitumor activity in mice

    Given the potency of our antigen-specific Th17 cells, we hypothesized that preconditioning may not be needed for its efficacy. We explored this idea via comparing two distinct preconditioning regimens: chemotherapy using CTX or TBI prior to ACT. This cell therapy experiment was done by comparing a chemotherapy to TBI or no preconditioning prior to the transfer of Th17 cells into mice with melanoma (figure 2A). Moreover, we tested the impact of different preconditioning methods of lymphodepletion on in vivo tumor growth and survival of mice.

    Figure 2
    Figure 2

    Th17 therapy mediated superior survival in melanoma-bearing mice preconditioned with TBI compared with unconditioned mice or mice preconditioned with CTX. (A) Mouse experimental model. C57BL/6 mice bearing B16F10 melanoma were preconditioned with 5 Gy total body irradiation, 200 mg/kg CTX, or no preconditioning 1-day prior to adoptive cell transfer of tyrosinase-related peptide-1 specific Th17 cells. (B) Tumor growth curves (n=2). Note that the dotted lines are from one independent experiment and the solid lines are from a second independent experiment. Tumors were measured as length×width. (C) Overlay of TBI preconditioned and CTX preconditioned mice given ACT for two independent experiments (n=32 mice). (D) Survival curve for TBI+ACT and CTX+ACT groups. Log-rank test comparison of the two groups resulted in p<0.0001. ACT, adoptive cellular therapy; CTX, cyclophosphamide; TBI, total body irradiation; Th17, T helper 17.

    In contrast to our hypothesis that preconditioning is dispensable for the antitumor effects of our potent Th17 ACT, we found that if mice were not preconditioned with some form of a lymphodepletion, then all but one animal had rapidly growing tumors and reached tumor endpoint within a month (figure 2B). Radiation or chemotherapy alone (without an infusion of Th17 cells) was not effective. However, when mice were pretreated with either CTX or TBI prior to TRP-1 Th17 therapy, mice in both cohorts achieved cures, although with different efficacies (figure 2B). For example, most animals given TBI (15/16 mice) experienced tumor regression (figure 2B) and curative responses when infused with antitumor Th17 cells. In contrast, CTX was less efficacious overall, as only a few mice were cured via ACT, with most (10/16) succumbing to disease between 2 and 4 weeks post infusion of Th17 cells. This stark contrast in response can be appreciated in the overlay of these two conditions as shown in figure 2C. The two lymphodepletion regimens differentially impacted survival as well (figure 2D), as evidenced by improved survival in mice receiving Th17 ACT that were preconditioned with TBI versus CTX (p<0.0001).

    A unique cytokine signature is induced in the serum of mice treated with TBI Th17 therapy

    Because TBI preconditioning of mice was more effective than CTX when treating with Th17 ACT, we next examined what factors might potentially differentiate these therapies. We hypothesized that the induction of a cytokine profile may be associated with improved efficacy. We therefore assessed levels of serum cytokines in these mice 10 days after ACT. IFN-γ and IL-17 (produced by transferred Th17 cells) were elevated in animals given either lymphodepletion method when infused with Th17 cells (figure 3A). Note, these two cytokines were only elevated in mice treated with a preconditioning regimen combined with Th17 ACT (figure 3A). In CTX preconditioned mice with ACT, there are some mice with high IL-17 expression (~50 pg/mL) and half with lower IL-17 (~5 pg/mL) (figure 3A). Interestingly, the mice in this first cohort with more serum IL-17 survived longer (nearly 2 months post ACT) when given CTX. Overall, these findings demonstrate that preconditioning increased the functionality of transferred Th17 cells.

    Figure 3
    Figure 3

    TBI potentiates unique inflammatory but not type 1 or type 17 cytokines in mice compared with CTX conditioning. (A) Observed concentration in pg/mL of IL-17 and IFN-γ. Statistics were calculated via one-way ANOVA. (B) Observed concentration in pg/mL of cytokines KC, G-CSF, EPO, MCP-1, IL-6, and IL-5 in serum collected from mice. Statistics were calculated via one-way ANOVA. (C) Radar plot displaying the fold change of TBI Th17 and CTX Th17 groups compared with Th17 alone control. ANOVA, analysis of variance; CTX, cyclophosphamide; EPO, erythropoietin; G-CSF, granulocyte colony stimulating factor; IFN, interferon; IL, interleukin; KC, keratinocyte chemoattractant; MCP-1, monocyte chemoattractant protein-1; TBI, total body irradiation; Th17, T helper 17.

    Additionally, levels of homeostatic cytokines, IL-7 and IL-15, were evaluated in mice preconditioned with TBI or CTX and receiving ACT, but were not significantly different between these two different preconditioning methods regardless of time point examined (online supplemental figure 1). However, differences in other cytokine profiles were evident in mice given CTX versus TBI prior to Th17 therapy. Keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), erythropoietin (EPO), IL-6, and IL-5 were vastly elevated in the serum of mice treated with Th17 cells and preconditioned with TBI, compared with those given CTX (figure 3B). Fold change increases in these cytokines are visualized in figure 3C, with a systemic cytokine induction ranging from 3 to 36 times higher in animals given TBI, versus non-preconditioned mice.

    Supplemental material

    Infused Th17 cells persist best in mice preconditioned with TBI

    Another parameter used as a biomarker for successful ACT therapy is the degree to which the infused T-cell products engraft and persist in patients. Compared with mice given Th17 therapy alone (without preconditioning), both lymphodepletion methods permitted improved initial engraftment of transferred donor CD4+ Th17 cells (day 7) (figure 4A). Those preconditioned with TBI and given ACT had significantly increased engraftment on day 7, compared with the CTX and ACT group (figure 4A). This also held true at endpoint (day 39 post-ACT), where more TRP-1 Th17 cells were detected across multiple different tissues if the animals were preconditioned with TBI compared with CTX (figure 4B). This difference is further illustrated by a radar plot (figure 4C). In summary, these data suggest the TBI and CTX differentially impact Th17 functionality and persistence, as well as their acute ability to engraft, with TBI+ACT leading to an increase in cytokines, enhanced survival, and better persistence of transferred cells. Our findings also underscore that either form of lymphodepletion improves the success of Th17 therapy in this well-established melanoma model system.

