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Co-transfer of tumor-specific effector and memory CD8+ T cells enhances the efficacy of adoptive melanoma immunotherapy in a mouse model

Co-transfer of tumor-specific effector and memory CD8+ T cells enhances the efficacy of adoptive... Background: Adoptive cell transfer (ACT) is a promising cancer immunotherapeutic strategy that remains ineffective for a large subset of patients. ACT with memory CD8+ T cells (T ) has been shown to have superior mem efficacy compared to traditional ACT with effector CD8+ T cells (T ). T and T have complementary eff eff mem physiological advantages for immunotherapy, but previous publications have not examined ACT using a combination of T and T . eff mem Methods: Splenocytes harvested from Ly5.1+/C57BL/6 mice during and after infection with lymphocytic choriomeningitis virus (LCMV) were used to generate bona fide effector and memory CD8+ T cells specific for the LCMV epitope peptide GP33. Congenic Ly5.2+/C57BL/6 mice were inoculated with B16F10 melanoma cells transfected to express very low levels of GP33, then treated with ACT 7 days later with GP33-specific T ,T ,ora eff mem combination of T +T . eff mem Results: Inhibition of melanoma growth was strongest in mice receiving combinatorial ACT. Although combinatorial ACT and memory ACT resulted in maximal intratumoral infiltration of CD8+ T cells, combinatorial ACT induced stronger infiltration of endogenous CD8+ T cells than T ACT and a stronger systemic T cell mem responsiveness to tumor antigen. In vitro assays revealed rapid but transient melanoma inhibition with T and eff gradual but prolonged melanoma inhibition with T ; the addition of T enhanced the ability of T to inhibit mem mem eff melanoma in a manner that could be reproduced using conditioned media from activated T and blocked by mem the addition of anti-IL-2 blocking antibody. Conclusions: These findings suggest that a novel combinatorial approach that takes advantage of the unique and complementary strengths of tumor-specific T and T may be a way to optimize the efficacy of adoptive eff mem immunotherapy. Keywords: Adoptive transfer, Immunotherapy, T cell, Memory, Effector, Melanoma, Cancer * Correspondence: cliffcho@med.umich.edu Amanda Contreras and Megan V. Beems contributed equally to this manuscript Equal contributors VA Ann Arbor Healthcare System, 2215 Fuller Road, Ann Arbor, MI 48105, USA Department of Surgery, University of Michigan Medical School, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 2 of 10 Background Methods Adoptive cell transfer (ACT) is a promising strategy for Mice cancer immunotherapy that involves the isolation, ex- Seven- to eight-week old female congenic C57BL/6 pansion, and infusion of tumor-specific CD8+ T cells. mice with allele Ly5.1+ (T cell donor mice) or allele When combined with interleukin-2 (IL-2) and lympho- Ly5.2+ (T cell recipient mice) were purchased from depletion, ACT has led to complete and durable tumor Taconic (Hudson, NY) and maintained in sterile hous- regression in up to 20% of patients with metastatic mel- ing. All mouse work was reviewed and approved by the anoma [1]. Despite this, ACT remains ineffective for a University of Wisconsin and William S. Middleton large subset of patients. Memorial VA Hospital and VA Ann Arbor Healthcare Traditionally, ACT involves the use of terminally Animal Care and Use Committees. differentiated CD8+ effector T cells (T ) collected eff from tumor-infiltrating lymphocytes (TILs). When Tumor cell lines and effector/memory cell generation naïve T cells encounter antigen, they clonally expand Viral infections and tumor inoculations were performed and differentiate into T that have the ability to rap- per our previously published protocols [7, 9, 10]. In eff idly clear cells expressing their cognate antigen [2, 3]. brief, Ly5.1+/C57BL/6 mice were infected with 2 × 10 Following antigen clearance, the majority of T then PFU of Armstrong strain lymphocytic choriomeningitis eff undergo apoptosis, while a small population of virus (LCMV; gift from Marulasiddappa Suresh, antigen-specific CD8+ memory T cells (T ) persists University of Wisconsin) via intraperitoneal injection to mem long-term and can rapidly proliferate upon antigen generate CD8+ T cells specific for GP33, a class re-exposure [2, 3]. I MHC-restricted LCMV surface glycoprotein. Spleno- The use of different T cell subsets is one potential way cytes were harvested 8 days after infection (at which to improve the efficacy of ACT. Less-differentiated ef- time LCMV peptide-specific CD8+ T cells are of effector fector cells have been shown to have superior antitumor differentiation) to obtain bona fide T (as evidenced by eff low high immunity compared to terminally-differentiated cells, CD127 /KLRG1 expression) and 50–80 days after potentially due to a number of mechanisms such as de- infection (at which time LCMV peptide-specific CD8+ T creased IL-2 production and increased apoptosis with cells are of memory differentiation) to obtain bona fide high low further differentiation [4, 5]. Our laboratory and others T (as evidenced by CD127 /KLRG1 expression) mem have also demonstrated that ACT with T is more ef- [7, 8]. The yield of GP33-specific memory CD8+ T cells mem fective than ACT with T or naïve T cells [6, 7]. T was increased by adoptively transferring Ly5.1+/C57BL/ eff mem appear to be uniquely resistant to melanoma-induced 6 mice with 10 Ly5.1+/CD8+ T cells derived from sple- suppression and generate a stronger intratumoral im- nocytes of Ly5.1+/P14 TCR transgenic mice (C57BL/6 mune response [7, 8]. background mice with CD8+ T cell specificity for GP33 No previous publications have explored performing [7]. The spontaneous mouse melanoma cell line B16F10 ACT with a combination of T and T . Although T was transfected with a plasmid encoding GP33, and mem eff eff are prone to apoptosis, we hypothesized that this limita- poorly immunogenic clones with low levels of GP33 ex- tion could be offset by the innate proliferative ability of pression were selected to create a new B16GP33 cell line, T , with T contributing to initial tumor control and which we have previously demonstrated to be of compar- mem eff T to later tumor control. In a murine model, we ably low immunogenicity as parental B16F10 [9, 10]. This mem compared the efficacy of ACT using bona fide T , line was cultured in RPMI-1640 medium (Mediatech, eff T , and a combination of both. We utilized a novel Herndon, VA) with 10% fetal bovine serum, 2 mM L-gluta- mem melanoma tumor expressing very low levels of a viral mine, 100 μg/mL streptomycin, and 100 U/mL penicillin peptide in order to dissect the biological effects of (Life Technologies, Inc., Grand Island, NY). Ly5.2 peptide-specific CD8+ T cells of true effector and mem- +/C57BL/6 mice received subcutaneous injections of 1 × ory differentiation. This methodology enables us to 10 B16GP33 cells suspended in serum-free RPMI-1640 examine the therapeutic implications of bona fide ef- to generate flank melanoma tumors. fector and memory tumor-specific CD8+ T cells in a manner not possible with standard murine melanoma Adoptive cell transfer targets like GP100. Using in vitro experiments, we ex- Adoptive cell transfer was performed per our previously plored the temporal cell killing patterns and paracrine published protocol [7, 8, 10]. As previously observed, effects of these T cell subsets. We discovered that the GP33-specific CD8+ T cells typically comprised approxi- combination of T +T led to the strongest control mately 8–10% of splenocytes harvested from effector eff mem of melanoma tumor growth, potentially due to comple- and memory T cell donor mice [7, 8]. After obtaining mentary cell killing patterns and local production of splenocytes from mice infected with LCMV, magnetic IL-2 by T . bead separation columns (Miltenyi, Auburn, CA) were mem Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 3 of 10 used to isolate CD8+ T cells. Flow cytometry was per- Statistical analysis formed to quantify the percentage of tumor-specific Experimental data was analyzed using IBM SPSS statis- GP33-specific CD8+ T cells. 