    Figure 4
    Figure 4

    Adoptively transferred Th17 cells prevail best when mice are preconditioned with total body irradiation (A) Th17 cells from adoptive cellular therapy identified via Vβ14 positivity via flow. Gated on live/CD3+/CD4+/Vβ14+. Displayed as a per cent of live cells that are donor cells (CD4+ and Vβ14+) in the peripheral blood on day 7 in mice with 5 Gy TBI, CTX, or no preconditioning. Analysis via one-way analysis of variance. (B) Persistence at endpoint (D39) identified via percentage of CD4+T cells that are adoptively transferred (Vβ14+). Gated on live/CD3+/CD4+ in the peripheral blood, lymph node, skin, and spleen. Analysis via Mann-Whitney test. (C) Radar plot displaying the average percentage CD4+T cells that are adoptively transferred in the blood, spleen, lymph node, and skin at endpoint (D39). CTX, cyclophosphamide; TBI, total body irradiation; Th17, T helper 17.

    Dual chemotherapy improves ACT efficacy, comparable to that of TBI

    Patients with melanoma are often given a dual CTX and fludarabine lymphodepletion regimen prior to receiving ACT.46 Thus, we tested how CTX+FLU would impact Th17 therapy. To evaluate this question, we preconditioned mice with either TBI, single-agent chemotherapy (FLU or CTX), or dual chemotherapy (FLU+CTX). As a negative control, mice were not preconditioned. Mice were then infused with antigen-specific Th17 cells and monitored over time.

    As demonstrated in figure 5A, conditioning mice with either TBI or dual CTX+FLU regimens augments ACT, resulting in a significant reduction in tumor burden and improved overall survival. This finding is in contrast to mice receiving either CTX or FLU alone prior to ACT, which had more moderate tumor reduction but still had improved survival compared with animals given ACT without host preconditioning. Notably, survival between mice given Th17 therapy is not significantly different when preconditioned with either TBI or dual chemotherapy and ACT (figure 5B), indicating that the addition of a secondary chemotherapy agent can improve ACT and be equally efficacious.

    Figure 5
    Figure 5

    Addition of fludarabine to cyclophosphamide increases the antitumor activity, proliferation, and persistence of adoptively transferred Th17 cells comparable to TBI. (A) Tumor growth curves. Tumors were measured as length×width. (B) Survival curve of TBI+ACT, CTX+ACT, FLU+ACT, CTX+FLU+ACT, and the ACT alone groups. NS denotes survival differences between TBI+ACT and CTX+FLU+ACT are not significant. (C) Proliferation via Ki-67 expression. Peripheral blood was taken from mice on day 7 after ACT. Gated on live/CD3+/CD4+/Vβ14+/Ki-67+. Analysis via one-way analysis of variance. Representative flow plots are also displayed. (D) Engraftment of adoptively transferred cells in the peripheral blood on day 7 post-ACT. Represented as the percentage of live cells that are CD4+Vβ14+. Analysis via Mann-Whitney test. (E) Engraftment of adoptively transferred cells at endpoint (D42) in the peripheral blood, lymph node, and spleen. Represented as the percentage of live cells that are CD4+Vβ14+. Analysis via Mann-Whitney test. ACT, adoptive cellular therapy; CTX, cyclophosphamide; ns, not significant; TBI, total body irradiation; Th17, T helper 17.

    To further investigate how this dual chemotherapy regimen impacted Th17 therapy, mice were bled 1 week after ACT and evaluated for Th17 engraftment and proliferation (figure 5C,D). Th17 proliferation was significantly increased in the mice preconditioned with TBI, CTX+FLU, as well as CTX, indicating that these cells were expanding to a similar degree early on after transfer (figure 5C). However, transferred Th17 cells in the FLU or no preconditioning groups proliferated poorly. The engraftment of these cells was also examined on Day 7. Interestingly, we found more adoptively transferred Th17 cells in mice preconditioned with TBI compared with CTX+FLU (figure 5D). This early engraftment benefit does not appear to be significant long-term, however. When the blood, spleen, and lymph node were examined for persistence of adoptively transferred cells at endpoint (day 42), there was no difference in the number of donor Th17 cells in any of these organs between the TBI and CTX+FLU and ACT groups (figure 5E). Overall, these results indicate that both TBI and CTX+FLU aid in the proliferation and persistence of adoptively transferred Th17 cells.

    Summarized in figure 6, we demonstrate that antigen-specific Th17 cells are potent regressors of melanoma tumors and are a promising ACT strategy. We sought to investigate the important question of which preconditioning method would be optimal for CD4+ ACT models, a currently uninvestigated question—finding that TBI was superior to CTX or no preconditioning and allows for increased inflammatory cytokines, cell persistence, and antitumor activity. We found that a common method of patient preconditioning (CTX+FLU) similarly allowed these transferred cells to persist and mediate tumor regression when compared with TBI.

    Figure 6
    Figure 6

    Summary figure shows that CD4+T helper 17 ACT has superior efficacy when mice are preconditioned with total body irradiation compared with single-agent chemotherapy. This antitumor activity can be rescued with the addition of a second chemotherapy agent. There is differential induction of cytokines, enhanced survival, and increased persistence of adoptively transferred cells when using these different preconditioning methods. Made using BioRender. ACT, adoptive cellular therapy; CTX, cyclophosphamide; FLU, fludarabine.

    Discussion

    Cellular therapy has revolutionized medicine for patients.2 47 48 Yet ACT therapy is less effective in treating patients with solid tumors for several reasons, including (1) difficulty in T-cell trafficking to the tumor, (2) decreased T-cell fitness, and (3) immunosuppression in the tumor microenvironment (TME).2 49–51 Regarding the TME, regulatory T cells, fibroblasts, and myeloid-derived suppressor cells (MDSCs) blunt T-cell survival.52 However, despite the challenges faced in the treatment of solid tumors with cellular therapy, one key solution to this problem may be to refine how a patient is preconditioned prior to ACT therapy.

    Fundamentally, there is a great need to understand how transferred T cells interact with the tumor and with host immune cells. In essence, this knowledge could improve our application of preconditioning to maximize ACT efficacy, including Th17-based cell products. A wealth of data has been reported on how manipulating the host via lymphodepletion can augment infused T cells.19 22 53 Lymphodepletion augments ACT therapy in a multifactorial fashion—depleting host Regulatory T cells (Tregs) and MDSCs, stimulating the innate immune system, and enhancing T-cell activation.14 53 Finally, lymphodepletion reduces host cells that consume IL-7 and IL-15, in turn empowering the transferred T cells to consume these instead.22 Consistent with these data, these homeostatic cytokines gradually became detectable post-lymphodepletion in our model, but did not appear to be differentially impacted by modality of lymphodepletion. Yet it is unknown how host lymphodepletion impacts the effectiveness of antitumor CD4+ T helper cells, which have emerged in the clinic as a promising therapy.54–56

    Th17 cells have been a major focus of our group for many years, as they are potent regressors of melanoma tumors.40 57 Th17 cells have many features that make them desirable for ACT therapy, including their stem-like properties,58 polyfunctional cytokine nature,38 and capacity to persist long-term in vivo.59 In addition to their direct effects against the tumor, Th17 cells also engage other host cell types - activating antigen-specific CD8+ T cells60 and promoting B-cell activation and antibody production.61 Because Th17 cells have superior antitumor responses to that of Th1 cells and mediate cures in mice with melanoma when directed against relevant tumor antigens, we explored how preconditioning impacts this potent ACT product.

    We discovered that both forms of lymphodepletion (TBI and CTX) improved antitumor Th17 therapy. Although lymphodepletion with TBI best augmented Th17 therapy, resulting in more cures in animals. Tumor-bearing animals who were untreated died quickly, underscoring that host preconditioning only slightly delays tumor growth and that Th17 therapy is needed to mediate durable immunity. Several clues on the cytokines that were distinctly present in animals given TBI+Th17 might inform the mechanisms for why this therapy was more effective. Key cytokines that regulate neutrophils (KC, G-CSF), mature eosinophils (IL-5), prevent apoptosis of red blood cells (EPO) and mediate a “cytokine storm” (MCP-1, IL-6), were heightened in animals given TBI+Th17 therapy versus CTX+Th17 therapy.62–65 Future studies could test if administering these cytokines to animals given CTX could augment antitumor Th17 therapy to the degree seen in animals given TBI+Th17 therapy.

    We consistently found that infused TRP-1 Th17 cells persisted better in multiple organs of animals that were given TBI compared with CTX, and this factor might explain why this therapy was more effective. Future studies to elaborate on the specific host cells present in mice given either TBI or CTX are needed to better define how these host elements might regulate transferred Th17 cells. Collectively, this new body of work provides deeper insight into host responses to Th17 therapy, implying novel mechanisms that might promote effective ACT. For example, we detect resident memory Th17 cells in animals given TBI therapy, which might play a role in producing protective immunity.33 Perhaps IL-6 contributes to the durable memory of infused Th17 cells, as we reported that blocking it impaired the antitumor efficacy of this ACT approach.33 Neutrophils can also play a positive role in TRP-1 CD4+ T-cell therapy.66 As KC and G-CSF induce neutrophils, they might be responsible for augmenting the TBI+Th17 therapy. Future studies in our laboratory will test this idea.

    We discovered that combining fludarabine with CTX augmented Th17 therapy in a manner comparable to that of TBI. This finding has vast clinical implications, as it reveals modulating the type of lymphodepletion by using dual chemotherapeutic agents can recapitulate the benefit of TBI to augment the efficacy of CD4-based ACT therapies that are emerging in clinical development pipelines. The level of lymphodepletion induced by TBI, FLU, CTX, and CTX+FLU in combination has been previously explored in depth—indicating that indeed, CTX+FLU and TBI achieve similar levels of lymphodepletion, improved from that of CTX or FLU alone in these models.17 Notably, TBI did lead to slower immune reconstitution compared with chemotherapeutic agents though, despite comparable initial levels of lymphodepletion.17 However, while the level of lymphodepletion does appear to be correlated with CD4 ACT success, it is important to note that there are side effects associated with each of these regimens, and the addition of multiple agents might also decrease the tolerability of these regimens for patients. Future studies will continue to explore how these regimens impact host cells and ACT success.

    Additionally, while preconditioning mice with TBI prior to Th17 therapy was more potent that conditioning with CTX or FLU alone, we are not advocating the use of TBI in human patients given cellular therapy. Rather, our work reveals that it is important to further disentangle the mechanisms by which TBI mediates robust antitumor responses and use this insight to improve next-generation ACT protocols. Given the historic and widespread use of TBI in animal studies, this work can inform our interpretation of these preclinical results, which are often used as a rationale for translating cell therapy products into patients. CTX and CTX+FLU are used frequently in patients as a means of preconditioning for several important reasons: TBI can have many detrimental side effects and has only been conducted comprehensively in one investigation (the Surgery Branch NCI) in patients with melanoma treated with autologous TIL products.9 34 67 Also, this study, when comparing CTX and TBI in a melanoma TIL ACT setting, found that chemotherapy regimens with or without the addition of TBI all yielded similar results, with complete responses of around 24% in both groups.9 In contrast, mice given increasing TBI concentrations and then infused with antitumor CD8+ T cells experienced improved long-term survival, particularly when the mouse was given one myeloablative dose of TBI of 9 Gy with stem-cell support compared with its delivery in the fractionated format.35 These discrepancies additionally highlight the necessary investigation into the impact of these different preconditioning methods on different forms of ACT.