10 GP33-specific CD8+ tical software version 23 (Armonk, NY). T-test was used 4 4 T ,T ,or5×10 T +5×10 T were adoptively to compare groups and a repeated-measures analysis of eff mem eff mem transferred via retro-orbital intravenous injections into variance (ANOVA) with pair-wise comparisons per- B16F10- or B16GP33 melanoma-bearing mice at specific formed using Fisher’s protected least significant differ- time points after tumor inoculation. ence tests were used to compare multiple groups. Significance was defined as p < 0.05, and error bars rep- resent standard errors of the mean. MTT assay For multiple timepoints, a total number of 2 × 10 GP33-specific CD8+ T and T or both were separ- eff mem Results ately co-cultured with 2 × 10 B16GP33 melanoma cells Co-ACT of T +T is more effective at inhibiting in eff mem at an overall effector:target ratio of 1:1 in 48-well plates vivo melanoma growth than ACT of either T cell subset in the presence of 10 ng/mL recombinant human IL-2 alone (BD Biosciences, San Jose, CA) for MTT assays as previ- To verify the peptide specificity of our ACT model, ously described [7]. T cells were characterized and iso- C57BL/6 mice were treated with 10 GP33-specific T eff lated using the methodology described for adoptive cell one day after subcutaneous inoculation with B16GP33 transfer. Viable B16GP33 melanoma cells were quanti- melanoma or parental B16F10 melanoma (which does fied using the Celltiter 96 non-radioactive MTT assay kit not express GP33). As shown in Fig. 1a-b, early ACT (Promega, Madison, WI) following the manufacturer’s strongly suppressed the growth of B16GP33 melanoma instructions. but not B16F10 melanoma, suggesting that the effect of ACT was peptide-specific. Moreover, the kinetics of Conditioned media assay B16GP33 tumor growth in immunocompetent mice was For 5 h, GP33-specific CD8+ T and T were cul- eff mem comparable to those of parental B16F10 tumor. To tured separately or together in RPMI-1640 with 10 ng/ compare the efficacy of ACT with different T cell mL IL-2 and 0.1 μg/mL GP33 peptide (gift from Marula- subsets, Ly5.2+/C57BL/6 mice received ACT of 10 siddappa Suresh, University of Wisconsin). Conditioned GP33-specific CD8+ T ,T ,or T +T on day 7 eff mem eff mem media was collected by centrifuging wells and collecting after subcutaneous inoculation with 10 B16GP33 cells. the supernatant. For 12 h, GP33-specific CD8+ T and eff As shown in Fig. 1c-d, memory ACT resulted in signifi- T were then co-cultured with B16GP33 melanoma mem cantly greater inhibition of tumor growth compared to cells with or without conditioned media and anti-IL-2 effector ACT as previously described. Combinatorial (BD Biosciences, San Jose, CA) for MTT assays as de- ACT with T +T had significantly greater inhibition eff mem scribed above. of tumor growth compared to ACT with T or T eff mem separately, making this combination the most effective Flow cytometry strategy for ACT. Flow cytometric analysis was performed per our previ- ously published protocol [7–10]. Once harvested, tumor samples were processed into single-cell suspensions with Co-ACT of T +T promotes more potent intratumoral eff mem mechanical fractionation on a fine wire mesh followed CD8 + T cell infiltration than ACT of either T cell subset by lymphocyte isolation using Histopaque (Sigma Al- alone drich, St. Louis, MO). Splenocytes before ACT and To determine if the efficacy of combinatorial ACT was tumor-infiltrating lymphocytes (TIL) 4 to 14 days after associated with stronger intratumoral T cell infiltration, ACT were stained with APC-labeled MHC class I tetra- mice were euthanized 4 and 14 days after ACT to allow mers loaded with GP33 peptide (NIH Tetramer Facility for tumor harvesting and flow cytometric analysis of TIL Core, Atlanta, GA), PerCP-labeled anti-CD8 mAb, populations. Representative data shown in Fig. 2a dem- PE-labeled anti-Ly5.1 mAb, and FITC-labeled anti-CD44 onstrate that CD8+ T cells comprised a higher percent- mAb (BD Biosciences, San Jose, CA). Intracellular stain- age of TIL in mice treated with ACT in comparison ing required stimulation of lymphocytes with media with untreated controls. As shown in Fig. 2b, total num- alone or with GP33 peptide (1 μg/mL), brefeldin A, and bers of intratumoral CD8+ were transiently elevated on recombinant human IL-2 (10 ng/mL) at 37°C for 5 h. A day 4 in mice treated with effector ACT, but this effect BD LSRFortessa flow cytometer (BD Biosciences, San was short-lived; in contrast, intratumoral CD8+ T cells Jose, CA) was used and data were analyzed using FlowJo remain elevated on day 14 in mice treated with memory software (Tree Star Inc., Ashland, OR). and combinatorial ACT. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 4 of 10 Fig. 1 ACT with a combination of T +T resulted in optimal control of melanoma growth. Mice bearing B16GP33 (a)or B16F10 (b)melanoma eff mem were untreated (control) or treated with ACT of 10 GP33-specific CD8+ T cells one day after tumor inoculation. Serial tumor measurements confirmed that ACT resulted in a GP33-specific inhibition of melanoma tumor growth. (c) Mice bearing B16GP33 melanoma were treated 7 days after tumor 5 5 4 4 inoculation with no treatment (control) or 10 T ,10 T ,or 5 × 10 T +5× 10 T . Comparison of treated groups (d)demonstrates optimal eff mem eff mem control of melanoma growth with combinatorial ACT. This experiment was repeated three times with similar results, 4–5 mice per group. (+ p <0.05 compared with control; * p < 0.05 compared with effector ACT; Ɨ p < 0.05 compared with memory ACT) The more potent intratumoral CD8+ T cell infiltration Combinatorial ACT results in a stronger systemic CD8+ T seen with combinatorial ACT is the result of a stronger cell response against tumor antigen endogenous immune response To determine if combinatorial ACT improved sys- To characterize the increase in intratumoral CD8+ T cell temic antitumor responsiveness in addition to local infiltration seen following ACT, flow cytometric analysis antitumor activity, splenocytes harvested 14 days after was used to quantify the presence of Ly5.1+ adoptively ACT were stimulated with or without GP33 peptide transferred CD8+ T cells and Ly5.2+ endogenous CD8+ T in the presence of IL-2 and brefeldin A, and stained cells. Representative flow cytometric data are shown as to assess intracellular levels of IFNγ expression. Rep- percentages in Fig. 3a; overall data are shown as total cell resentative flow cytometric data are shown as per- numbers in Fig. 3b-c. As shown in Fig. 3b,Ly5.1+ adop- centages in Fig. 4a;overall data areshownastotal tively transferred CD8+ T cells comprised a small and cell numbers in Fig. 4b.As shown in Fig. 4b,memory short-lived fraction of TIL after effector ACT but were ACT resulted in stronger splenocyte reactivity to present in substantial and stable numbers after memory GP33 than effector ACT, as we have previously de- ACT; combinatorial ACT, which used half the number of scribed. However, combinatorial ACT produced sig- T used in memory ACT, resulted in smaller numbers nificantly greater IFNγ expression among splenocytes mem of intratumoral Ly5.1+/CD8+ TIL. In contrast, as shown than memory ACT, suggesting that combinatorial in Fig. 3c, combinatorial ACT resulted in the strongest ACT may induce a much stronger level of CD8+ T intratumoral infiltration of endogenous CD8+ T cells. cell responsivenesstotumor antigen. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 5 of 10 Fig. 2 Combinatorial ACT results in a stronger intratumoral T cell response. Seven days after tumor inoculation, Ly5.2+ mice were treated with 5 5 4 4 10 T ,10 T ,or5 × 10 T +5× 10 T derived from Ly5.1+ mice. TIL were harvested from melanoma tumors resected 4 or 14 days after eff mem eff mem ACT and stained for flow cytometric analysis. (a) Representative TIL data (gated on lymphocyte populations) demonstrate that CD8+ T cells comprised the highest percentage of TIL after effector ACT on day 4 and after combinatorial ACT on day 14. (b) Overall comparison of total numbers of intratumoral CD8+ T cells on days 4 and 14 demonstrated a transient increase after effector ACT and a sustained increase after memory ACT and combinatorial ACT. This experiment was repeated twice with similar results, 4–5 mice per group. (* p < 0.05 compared with control condition; † p < 0.05 compared with day 4) Combinatorial ACT may benefit from temporal differences During combinatorial ACT, tumor cell caused by by T eff in melanoma cell killing between T and T may be enhanced by IL-2 production from T eff mem mem To further explore the improved efficacy of combinator- To determine if the improved efficacy of combinatorial ial ACT, T and T were co-cultured in vitro with ACT was attributable to local interactions between cell eff mem B16GP33 melanoma cells in the presence of IL-2 for types, T ,T ,and T +T were co-cultured with eff mem eff mem 72 h. As shown in Fig. 5, serial MTT assays demon- B16GP33 cells in the presence of IL-2 for 8 h. As shown strated that T inhibited melanoma more potently than in Fig. 6a, whereas T and T resulted in comparable eff eff mem T at 6 h. However, whereas the ability of T to in- levels of melanoma inhibition at this time point, the mem eff hibit melanoma was lost at later timepoints, T con- combination of T +T resulted in a stronger level mem eff mem tinued to inhibit melanoma for the duration of time of melanoma inhibition. To determine if this enhance- points tested (up to 72 h), suggesting that the enhanced ment was attributable to paracrine effects, T and T eff mem therapeutic efficacy of combinatorial ACT could be due were separately stimulated with GP33 peptide for 5 h, to the temporal combination of early melanoma inhib- after which cells were pelleted by centrifugation and ition mediated by T plus delayed but durable melan- conditioned media were collected. As shown in Fig. 6b, eff oma inhibition mediated by T . the addition of conditioned media from T enhanced mem mem Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 6 of 10 Fig. 3 The stronger local CD8+ T cell response seen with combinatorial ACT is largely the result of endogenous T cell recruitment. Seven days 5 5 4 4 after tumor inoculation, Ly5.2+ mice were treated with 10 T ,10 T ,or5 × 10 T +5× 10 T derived from Ly5.1+ mice. Melanoma eff mem eff mem tumors were resected 4 or 14 days after ACT and TIL were harvested for flow cytometric analysis. (a) Representative data demonstrate higher percentages of Ly5.1+/CD8+ T cells in tumors after memory and combinatorial ACT. (b) Analysis of total numbers of Ly5.1+/CD8+ T cell populations showed that significant and durable infiltration of adoptively transferred cells was observed after memory ACT; combinatorial ACT, which used half the number of adoptively transferred T as memory ACT, resulted in smaller numbers of intratumoral Ly5.1+/CD8+ T cells. (c) mem Analysis of total numbers of Ly5.2+/CD8+ T cell populations showed that memory ACT resulted in a significant recruitment of endogenously- derived CD8+ T cells that persisted on day 14; despite using half the number of T , combinatorial ACT also resulted in durable infiltration of mem endogenously-derived CD8+ T cells. This experiment was repeated twice with similar results, 4–5 mice per group. (* p < 0.05 compared with control condition; † p < 0.05 compared with day 4) Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 7 of 10 Fig. 4 Combinatorial ACT results in the strongest level of systemic CD8+ T cell responsiveness to tumor antigen. Seven days after tumor inoculation, 5 5 4 4 Ly5.2+ mice were treated with 10 T ,10 T ,or 5× 10 T +5×10 T derived from Ly5.1+ mice. Spleens were resected 14 days after ACT and eff mem eff mem splenocytes were incubated with media alone (control) or with tumor antigen (GP33). (a) Representative data demonstrate negligible levels of IFNγ expression by splenocytes in response to GP33 stimulation after no ACT or effector ACT, with increasing levels seen after memory ACT and combinatorial ACT, respectively. (b) The highest levels of IFNγ expression by splenocytes in response to GP33 stimulation were seen after combinatorial ACT. This experiment was repeated with similar results, 4 mice per group. (* p < 0.05 compared with control condition; † p <0.05 compared with effector ACT, ‡ p < 0.05 compared with memory ACT) the ability of T to inhibit melanoma to a comparable caused by T . The addition of anti-IL-2 blocking anti- eff eff level seen with T +T . The addition of conditioned body alone did not affect the ability of T to inhibit eff mem eff media from T did not enhance the ability of T to melanoma. eff mem inhibit melanoma (data not shown). When anti-IL-2 blocking antibody was added to conditioned media from Discussion T , this effect was lost, suggesting that paracrine re- For the first time, we have shown that ACT using a mem lease of IL-2 from T may potentiate tumor cell death combination of T +T leads to even more robust mem eff mem Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 8 of 10 cytokine/chemokine release, antigen presentation, and priming of endogenous tumor-specific T cells [13], ul- timately promoting stronger systemic anti-tumor immunity. Given the innate predisposition of effector cells to rap- idly target antigen, and the ability of memory cells to persist and expand, we hypothesized that the ability of T and T to engage and inhibit melanoma would eff mem exhibit temporal differences. Indeed, in vitro assays dem- onstrated that T cause immediate but transient melan- eff oma inhibition, while T cause gradual but persistent mem inhibition. This pattern is further reflected by the fact that, whereas ACT with T resulted in a decline in eff intratumoral adoptively transferred Ly5.1+/CD8+ T cells Fig. 5 T and T have different temporal patterns of melanoma mem eff at 14 days, ACT with T resulted in stable numbers mem inhibition in vitro. T and T were co-cultured with B16GP33 eff mem of intratumoral transferred T cells. Thus, the activity of melanoma cells in the presence of 10 ng/mL recombinant human IL-2 for 72 h. Control wells contained no T cells. Serial MTT assays effector and memory cells may be complementary over were performed every 6 h. Over approximately the first 8 h, T eff time, leading to stronger and more prolonged melanoma produced significantly greater inhibition of B16GP33. At all later time suppression. points, T demonstrated significantly greater suppression of mem We found that the intrinsic ability of T to inhibit eff tumor growth. This experiment was repeated twice with similar melanoma may also be enhanced by local production of results, 3 samples per