    Overall, our findings have important implications. We demonstrate that single-agent chemotherapy and radiation-based lymphodepletion methods are not equal and have different effects on cytokine induction and engraftment of CD4+ T cells. We find that antitumor responses vary between these methods but that the addition of a second, clinically-relevant chemotherapy agent, such as FLU, to CTX allows for ACT improvement comparable to that of TBI. These regimens may be further improved by supplemental therapies. Incorporating supplemental therapies along with ACT could encompass agents that would increase neutrophils, or reduce the number of host cells that act as suppressor cells. In particular, co-administering cancer vaccines and/or checkpoint blockade therapy deserves further investigation as a means to further augment the efficacy of ACT. Overall, our data reveal new mechanisms underlying how the host immune system can be altered to augment potent Th17-based immunotherapies for cancer.

    Methods

    Mice and tumor lines

    These studies were conducted using C57BL/6 and TRP-1 TCR transgenic mice (Rag−/− BWTRP-1 TCR) purchased from Jackson Laboratories. The TRP-1 TCR transgenic mice (Rag−/− BWTRP-1 TCR) are bred in-house at Emory University, while C57BL/6 mice were purchased from the Jackson Laboratory, and tumor experiments are conducted with mice ages 6–10 weeks. Three independent mouse experiments were performed, with experimental groups (TBI+ACT, CTX+ACT, FLU+ACT, CTX+FLU+ACT, ACT alone, TBI alone, CTX alone, FLU alone, CTX+FLU alone, and no treatment). Mice were randomized prior to treatment with no exclusion/inclusion criteria set. Randomization was performed by grouping mice based on small, medium, or large tumor size on day 6 post tumor injection, and then randomly assigning an equal number of each to the various treatment groups. Treatments and tumor measurements were always performed on the same day and relative time to avoid potential effects of confounders, and individuals were blinded to the treatment group of each mouse. The Institutional Animal Care and Use Committee at Emory University approved the animal work, and we additionally have the support of Emory’s Division of Laboratory and Animal Resources. Initial experiments were also previously conducted at the Medical University of South Carolina (MUSC) with support from the Institutional Animal Care and Use Committee at MUSC and support of MUSC’s Division of Laboratory and Animal Resources. B16F10 tumors were obtained from the laboratory of Dr Nicholas Restifo and were validated and confirmed pathogen and Mycoplasma-free via PCR screen.

    T-cell cultures

    TRP-1 cells

    Transgenic TRP-1 T cells were cultured in complete media (CM) using Th17 polarizing conditions (IL-6, IL-21, IL-1β, TGF-β, anti-Interferon-γ, anti-IL-4, anti-IL-2) as described previously.33 They were activated with 1 µmol/L TRP-1106–130 peptide (SGHNCGTCRPGWRGAACNQKILTVR) in the presence of splenocytes that were irradiated at 10 Gy in a 1:5 cell to splenocyte ratio and grown at a concentration of 1–1.5×106 cells/mL. 20 ng/mL of IL-23 (BioLegend) was added to the culture on days 2 and 3, and on day 4 cells were washed and resuspended in sterile phosphate-buffered saline (PBS) for ACT at a concentration of either 800,000 cells in 200 µL PBS per mouse or 400,000 cells in 200 µL PBS per mouse. For Th1 cells displayed in figure 1, TRP-1 T cells were cultured in the presence of IL-12 (3 ng/mL), αIL-4 (10 µg/mL), and IL-2 (100 IU/mL) for 1 week, splitting as needed with the addition of 100 IU/mL of IL-2 each time cells were split. These cells were injected at a concentration of 1 million cells in 200 µL of PBS per mouse. Th17 cells displayed in figure 1 were cultured using the same conditions described above but for 1 week instead of 4 days and injected at a concentration of 1 million cells in 200 µL of PBS per mouse.

    Adoptive cell transfer

    Previously, C57BL/6 mice were given subcutaneous B16F10 melanoma tumors by injection of 200 µL of 5×105 B16F10 cells in sterile PBS. Tumors were grown for 1-week prior to ACT. One-day prior to ACT, mice received either 5 Gy of TBI or 200 mg/kg (4 mg/mouse) of CTX, 200 mg/kg (4 mg/mouse) of fludarabine phosphate, a combination of 200 mg/kg (4 mg/mouse) of CTX and 200 mg/kg (4 mg/mouse) of fludarabine phosphate, or no preconditioning. ACT of the Th17 polarized TRP-1 cells as described above was performed by tail vein injection 1 week after tumor injection.

    Cytokine multiplex assay

    Serum from mice was collected on day 10 and was then stored at −80°C prior to analysis using the Eve Technologies 44-plex Mouse Cytokine Array Discovery Assay. Data from 1online supplemental figure 1 was collected on days 7, 12, and 16 and the serum used for the same assay.

    Tissue collection and processing

    On day 7, peripheral blood was collected from the mandibular vein into 0.125 mol/L EDTA, spun down, and resuspended in red blood cell lysis buffer (BioLegend) for 5 min, and then assayed using flow cytometry using the antibodies depicted in 1online supplemental table 1. Peripheral blood was collected again on day 10 and was spun down to collect serum that was sent for cytokine multiplex array (see above). Peripheral blood was also taken from a subset of mice on days 7, 12, and 16 to collect serum for data displayed in online supplemental figure 1 (see above). On day 39 (figure 4) or day 42 (figure 5), the lymph nodes, spleen, skin, and blood were collected from the mice after euthanasia. Lymph nodes were collected into CM media and then processed into a single cell suspension by mechanical dissociation over a 40 µm filter and assayed using flow cytometry. Spleens were also processed into single-cell suspension by mechanical dissociation over a 40 µm filter, then resuspended in red blood cell lysis buffer (BioLegend) for 5 min before assaying via flow cytometry. Blood was processed as described above and similarly assayed using flow cytometry. Skin was minced and incubated in buffer containing 3 mg/mL collagenase IV (Worthington Biochemical), and 0.2 mg/mL DNase (Sigma) in Hank’s balanced salt solution at 37°C for 1 hour with stirring. Digestion was neutralized with RPMI containing 10% fetal b0vine serum (FBS) and 10 mmol/L EDTA. Digested and processed tissue was filtered prior to assay using flow cytometry. online supplemental table 2 contains the antibodies used for this analysis.

    Supplemental material

    Flow cytometry

    Flow cytometry was performed using BD FACSymphony A3 Cell Analyzer and analyzed using FlowJo software (BD Biosciences). For extracellular antibodies, the samples were resuspended in Fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FBS) and incubated in antibodies at a 1:500 dilution for 20 min. Intracellular staining was conducted using the FoxP3/Transcription Factor kit according to the manufacturer’s instructions and a 1:200 dilution of antibodies (eBioscience). Antibodies used are displayed in online supplemental tables 1 and 2.

    Statistical analysis

    Comparisons of cytokines collected on day 10 were performed using one-way analysis of variance (ANOVA). Comparison of cytokines from day 7, 12, and 16 in online supplemental figure 1 was performed using the Mann-Whitney test. Day 7 engraftment data was compared using the Mann-Whitney test (figure 5) or one-way ANOVA (figure 4). Endpoint engraftment in all organs was compared using the Mann-Whitney test. Ki-67 levels were compared using one-way ANOVA. P values are depicted numerically on the figure with values less than 0.05 considered significant and ns denoting values that are not significant. For the IFN-γ and IL-17 comparison in figure 1, a t-test was used. Kaplan-Meier survival curves were compared between treatment group pairs using the log-rank test.

    Data availability statement

    Data are available upon reasonable request.

    Ethics statements

    Patient consent for publication

    Ethics approval

    Not applicable.

    Acknowledgments

    We would like to acknowledge the cores both at Medical University of South Carolina and the Winship Cancer Institute/Emory University that made this research possible including the Pediatric/Winship Flow Cytometry Shared Resource, Winship Cancer Animal Models Shared Resource (NIH/NCI award number P30CA138292), and FACS Shared Resource at Hollings Cancer Center, Medical University of South Carolina (P30CA138313).