group. († p < 0.05 compared with T ) eff IL-2 by T . Unlike effector T ,T are uniquely mem eff mem capable of elaborating IL-2 in response to antigen stimu- lation [2, 3]. IL-2 supplementation has long been associ- control of melanoma growth than ACT using T or ated with improved outcomes following ACT [14]. eff T alone. Our findings suggest that the advantage Systemic and local delivery of IL-2 in combination with mem of combinatorial ACT may result from the induction ACT has been shown to increase CD8+ tumor infiltra- of stronger infiltration of endogenous CD8+ T cells tion and suppression of tumor growth [1, 14–17]. With into tumor, stronger systemic CD8+ T cell responsive- combinatorial ACT, the local, paracrine production of ness to tumor antigen, temporal differences in melan- IL-2 by T may mitigate the need for exogenous IL-2 mem oma cell killing, and paracrine IL-2 effects. One administration, decreasing the risk of systemic inflam- recent publication addressed the effects of combining matory side effects. T cell subsets prior to ACT, but this involved By utilizing a melanoma expressing low levels of a co-culturing naïve cells with T for 6 days prior to viral peptide, we are able to examine the therapeutic mem ACT [11]. That approach led to a loss of effects of bona fide effector and memory differenti- less-differentiated T cell subsets and impaired in vivo ation tumor-specific CD8+ T cells in ways not per- tumor regression [11]. By combining T +T at missible with the use of traditional tumor antigens eff mem the time of adoptive transfer, we were able to retain (e.g., GP100). Although our B16GP33 tumor model the innate advantages of each T cell subset. does not appear to be more intrinsically immunogenic We observed that combinatorial ACT generated stron- than parental B16F10 melanoma) [10], further work ger intratumoral CD8+ T cell infiltration than ACT with will be necessary to validate our observations using T or T alone; this was not due to greater traffick- effector and memory CD8+ T cells directed against eff mem ing of adoptively transferred cells into tumor, but a re- naturally occurring melanoma antigens. Our observa- sult of more potent induction of endogenous immune tions suggest that a combinatorial approach to ACT responses. This stimulation of ongoing endogenous CD8 using both T and T may beameanstooptimize eff mem + T cell responses may contribute to the heightened effi- the efficacy of adoptive melanoma immunotherapy. cacy of combinatorial ACT compared to memory ACT. While T can be readily expanded from TIL cultured eff We previously found that combining ACT with check- in IL-2, it is difficult to prepare large quantities of point inhibition led to a similar induction of endogenous T . There is a growing body of literature character- mem TILs that was also associated with greater tumor sup- izing the metabolic determinants of effector versus pression, stronger systemic T cell responsiveness to memory CD8+ T cell differentiation, suggesting that it tumor antigen, and a more potent long-term anti-tumor may become feasible to develop this combinatorial immunity [12]. Enhanced melanoma cell death resulting approach for clinical evaluation [18–25]. This is an from combinatorial ACT may promote stronger local area of ongoing investigation in our laboratory. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 9 of 10 Fig. 6 Paracrine release of IL-2 from T may potentiate melanoma inhibition by T . The local interaction of T and T was examined by mem eff eff mem performing in vitro melanoma inhibition assays. (a)T and T and T +T were co-cultured with B16GP33 for 8 h. Although melanoma eff mem eff mem inhibition was comparable between T and T , significantly stronger inhibition of melanoma was observed with the combination of T + eff mem eff T .(b) Conditioned media (CM) was collected from T stimulated with GP33 for 5 h. The addition of CM enhanced the efficacy of T to a mem mem eff similar degree as the addition of T . The effect of CM was negated by the addition of anti-IL-2 blocking antibody; the addition of anti-IL-2 mem blocking antibody alone did not affect the ability of T to inhibit melanoma. The addition of anti-IL-2 blocking antibody blunted the ability of eff T +T to inhibit melanoma, but it could not be determined if this effect was exerted on T or T (data not shown). This experiment was eff mem eff mem repeated twice with similar results, 3 samples per group. (p-values as shown on horizontal bars) Conclusions complementary differences in temporal killing of Traditional approaches to adoptive immunotherapy tumor cells by effector and memory CD8+ T cells, as have used effector CD8+ T cells for their rapid prolif- well as local paracrine potentiation of effector CD8+ erative capacity and ability to engage and clear tumor T cell function by memory T cell-secreted IL-2. These cells. Experimental evidence suggests that memory findings suggest that combinatorial approaches to CD8+ T cells may have physiological advantages over cell-based immunotherapy harnessing diverse states of effector CD8+ T cells. Our observations indicate that T cell differentiation may open the door to even a combinatorial approach to adoptive immunotherapy greater therapeutic benefit. using effector and memory CD8+ T cells concurrently Abbreviations results in superior tumor control associated with ACT: Adoptive cell transfer; LCMV: Lymphocytic choriomeningitis virus; maximal induction of endogenous T cell responses to T : Effector CD8+ T cell; TIL: Tumor infiltrating lymphocyte; T : Memory eff mem tumor. This strategy appears to take advantage of CD8+ T cell Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 10 of 10 Funding memory T cell-based adoptive immunotherapy in mice. Cancer Immunol This work was supported by grant support from the Department of Veterans Immunother. 2016;65:601–11. Affairs CDA-2 and Merit Review Award (1I01BX001619-01A1) to CSC, National 8. Wentworth L, Meyers JV, Alam S, Russ AJ, Suresh M, Cho CS. Memory T cells Institutes of Health T32 (CA090217) to MVB, National Institutes of Health T32 are uniquely resistant to melanoma-induced suppression. Cancer Immunol (GM081061 and AI55397) to AC, and National Institutes of Health AI48785 to Immunother. 2013;62:149–59. MS. The content is solely the responsibility of the authors and does not 9. Russ AJ, Wentworth L, Xu K, Seroogy CM, Rakhmilevich A, Sondel PM, et al. represent the views of the Department of Veterans Affairs or the United Suppression of T-cell expansion by melanoma is exerted on resting cells. States Government or the National Institutes of Health. Ann Surg Oncol. 2011;18:3848–57. 10. Russ AJ, Xu K, Wentworth L, Alam S, Meyers JV, Macklin MD, et al. Availability of data and materials Melanoma-induced suppression of tumor antigen-specific T cell expansion The datasets used and analyzed during the current study are available from is comparable to suppression of global T cell expansion. Cell Immunol. the corresponding author on reasonable request. 2011;271:104–9. 11. Klebanoff CA, Scott CD, Leonardi AJ, Yamamoto TN, Cruz AC, Ouyang C, Authors’ contributions et al. Memory T cell-driven differentiation of naïve cells impairs adoptive AC and MVB performed the mouse and lymphocyte experiments and immunotherapy. J Clin Invest. 2016;126:318–34. analyzed and interpreted the data and contributed equally to the writing of 12. Mahvi DA, Meyers JV, Tatar AJ, Contreras A, Suresh M, Leverson GE, et al. this manuscript. AJT, SS and PS performed the mouse and lymphocytes CTLA-4 blockade plus adoptive T cell transfer promotes optimal melanoma experiments. MS interpreted the data. TKL performed the co-culture experiments immunity in mice. J Immunother. 2015;38(2):54–61. and contributed to the writing of this manuscript. CSC analyzed and interpreted 13. Schiavoni G, Gabriele L, Mattei F. The tumor microenvironment: a pitch for the data and contributed to the writing of this manuscript. All authors read and multiple players. Front Oncol. 2013;3:1–15. approved the final manuscript. 14. Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol. 2014;192:5451–8. Ethics approval and consent to participate 15. Heemskerk B, Liu K, Dudley ME, Johnson LA, Kaiser A, Downey S, et al. All mouse work was reviewed and approved by the University of Wisconsin Adoptive cell transfer for patients with melanoma, using tumor-infiltrating and William S. Middleton Memorial VA Hospital and VA Ann Arbor lymphocytes genetically engineered to secrete interleukin-2. Hum Gene Healthcare Animal Care and Use Committees. Ther. 2008;19:496–510. 16. Tahtinen S, Kaikkonen S, Merisalo-Soikkeli M, Gronerg-Vaha-Koskela S, Competing interests Kanerva A, Parviainen S, et al. 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Kaiser AD, Gadiot J, Guislain A, Blank CU. Mimicking homeostatic Received: 25 April 2018 Accepted: 15 May 2018 proliferation in vitro generates T cells with high anti-tumor function in non- lymphopenic hosts. Cancer Immunol Immunother. 2013;62:503–15. 22. Tejera MM, Kim EH, Sullivan JA, Plisch EH, Suresh M. FoxO1 controls References effector-to-memory transition and maintenance of functional CD8 T cell 1. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, memory. J Immunol. 2013;191:187–99. et al. Durable complete responses in heavily pretreated patients with 23. Zoon CK, Seitelman E, Keller S, Graham L, Blevins TL, Dumur CI, et al. metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. Expansion of melanoma-specific lymphocytes in alternative gamma chain 2011;17:4550–7. cytokines: gene expression variances between T cells and T-cell subsets 2. Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: exposed to IL-2 versus IL-7/15. Cancer Gene Ther. 2014;21:441–7. implications for vaccine development. Nat Rev Immunol. 2002;2:251–62. 24. Pollizzi KN, Patel CH, Sun IH, Oh MH, Waickman AT, Wen J, et al. mTORC1 3. Kaech SM, Wherry EJ. Heterogeneity and cell-fate decisions in effector and and mTORC2 selectively regulate CD8+ T cell differentiation. J Clin Invest. memory CD8+ T cell differentiation during viral infection. Immunity. 2007; 2015;125:2090–108. 27:393–405. 25. Zhang L, Tschumi BO, Lopez-Mejia IC, Oberle SG, Meyer M, Samson G, et al. 4. Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, et al. Mammalian target of rapamycin complex 2 controls CD8 T cell memory Acquisition of full effector function in vitro paradoxically impairs the in vivo differentiation in a Foxo1-dependent manner. Cell Rep. 2016;14:1206–17. antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest. 2005; 115:1616–26. 5. Klebanoff CA, Gattinoni L, Restifo NP. Sorting through subsets. Which T cell populations mediate highly effective adoptive immunotherapy? J Immunother. 2012;35:651–60. 6. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE, et al. Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci U S A. 2005;102:9571–6. 7. Contreras A, Sen S, Tatar AJ, Mahvi DA, Meyers JV, Srinand P, et al. Enhanced local and systemic anti-melanoma CD8+ T cell responses after http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

Co-transfer of tumor-specific effector and memory CD8+ T cells enhances the efficacy of adoptive melanoma immunotherapy in a mouse model

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Medicine & Public Health; Oncology; Immunology
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Abstract

Background: Adoptive cell transfer (ACT) is a promising cancer immunotherapeutic strategy that remains ineffective for a large subset of patients. ACT with memory CD8+ T cells (T ) has been shown to have superior mem efficacy compared to traditional ACT with effector CD8+ T cells (T ). T and T have complementary eff eff mem physiological advantages for immunotherapy, but previous publications have not examined ACT using a combination of T and T . eff mem Methods: Splenocytes harvested from Ly5.1+/C57BL/6 mice during and after infection with lymphocytic choriomeningitis virus (LCMV) were used to generate bona fide effector and memory CD8+ T cells specific for the LCMV epitope peptide GP33. Congenic Ly5.2+/C57BL/6 mice were inoculated with B16F10 melanoma cells transfected to express very low levels of GP33, then treated with ACT 7 days later with GP33-specific T ,T ,ora eff mem combination of T +T . eff mem Results: Inhibition of melanoma growth was strongest in mice receiving combinatorial ACT. Although combinatorial ACT and memory ACT resulted in maximal intratumoral infiltration of CD8+ T cells, combinatorial ACT induced stronger infiltration of endogenous CD8+ T cells than T ACT and a stronger systemic T cell mem responsiveness to tumor antigen. In vitro assays revealed rapid but transient melanoma inhibition with T and eff gradual but prolonged melanoma inhibition with T ; the addition of T enhanced the ability of T to inhibit mem mem eff melanoma in a manner that could be reproduced using conditioned media from activated T and blocked by mem the addition of anti-IL-2 blocking antibody. Conclusions: These findings suggest that a novel combinatorial approach that takes advantage of the unique and complementary strengths of tumor-specific T and T may be a way to optimize the efficacy of adoptive eff mem immunotherapy. Keywords: Adoptive transfer, Immunotherapy, T cell, Memory, Effector, Melanoma, Cancer * Correspondence: cliffcho@med.umich.edu Amanda Contreras and Megan V. Beems contributed equally to this manuscript Equal contributors VA Ann Arbor Healthcare System, 2215 Fuller Road, Ann Arbor, MI 48105, USA Department of Surgery, University of Michigan Medical School, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 2 of 10 Background Methods Adoptive cell transfer (ACT) is a promising strategy for Mice cancer immunotherapy that involves the isolation, ex- Seven- to eight-week old female congenic C57BL/6 pansion, and infusion of tumor-specific CD8+ T cells. mice with allele Ly5.1+ (T cell donor mice) or allele When combined with interleukin-2 (IL-2) and lympho- Ly5.2+ (T cell recipient mice) were purchased from depletion, ACT has led to complete and durable tumor Taconic (Hudson, NY) and maintained in sterile hous- regression in up to 20% of patients with metastatic mel- ing. All mouse work was reviewed and approved by the anoma [1]. Despite this, ACT remains ineffective for a University of Wisconsin and William S. Middleton large subset of patients. Memorial VA Hospital and VA Ann Arbor Healthcare Traditionally, ACT involves the use of terminally Animal Care and Use Committees. differentiated CD8+ effector T cells (T ) collected eff from tumor-infiltrating lymphocytes (TILs). When Tumor cell lines and effector/memory cell generation naïve T cells encounter antigen, they clonally expand Viral infections and tumor inoculations were performed and differentiate into T that have the ability to rap- per our previously published protocols [7, 9, 10]. In eff idly clear cells expressing their cognate antigen [2, 3]. brief, Ly5.1+/C57BL/6 mice were infected with 2 × 10 Following antigen clearance, the majority of T then PFU of Armstrong strain lymphocytic choriomeningitis eff undergo apoptosis, while a small population of virus (LCMV; gift from Marulasiddappa Suresh, antigen-specific