    References

    1. ↵
      1. Giordano Attianese GMP,
      2. Ash S,
      3. Irving M
      . Coengineering specificity, safety, and function into T cells for cancer immunotherapy. Immunol Rev 2023;320:166–98. doi:10.1111/imr.13252
      OpenUrl
    2. ↵
      1. Knochelmann HM,
      2. Smith AS,
      3. Dwyer CJ, et al
      . CAR T cells in solid tumors: blueprints for building effective therapies. Front Immunol 2018;9:1740. doi:10.3389/fimmu.2018.01740
    3. ↵
      1. Hossian A,
      2. Hackett CS,
      3. Brentjens RJ, et al
      . Multipurposing cars: same engine, different vehicles. Mol Ther 2022;30:1381–95. doi:10.1016/j.ymthe.2022.02.012
      OpenUrl
    4. ↵
      1. Akce M,
      2. Zaidi MY,
      3. Waller EK, et al
      . The potential of CAR T cell therapy in Pancreatic cancer. Front Immunol 2018;9:2166. doi:10.3389/fimmu.2018.02166
    5. ↵
      1. Rosenberg SA,
      2. Dudley ME
      . Adoptive cell therapy for the treatment of patients with metastatic Melanoma. Curr Opin Immunol 2009;21:233–40. doi:10.1016/j.coi.2009.03.002
      OpenUrlCrossRefPubMedWeb of Science
    6. ↵
      1. Rohaan MW,
      2. Wilgenhof S,
      3. Haanen JBAG
      . Adoptive cellular therapies: the current landscape. Virchows Arch 2019;474:449–61. doi:10.1007/s00428-018-2484-0
      OpenUrlCrossRef
    7. ↵
      1. Porter DL,
      2. Levine BL,
      3. Kalos M, et al
      . Chimeric antigen receptor-modified T cells in chronic Lymphoid leukemia. N Engl J Med 2011;365:725–33. doi:10.1056/NEJMoa1103849
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Maude SL,
      2. Frey N,
      3. Shaw PA, et al
      . Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014;371:1507–17. doi:10.1056/NEJMoa1407222
      OpenUrlCrossRefPubMedWeb of Science
    9. ↵
      1. Goff SL,
      2. Dudley ME,
      3. Citrin DE, et al
      . Prospective evaluation comparing intensity of Lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic Melanoma. J Clin Oncol 2016;34:2389–97. doi:10.1200/JCO.2016.66.7220
      OpenUrlAbstract/FREE Full Text
    10. ↵
      1. Leidner R,
      2. Sanjuan Silva N,
      3. Huang H, et al
      . Neoantigen T-cell receptor gene therapy in Pancreatic cancer. N Engl J Med 2022;386:2112–9. doi:10.1056/NEJMoa2119662
      OpenUrlCrossRefPubMed
    11. ↵
      1. Paulos CM,
      2. Kaiser A,
      3. Wrzesinski C, et al
      . Toll-like receptors in tumor immunotherapy. Clin Cancer Res 2007;13:5280–9. doi:10.1158/1078-0432.CCR-07-1378
      OpenUrlAbstract/FREE Full Text
    12. ↵
      1. Gattinoni L,
      2. Powell DJ Jr,
      3. Rosenberg SA, et al
      . Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol 2006;6:383–93. doi:10.1038/nri1842
      OpenUrlCrossRefPubMedWeb of Science
    13. ↵
      1. Salem ML,
      2. Kadima AN,
      3. El-Naggar SA, et al
      . Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+ T-cell response to peptide vaccination: creation of a beneficial host Microenvironment involving type I Ifns and myeloid cells. J Immunother 2007;30:40–53. doi:10.1097/01.cji.0000211311.28739.e3
      OpenUrlCrossRefPubMed
    14. ↵
      1. Paulos CM,
      2. Wrzesinski C,
      3. Kaiser A, et al
      . Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling. J Clin Invest 2007;117:2197–204. doi:10.1172/JCI32205
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      1. Oh DY,
      2. Fong L
      . Cytotoxic CD4(+) T cells in cancer: expanding the immune effector toolbox. Immunity 2021;54:2701–11. doi:10.1016/j.immuni.2021.11.015
      OpenUrlCrossRefPubMed
    16. ↵
      1. Nelson MH,
      2. Knochelmann HM,
      3. Bailey SR, et al
      . Identification of human CD4(+) T cell populations with distinct antitumor activity. Sci Adv 2020;6:27. doi:10.1126/sciadv.aba7443
      OpenUrlCrossRef
    17. ↵
      1. Johnson CB,
      2. May BR,
      3. Riesenberg BP, et al
      . Enhanced Lymphodepletion is insufficient to replace exogenous IL2 or IL15 therapy in augmenting the efficacy of adoptively transferred effector CD8(+) T cells. Cancer Res 2018;78:3067–74. doi:10.1158/0008-5472.CAN-17-2153
      OpenUrlAbstract/FREE Full Text
    18. ↵
      1. Bronte V,
      2. Wang M,
      3. Overwijk WW, et al
      . Apoptotic death of CD8+ T lymphocytes after immunization: induction of a suppressive population of Mac-1+/Gr-1+ cells. J Immunol 1998;161:5313–20.
      OpenUrlAbstract/FREE Full Text
    19. ↵
      1. Antony PA,
      2. Piccirillo CA,
      3. Akpinarli A, et al
      . CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J Immunol 2005;174:2591–601. doi:10.4049/jimmunol.174.5.2591
      OpenUrlAbstract/FREE Full Text
    20. ↵
      1. Turk MJ,
      2. Guevara-Patiño JA,
      3. Rizzuto GA, et al
      . Concomitant tumor immunity to a poorly Immunogenic Melanoma is prevented by regulatory T cells. J Exp Med 2004;200:771–82. doi:10.1084/jem.20041130
      OpenUrlAbstract/FREE Full Text
    21. ↵
      1. Wallen H,
      2. Thompson JA,
      3. Reilly JZ, et al
      . Fludarabine modulates immune response and extends in vivo survival of adoptively transferred CD8 T cells in patients with metastatic Melanoma. PLoS One 2009;4:e4749. doi:10.1371/journal.pone.0004749
    22. ↵
      1. Gattinoni L,
      2. Finkelstein SE,
      3. Klebanoff CA, et al
      . Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med 2005;202:907–12. doi:10.1084/jem.20050732
      OpenUrlAbstract/FREE Full Text
    23. ↵
      1. Citrin DE
      . Recent developments in radiotherapy. N Engl J Med 2017;377:1065–75. doi:10.1056/NEJMra1608986
      OpenUrl
    24. ↵
      1. Viaud S,
      2. Flament C,
      3. Zoubir M, et al
      . Cyclophosphamide induces differentiation of Th17 cells in cancer patients. Cancer Res 2011;71:661–5. doi:10.1158/0008-5472.CAN-10-1259
      OpenUrlAbstract/FREE Full Text
    25. ↵
      1. Schiavoni G,
      2. Mattei F,
      3. Di Pucchio T, et al
      . Cyclophosphamide induces type I interferon and augments the number of CD44(Hi) T lymphocytes in mice: implications for strategies of chemoimmunotherapy of cancer. Blood 2000;95:2024–30.
      OpenUrlAbstract/FREE Full Text
    26. ↵
      1. Gandhi V,
      2. Plunkett W
      . Cellular and clinical pharmacology of fludarabine. Clin Pharmacokinet 2002;41:93–103. doi:10.2165/00003088-200241020-00002
      OpenUrlCrossRefPubMedWeb of Science
    27. ↵
      1. Owen K,
      2. Ghaly R,
      3. Shohdy KS, et al
      . Lymphodepleting chemotherapy practices and effect on safety and efficacy outcomes in patients with solid tumours undergoing T cell receptor-engineered T cell (TCR-T) therapy: a systematic review and meta-analysis. Cancer Immunol Immunother 2023;72:805–14. doi:10.1007/s00262-022-03287-1
      OpenUrl
    28. ↵
      1. Di Roio A,
      2. Hubert M,
      3. Besson L, et al
      . Mdr1-expressing CD4(+) T cells with Th1.17 features resist to neoadjuvant chemotherapy and are associated with breast cancer clinical response. J Immunother Cancer 2023;11:e007733. doi:10.1136/jitc-2023-007733
    29. ↵
      1. Hanada K-I,
      2. Yu Z,
      3. Chappell GR, et al
      . An effective mouse model for adoptive cancer immunotherapy targeting neoantigens. JCI Insight 2019;4:e124405. doi:10.1172/jci.insight.124405
    30. ↵
      1. Ward-Kavanagh LK,
      2. Zhu J,
      3. Cooper TK, et al
      . Whole-body irradiation increases the magnitude and persistence of adoptively transferred T cells associated with tumor regression in a mouse model of prostate cancer. Cancer Immunol Res 2014;2:777–88. doi:10.1158/2326-6066.CIR-13-0164
      OpenUrlAbstract/FREE Full Text
    31. ↵
      1. Zhu EF,
      2. Gai SA,
      3. Opel CF, et al
      . Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2. Cancer Cell 2015;27:489–501. doi:10.1016/j.ccell.2015.03.004
      OpenUrlCrossRefPubMed
    32. ↵
      1. Xie Y,
      2. Akpinarli A,
      3. Maris C, et al
      . Naive tumor-specific CD4(+) T cells differentiated in vivo eradicate established Melanoma. J Exp Med 2010;207:651–67. doi:10.1084/jem.20091921
      OpenUrlAbstract/FREE Full Text
    33. ↵
      1. Knochelmann HM,
      2. Dwyer CJ,
      3. Smith AS, et al
      . IL6 fuels durable memory for Th17 cell-mediated responses to tumors. Cancer Res 2020;80:3920–32. doi:10.1158/0008-5472.CAN-19-3685
      OpenUrlAbstract/FREE Full Text
    34. ↵
      1. Dudley ME,
      2. Yang JC,