CD8+ memory T cells (T ) persists University of Wisconsin) via intraperitoneal injection to mem long-term and can rapidly proliferate upon antigen generate CD8+ T cells specific for GP33, a class re-exposure [2, 3]. I MHC-restricted LCMV surface glycoprotein. Spleno- The use of different T cell subsets is one potential way cytes were harvested 8 days after infection (at which to improve the efficacy of ACT. Less-differentiated ef- time LCMV peptide-specific CD8+ T cells are of effector fector cells have been shown to have superior antitumor differentiation) to obtain bona fide T (as evidenced by eff low high immunity compared to terminally-differentiated cells, CD127 /KLRG1 expression) and 50–80 days after potentially due to a number of mechanisms such as de- infection (at which time LCMV peptide-specific CD8+ T creased IL-2 production and increased apoptosis with cells are of memory differentiation) to obtain bona fide high low further differentiation [4, 5]. Our laboratory and others T (as evidenced by CD127 /KLRG1 expression) mem have also demonstrated that ACT with T is more ef- [7, 8]. The yield of GP33-specific memory CD8+ T cells mem fective than ACT with T or naïve T cells [6, 7]. T was increased by adoptively transferring Ly5.1+/C57BL/ eff mem appear to be uniquely resistant to melanoma-induced 6 mice with 10 Ly5.1+/CD8+ T cells derived from sple- suppression and generate a stronger intratumoral im- nocytes of Ly5.1+/P14 TCR transgenic mice (C57BL/6 mune response [7, 8]. background mice with CD8+ T cell specificity for GP33 No previous publications have explored performing [7]. The spontaneous mouse melanoma cell line B16F10 ACT with a combination of T and T . Although T was transfected with a plasmid encoding GP33, and mem eff eff are prone to apoptosis, we hypothesized that this limita- poorly immunogenic clones with low levels of GP33 ex- tion could be offset by the innate proliferative ability of pression were selected to create a new B16GP33 cell line, T , with T contributing to initial tumor control and which we have previously demonstrated to be of compar- mem eff T to later tumor control. In a murine model, we ably low immunogenicity as parental B16F10 [9, 10]. This mem compared the efficacy of ACT using bona fide T , line was cultured in RPMI-1640 medium (Mediatech, eff T , and a combination of both. We utilized a novel Herndon, VA) with 10% fetal bovine serum, 2 mM L-gluta- mem melanoma tumor expressing very low levels of a viral mine, 100 μg/mL streptomycin, and 100 U/mL penicillin peptide in order to dissect the biological effects of (Life Technologies, Inc., Grand Island, NY). Ly5.2 peptide-specific CD8+ T cells of true effector and mem- +/C57BL/6 mice received subcutaneous injections of 1 × ory differentiation. This methodology enables us to 10 B16GP33 cells suspended in serum-free RPMI-1640 examine the therapeutic implications of bona fide ef- to generate flank melanoma tumors. fector and memory tumor-specific CD8+ T cells in a manner not possible with standard murine melanoma Adoptive cell transfer targets like GP100. Using in vitro experiments, we ex- Adoptive cell transfer was performed per our previously plored the temporal cell killing patterns and paracrine published protocol [7, 8, 10]. As previously observed, effects of these T cell subsets. We discovered that the GP33-specific CD8+ T cells typically comprised approxi- combination of T +T led to the strongest control mately 8–10% of splenocytes harvested from effector eff mem of melanoma tumor growth, potentially due to comple- and memory T cell donor mice [7, 8]. After obtaining mentary cell killing patterns and local production of splenocytes from mice infected with LCMV, magnetic IL-2 by T . bead separation columns (Miltenyi, Auburn, CA) were mem Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 3 of 10 used to isolate CD8+ T cells. Flow cytometry was per- Statistical analysis formed to quantify the percentage of tumor-specific Experimental data was analyzed using IBM SPSS statis- GP33-specific CD8+ T cells. 10 GP33-specific CD8+ tical software version 23 (Armonk, NY). T-test was used 4 4 T ,T ,or5×10 T +5×10 T were adoptively to compare groups and a repeated-measures analysis of eff mem eff mem transferred via retro-orbital intravenous injections into variance (ANOVA) with pair-wise comparisons per- B16F10- or B16GP33 melanoma-bearing mice at specific formed using Fisher’s protected least significant differ- time points after tumor inoculation. ence tests were used to compare multiple groups. Significance was defined as p < 0.05, and error bars rep- resent standard errors of the mean. MTT assay For multiple timepoints, a total number of 2 × 10 GP33-specific CD8+ T and T or both were separ- eff mem Results ately co-cultured with 2 × 10 B16GP33 melanoma cells Co-ACT of T +T is more effective at inhibiting in eff mem at an overall effector:target ratio of 1:1 in 48-well plates vivo melanoma growth than ACT of either T cell subset in the presence of 10 ng/mL recombinant human IL-2 alone (BD Biosciences, San Jose, CA) for MTT assays as previ- To verify the peptide specificity of our ACT model, ously described [7]. T cells were characterized and iso- C57BL/6 mice were treated with 10 GP33-specific T eff lated using the methodology described for adoptive cell one day after subcutaneous inoculation with B16GP33 transfer. Viable B16GP33 melanoma cells were quanti- melanoma or parental B16F10 melanoma (which does fied using the Celltiter 96 non-radioactive MTT assay kit not express GP33). As shown in Fig. 1a-b, early ACT (Promega, Madison, WI) following the manufacturer’s strongly suppressed the growth of B16GP33 melanoma instructions. but not B16F10 melanoma, suggesting that the effect of ACT was peptide-specific. Moreover, the kinetics of Conditioned media assay B16GP33 tumor growth in immunocompetent mice was For 5 h, GP33-specific CD8+ T and T were cul- eff mem comparable to those of parental B16F10 tumor. To tured separately or together in RPMI-1640 with 10 ng/ compare the efficacy of ACT with different T cell mL IL-2 and 0.1 μg/mL GP33 peptide (gift from Marula- subsets, Ly5.2+/C57BL/6 mice received ACT of 10 siddappa Suresh, University of Wisconsin). Conditioned GP33-specific CD8+ T ,T ,or T +T on day 7 eff mem eff mem media was collected by centrifuging wells and collecting after subcutaneous inoculation with 10 B16GP33 cells. the supernatant. For 12 h, GP33-specific CD8+ T and eff As shown in Fig. 1c-d, memory ACT resulted in signifi- T were then co-cultured with B16GP33 melanoma mem cantly greater inhibition of tumor growth compared to cells with or without conditioned media and anti-IL-2 effector ACT as previously described. Combinatorial (BD Biosciences, San Jose, CA) for MTT assays as de- ACT with T +T had significantly greater inhibition eff mem scribed above. of tumor growth compared to ACT with T or T eff mem separately, making this combination the most effective Flow cytometry strategy for ACT. Flow cytometric analysis was performed per our previ- ously published protocol [7–10]. Once harvested, tumor samples were processed into single-cell suspensions with Co-ACT of T +T promotes more potent intratumoral eff mem mechanical fractionation on a fine wire mesh followed CD8 + T cell infiltration than ACT of either T cell subset by lymphocyte isolation using Histopaque (Sigma Al- alone drich, St. Louis, MO). Splenocytes before ACT and To determine if the efficacy of combinatorial ACT was tumor-infiltrating lymphocytes (TIL) 4 to 14 days after associated with stronger intratumoral T cell infiltration, ACT were stained with APC-labeled MHC class I tetra- mice were euthanized 4 and 14 days after ACT to allow mers loaded with GP33 peptide (NIH Tetramer Facility for tumor harvesting and flow cytometric analysis of TIL Core, Atlanta, GA), PerCP-labeled anti-CD8 mAb, populations. Representative data shown in Fig. 2a dem- PE-labeled anti-Ly5.1 mAb, and FITC-labeled anti-CD44 onstrate that CD8+ T cells comprised a higher percent- mAb (BD Biosciences, San Jose, CA). Intracellular stain- age of TIL in mice treated with ACT in comparison ing required stimulation of lymphocytes with media with untreated controls. As shown in Fig. 2b, total num- alone or with GP33 peptide (1 μg/mL), brefeldin A, and bers of intratumoral CD8+ were transiently elevated on recombinant human IL-2 (10 ng/mL) at 37°C for 5 h. A day 4 in mice treated with effector ACT, but this effect BD LSRFortessa flow cytometer (BD Biosciences, San was short-lived; in contrast, intratumoral CD8+ T cells Jose, CA) was used and data were analyzed using FlowJo remain elevated on day 14 in mice treated with memory software (Tree Star Inc., Ashland, OR). and combinatorial ACT. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 4 of 10 Fig. 1 ACT with a combination of T +T resulted in optimal control of melanoma growth. Mice bearing B16GP33 (a)or B16F10 (b)melanoma eff mem were untreated (control) or treated with ACT of 10 GP33-specific CD8+ T cells one day after tumor inoculation. Serial tumor measurements confirmed that ACT resulted in a GP33-specific inhibition of melanoma tumor growth. (c) Mice bearing B16GP33 melanoma were treated 7 days after tumor 5 5 4 4 inoculation with no treatment (control) or 10 T ,10 T ,or 5 × 10 T +5× 10 T . Comparison of treated groups (d)demonstrates optimal eff mem eff mem control of melanoma growth with combinatorial ACT. This experiment was repeated three times with similar results, 4–5 mice per group. (+ p <0.05 compared with control; * p < 0.05 compared with effector ACT; Ɨ p < 0.05 compared with memory ACT) The more potent intratumoral CD8+ T cell infiltration Combinatorial ACT results in a stronger systemic CD8+ T seen with combinatorial ACT is the result of a stronger cell response against tumor antigen endogenous immune response To determine if combinatorial ACT improved sys- To characterize the increase in intratumoral CD8+ T cell temic antitumor responsiveness in addition to local infiltration seen following ACT, flow cytometric analysis antitumor activity, splenocytes harvested 14 days after was used to quantify the presence of Ly5.1+ adoptively ACT were stimulated with or without GP33 peptide transferred CD8+ T cells and Ly5.2+ endogenous CD8+ T in the presence of IL-2 and brefeldin A, and stained cells. Representative flow cytometric data are shown as to assess intracellular levels of IFNγ expression. Rep- percentages in Fig. 3a; overall data are shown as total cell resentative flow cytometric data are shown as per- numbers in Fig. 3b-c. As shown in Fig. 3b,Ly5.1+ adop- centages in Fig. 4a;overall data areshownastotal tively transferred CD8+ T cells comprised a small and cell numbers in Fig. 4b.As shown in Fig. 4b,memory short-lived fraction of TIL after effector ACT but were ACT resulted in stronger splenocyte reactivity to present in substantial and stable numbers after memory GP33 than effector ACT, as we have previously de- ACT; combinatorial ACT, which used half the number of scribed. However, combinatorial ACT produced sig- T used in memory ACT, resulted in smaller numbers nificantly greater IFNγ expression among splenocytes mem of intratumoral Ly5.1+/CD8+ TIL. In contrast, as shown than memory ACT, suggesting that combinatorial in Fig. 3c, combinatorial ACT resulted in the strongest ACT may induce a much stronger level of CD8+ T intratumoral infiltration of endogenous CD8+ T cells. cell responsivenesstotumor antigen. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 5 of 10 Fig. 2 Combinatorial ACT results in a stronger intratumoral T cell response. Seven days after tumor inoculation, Ly5.2+ mice were treated with 5 5 4 4 10 T ,10 T ,or5 × 10 T +5× 10 T derived from Ly5.1+ mice. TIL were harvested from melanoma tumors resected 4 or 14 days after eff mem eff mem ACT and stained for flow cytometric analysis. (a) Representative TIL data (gated on lymphocyte populations) demonstrate that CD8+ T cells comprised the highest percentage of TIL after effector ACT on day 4 and after combinatorial ACT on day 14. (b) Overall comparison of total numbers of intratumoral CD8+ T cells on days 4 and 14 demonstrated a transient increase after effector ACT and a sustained increase after memory ACT and combinatorial ACT. This experiment was repeated twice with similar results, 4–5 mice per group. (* p < 0.05 compared with control condition; † p < 0.05 compared with day 4) Combinatorial ACT may benefit from temporal differences During combinatorial ACT, tumor cell caused by by T eff in melanoma cell killing between T and T may be enhanced by IL-2 production from T eff mem mem To further explore the improved efficacy of combinator- To determine if the improved efficacy of combinatorial ial ACT, T and T were co-cultured in vitro with ACT was attributable to local interactions between cell eff mem B16GP33 melanoma cells in the presence of IL-2 for types, T ,T ,and T +T were co-cultured with eff mem eff mem 72 h. As shown in Fig. 5, serial MTT assays demon- B16GP33 cells in the presence of IL-2 for 8 h. As shown strated that T inhibited melanoma more potently than in Fig. 6a, whereas T and T resulted in comparable eff eff mem T at 6 h. However, whereas the ability of T to in- levels of melanoma inhibition at this time point, the mem eff hibit melanoma was lost at later timepoints, T con- combination of T +T resulted in a stronger level mem eff mem tinued to inhibit melanoma for the duration of time of melanoma inhibition. To determine if this enhance- points tested (up to 72 h), suggesting that the enhanced ment was attributable to paracrine effects, T and T eff mem therapeutic efficacy of combinatorial ACT could be due were separately stimulated with GP33 peptide for 5 h, to the temporal combination of early melanoma inhib- after which cells were pelleted by centrifugation and ition mediated by T plus delayed but durable melan- conditioned media were collected. As shown in Fig. 6b, eff oma inhibition mediated by T . the addition of conditioned media from T enhanced mem mem Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 6 of 10 Fig. 3 The stronger local CD8+ T cell response seen with combinatorial ACT is largely the result of endogenous T cell recruitment. Seven days 5 5 4 4 after tumor inoculation, Ly5.2+ mice were treated with 10 T ,10 T ,or5 × 10 T +5× 10 T derived from Ly5.1+ mice. Melanoma eff mem eff mem tumors were resected 4 or 14 days after ACT and TIL were harvested for flow cytometric analysis. (a) Representative data demonstrate higher percentages of Ly5.1+/CD8+ T cells in tumors after memory and combinatorial ACT. (b) Analysis of total numbers of Ly5.1+/CD8+ T cell populations showed that significant and durable infiltration of adoptively transferred cells was observed after memory ACT; combinatorial ACT, which used half the number of adoptively transferred T as memory ACT, resulted in smaller numbers of intratumoral Ly5.1+/CD8+ T cells. (c) mem Analysis of total numbers of Ly5.2+/CD8+ T cell populations showed that memory ACT resulted in a significant recruitment of endogenously- derived CD8+ T cells that persisted on day 14; despite using half the number of T , combinatorial ACT also resulted in durable infiltration