      3. Sherry R, et al
      . Adoptive cell therapy for patients with metastatic Melanoma: evaluation of intensive Myeloablative Chemoradiation preparative regimens. J Clin Oncol 2008;26:5233–9. doi:10.1200/JCO.2008.16.5449
      OpenUrlAbstract/FREE Full Text
    35. ↵
      1. Wrzesinski C,
      2. Paulos CM,
      3. Kaiser A, et al
      . Increased intensity Lymphodepletion enhances tumor treatment efficacy of Adoptively transferred tumor-specific T cells. J Immunother 2010;33:1–7. doi:10.1097/CJI.0b013e3181b88ffc
      OpenUrlCrossRefPubMedWeb of Science
    36. ↵
      1. Dudley ME,
      2. Wunderlich JR,
      3. Yang JC, et al
      . Adoptive cell transfer therapy following non-Myeloablative but Lymphodepleting chemotherapy for the treatment of patients with refractory metastatic Melanoma. J Clin Oncol 2005;23:2346–57. doi:10.1200/JCO.2005.00.240
      OpenUrlAbstract/FREE Full Text
    37. ↵
      1. Bechman N,
      2. Maher J
      . Lymphodepletion strategies to potentiate adoptive T-cell Immunotherapy - what are we doing; where are we going Expert Opin Biol Ther 2021;21:627–37. doi:10.1080/14712598.2021.1857361
      OpenUrl
    38. ↵
      1. Paulos CM,
      2. Carpenito C,
      3. Plesa G, et al
      . The inducible Costimulator (ICOS) is critical for the development of human T(H)17 cells. Sci Transl Med 2010;2:55ra78. doi:10.1126/scitranslmed.3000448
    39. ↵
      1. Bailey SR,
      2. Nelson MH,
      3. Himes RA, et al
      . Th17 cells in cancer: the ultimate identity crisis. Front Immunol 2014;5:276. doi:10.3389/fimmu.2014.00276
    40. ↵
      1. Muranski P,
      2. Boni A,
      3. Antony PA, et al
      . Tumor-specific Th17-polarized cells eradicate large established Melanoma. Blood 2008;112:362–73. doi:10.1182/blood-2007-11-120998
      OpenUrlAbstract/FREE Full Text
    41. ↵
      1. Lai P,
      2. Chen X,
      3. Wang Y, et al
      . C3Ar Costimulation enhances the antitumor efficacy of CAR-T cell therapy through Th17 expansion and memory T cell induction. J Hematol Oncol 2022;15:68. doi:10.1186/s13045-022-01288-2
    42. ↵
      1. Bowers JS,
      2. Nelson MH,
      3. Majchrzak K, et al
      . Th17 cells are refractory to Senescence and retain robust antitumor activity after long-term ex vivo expansion. JCI Insight 2017;2:e90772. doi:10.1172/jci.insight.90772
    43. ↵
      1. Neitzke DJ,
      2. Bowers JS,
      3. Andrijauskaite K, et al
      . Murine Th17 cells utilize IL-2 receptor gamma chain Cytokines but are resistant to cytokine withdrawal-induced apoptosis. Cancer Immunol Immunother 2017;66:737–51. doi:10.1007/s00262-017-1965-3
      OpenUrl
    44. ↵
      1. Kryczek I,
      2. Zhao E,
      3. Liu Y, et al
      . Human Th17 cells are long-lived effector memory cells. Sci Transl Med 2011;3:104ra100. doi:10.1126/scitranslmed.3002949
    45. ↵
      1. Xu N,
      2. Palmer DC,
      3. Robeson AC, et al
      . STING agonist promotes CAR T cell trafficking and persistence in breast cancer. J Exp Med 2021;218:e20200844. doi:10.1084/jem.20200844
    46. ↵
      1. Lickefett B,
      2. Chu L,
      3. Ortiz-Maldonado V, et al
      . Lymphodepletion - an essential but undervalued part of the Chimeric antigen receptor T-cell therapy cycle. Front Immunol 2023;14:1303935. doi:10.3389/fimmu.2023.1303935
    47. ↵
      1. Kalos M,
      2. Levine BL,
      3. Porter DL, et al
      . T cells with Chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73. doi:10.1126/scitranslmed.3002842
    48. ↵
      1. Yang JC,
      2. Rosenberg SA
      . Adoptive T-cell therapy for cancer. Adv Immunol 2016;130:279–94. doi:10.1016/bs.ai.2015.12.006
      OpenUrlCrossRefPubMed
    49. ↵
      1. Wagner J,
      2. Wickman E,
      3. DeRenzo C, et al
      . CAR T cell therapy for solid tumors: bright future or dark reality? Mol Ther 2020;28:2320–39. doi:10.1016/j.ymthe.2020.09.015
      OpenUrlCrossRefPubMed
    50. ↵
      1. Kirtane K,
      2. Elmariah H,
      3. Chung CH, et al
      . Adoptive cellular therapy in solid tumor malignancies: review of the literature and challenges ahead. J Immunother Cancer 2021;9:e002723. doi:10.1136/jitc-2021-002723
    51. ↵
      1. Wang E,
      2. Cesano A,
      3. Butterfield LH, et al
      . Improving the therapeutic index in adoptive cell therapy: key factors that impact efficacy. J Immunother Cancer 2020;8:e001619. doi:10.1136/jitc-2020-001619
    52. ↵
      1. Liu Z,
      2. Zhou Z,
      3. Dang Q, et al
      . Immunosuppression in tumor immune microenvironment and its optimization from CAR-T cell therapy. Theranostics 2022;12:6273–90. doi:10.7150/thno.76854
      OpenUrl
    53. ↵
      1. Klebanoff CA,
      2. Khong HT,
      3. Antony PA, et al
      . Sinks, suppressors and antigen presenters: how Lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol 2005;26:111–7. doi:10.1016/j.it.2004.12.003
      OpenUrlCrossRefPubMedWeb of Science
    54. ↵
      1. Cachot A,
      2. Bilous M,
      3. Liu Y-C, et al
      . Tumor-specific Cytolytic CD4 T cells mediate immunity against human cancer. Sci Adv 2021;7:eabe3348. doi:10.1126/sciadv.abe3348
    55. ↵
      1. Kamphorst AO,
      2. Ahmed R
      . CD4 T-cell immunotherapy for chronic viral infections and cancer. Immunotherapy 2013;5:975–87. doi:10.2217/imt.13.91
      OpenUrlCrossRefPubMed
    56. ↵
      1. Bailey SR,
      2. Nelson MH,
      3. Majchrzak K, et al
      . Human CD26(High) T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence. Nat Commun 2017;8:1961. doi:10.1038/s41467-017-01867-9
    57. ↵
      1. Majchrzak K,
      2. Nelson MH,
      3. Bowers JS, et al
      . Beta-Catenin and Pi3Kdelta inhibition expands precursor Th17 cells with heightened Stemness and antitumor activity. JCI Insight 2017;2:e90547. doi:10.1172/jci.insight.90547
    58. ↵
      1. Wei S,
      2. Zhao E,
      3. Kryczek I, et al
      . Th17 cells have stem cell-like features and promote long-term immunity. Oncoimmunology 2012;1:516–9. doi:10.4161/onci.19440
      OpenUrlCrossRefPubMed
    59. ↵
      1. Boni A,
      2. Muranski P,
      3. Cassard L, et al
      . Adoptive transfer of allogeneic tumor-specific T cells mediates effective regression of large tumors across major histocompatibility barriers. Blood 2008;112:4746–54. doi:10.1182/blood-2008-07-169797
      OpenUrlAbstract/FREE Full Text
    60. ↵
      1. Martin-Orozco N,
      2. Muranski P,
      3. Chung Y, et al
      . T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 2009;31:787–98. doi:10.1016/j.immuni.2009.09.014
      OpenUrlCrossRefPubMedWeb of Science
    61. ↵
      1. Mitsdoerffer M,
      2. Lee Y,
      3. Jäger A, et al
      . Proinflammatory T helper type 17 cells are effective B-cell helpers. Proc Natl Acad Sci U S A 2010;107:14292–7. doi:10.1073/pnas.1009234107
      OpenUrlAbstract/FREE Full Text
    62. ↵
      1. De Filippo K,
      2. Dudeck A,
      3. Hasenberg M, et al
      . Mast cell and macrophage Chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation. Blood 2013;121:4930–7. doi:10.1182/blood-2013-02-486217
      OpenUrlAbstract/FREE Full Text
    63. ↵
      1. Theyab A,
      2. Algahtani M,
      3. Alsharif KF, et al
      . New insight into the mechanism of granulocyte colony-stimulating factor (G-CSF) that induces the mobilization of neutrophils. Hematology 2021;26:628–36. doi:10.1080/16078454.2021.1965725
      OpenUrlCrossRef
    64. ↵
      1. Leitch VD,
      2. Strudwick XL,
      3. Matthaei KI, et al
      . IL-5-overexpressing mice exhibit eosinophilia and altered wound healing through mechanisms involving prolonged inflammation. Immunol Cell Biol 2009;87:131–40. doi:10.1038/icb.2008.72
      OpenUrlCrossRefPubMed
    65. ↵
      1. Fisher JW
      . Landmark advances in the development of erythropoietin. Exp Biol Med (Maywood) 2010;235:1398–411. doi:10.1258/ebm.2010.010137
      OpenUrlCrossRefPubMed
    66. ↵
      1. Hirschhorn D,
      2. Budhu S,
      3. Kraehenbuehl L, et al
      . T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants. Cell 2023;186:1432–47. doi:10.1016/j.cell.2023.03.007
      OpenUrlCrossRef
    67. ↵
      1. Rosenberg SA,
      2. Yang JC,
      3. Sherry RM, et al
      . Durable complete responses in heavily pretreated patients with metastatic Melanoma using T-cell transfer Immunotherapy. Clin Cancer Res 2011;17:4550–7. doi:10.1158/1078-0432.CCR-11-0116
      OpenUrlAbstract/FREE Full Text

    Supplementary materials

    • Supplementary Data

      This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

    Footnotes

    • X @megen_wittling, @GuillermoORang1, @LesinskiLab, @PaulosLab

    • Contributors MCW and HMK: designed and conducted experiments, analyzed and interpreted data, and wrote and assembled the manuscript. CMP and GBL: reviewed data, edited/wrote the manuscript, conceptualization, funding acquisition, supervision, guarantor. ACC, MMW and GORR helped to perform experiments and contributed to figures and/or text in the manuscript.

    • Funding This work was supported by the Melanoma Research Foundation (to GORR, HMK, and MCW), T32 CA272392-01A1 (to MCW), ARCs Foundation (to ACC), NCI F30CA243307 (to HMK), NIH DE017551 (HMK), NIH R50CA233186 (to MMW); NCI R01CA228406, R21CA266088-01, R21CA270903 (to GBL), NCI R01CA175061, R01CA208514, R01CA275199, V Foundation, plus MUSC and Emory University Start Up Funds (to CP).

    • Competing interests The authors disclose no conflicts of interest in relation to the published work. CMP has previously received funds for consultancies/advisory boards/research contracts: Ares Immunotherapy, Lycera, Obsidian and Thermo Fisher. GBL has received research funding through a sponsored research agreement between Emory University and Merck and Co., Bristol-Myers Squibb, Boehringer Ingelheim, and Vaccinex.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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