of mem endogenously-derived CD8+ T cells. This experiment was repeated twice with similar results, 4–5 mice per group. (* p < 0.05 compared with control condition; † p < 0.05 compared with day 4) Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 7 of 10 Fig. 4 Combinatorial ACT results in the strongest level of systemic CD8+ T cell responsiveness to tumor antigen. Seven days after tumor inoculation, 5 5 4 4 Ly5.2+ mice were treated with 10 T ,10 T ,or 5× 10 T +5×10 T derived from Ly5.1+ mice. Spleens were resected 14 days after ACT and eff mem eff mem splenocytes were incubated with media alone (control) or with tumor antigen (GP33). (a) Representative data demonstrate negligible levels of IFNγ expression by splenocytes in response to GP33 stimulation after no ACT or effector ACT, with increasing levels seen after memory ACT and combinatorial ACT, respectively. (b) The highest levels of IFNγ expression by splenocytes in response to GP33 stimulation were seen after combinatorial ACT. This experiment was repeated with similar results, 4 mice per group. (* p < 0.05 compared with control condition; † p <0.05 compared with effector ACT, ‡ p < 0.05 compared with memory ACT) the ability of T to inhibit melanoma to a comparable caused by T . The addition of anti-IL-2 blocking anti- eff eff level seen with T +T . The addition of conditioned body alone did not affect the ability of T to inhibit eff mem eff media from T did not enhance the ability of T to melanoma. eff mem inhibit melanoma (data not shown). When anti-IL-2 blocking antibody was added to conditioned media from Discussion T , this effect was lost, suggesting that paracrine re- For the first time, we have shown that ACT using a mem lease of IL-2 from T may potentiate tumor cell death combination of T +T leads to even more robust mem eff mem Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 8 of 10 cytokine/chemokine release, antigen presentation, and priming of endogenous tumor-specific T cells [13], ul- timately promoting stronger systemic anti-tumor immunity. Given the innate predisposition of effector cells to rap- idly target antigen, and the ability of memory cells to persist and expand, we hypothesized that the ability of T and T to engage and inhibit melanoma would eff mem exhibit temporal differences. Indeed, in vitro assays dem- onstrated that T cause immediate but transient melan- eff oma inhibition, while T cause gradual but persistent mem inhibition. This pattern is further reflected by the fact that, whereas ACT with T resulted in a decline in eff intratumoral adoptively transferred Ly5.1+/CD8+ T cells Fig. 5 T and T have different temporal patterns of melanoma mem eff at 14 days, ACT with T resulted in stable numbers mem inhibition in vitro. T and T were co-cultured with B16GP33 eff mem of intratumoral transferred T cells. Thus, the activity of melanoma cells in the presence of 10 ng/mL recombinant human IL-2 for 72 h. Control wells contained no T cells. Serial MTT assays effector and memory cells may be complementary over were performed every 6 h. Over approximately the first 8 h, T eff time, leading to stronger and more prolonged melanoma produced significantly greater inhibition of B16GP33. At all later time suppression. points, T demonstrated significantly greater suppression of mem We found that the intrinsic ability of T to inhibit eff tumor growth. This experiment was repeated twice with similar melanoma may also be enhanced by local production of results, 3 samples per group. († p < 0.05 compared with T ) eff IL-2 by T . Unlike effector T ,T are uniquely mem eff mem capable of elaborating IL-2 in response to antigen stimu- lation [2, 3]. IL-2 supplementation has long been associ- control of melanoma growth than ACT using T or ated with improved outcomes following ACT [14]. eff T alone. Our findings suggest that the advantage Systemic and local delivery of IL-2 in combination with mem of combinatorial ACT may result from the induction ACT has been shown to increase CD8+ tumor infiltra- of stronger infiltration of endogenous CD8+ T cells tion and suppression of tumor growth [1, 14–17]. With into tumor, stronger systemic CD8+ T cell responsive- combinatorial ACT, the local, paracrine production of ness to tumor antigen, temporal differences in melan- IL-2 by T may mitigate the need for exogenous IL-2 mem oma cell killing, and paracrine IL-2 effects. One administration, decreasing the risk of systemic inflam- recent publication addressed the effects of combining matory side effects. T cell subsets prior to ACT, but this involved By utilizing a melanoma expressing low levels of a co-culturing naïve cells with T for 6 days prior to viral peptide, we are able to examine the therapeutic mem ACT [11]. That approach led to a loss of effects of bona fide effector and memory differenti- less-differentiated T cell subsets and impaired in vivo ation tumor-specific CD8+ T cells in ways not per- tumor regression [11]. By combining T +T at missible with the use of traditional tumor antigens eff mem the time of adoptive transfer, we were able to retain (e.g., GP100). Although our B16GP33 tumor model the innate advantages of each T cell subset. does not appear to be more intrinsically immunogenic We observed that combinatorial ACT generated stron- than parental B16F10 melanoma) [10], further work ger intratumoral CD8+ T cell infiltration than ACT with will be necessary to validate our observations using T or T alone; this was not due to greater traffick- effector and memory CD8+ T cells directed against eff mem ing of adoptively transferred cells into tumor, but a re- naturally occurring melanoma antigens. Our observa- sult of more potent induction of endogenous immune tions suggest that a combinatorial approach to ACT responses. This stimulation of ongoing endogenous CD8 using both T and T may beameanstooptimize eff mem + T cell responses may contribute to the heightened effi- the efficacy of adoptive melanoma immunotherapy. cacy of combinatorial ACT compared to memory ACT. While T can be readily expanded from TIL cultured eff We previously found that combining ACT with check- in IL-2, it is difficult to prepare large quantities of point inhibition led to a similar induction of endogenous T . There is a growing body of literature character- mem TILs that was also associated with greater tumor sup- izing the metabolic determinants of effector versus pression, stronger systemic T cell responsiveness to memory CD8+ T cell differentiation, suggesting that it tumor antigen, and a more potent long-term anti-tumor may become feasible to develop this combinatorial immunity [12]. Enhanced melanoma cell death resulting approach for clinical evaluation [18–25]. This is an from combinatorial ACT may promote stronger local area of ongoing investigation in our laboratory. Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 9 of 10 Fig. 6 Paracrine release of IL-2 from T may potentiate melanoma inhibition by T . The local interaction of T and T was examined by mem eff eff mem performing in vitro melanoma inhibition assays. (a)T and T and T +T were co-cultured with B16GP33 for 8 h. Although melanoma eff mem eff mem inhibition was comparable between T and T , significantly stronger inhibition of melanoma was observed with the combination of T + eff mem eff T .(b) Conditioned media (CM) was collected from T stimulated with GP33 for 5 h. The addition of CM enhanced the efficacy of T to a mem mem eff similar degree as the addition of T . The effect of CM was negated by the addition of anti-IL-2 blocking antibody; the addition of anti-IL-2 mem blocking antibody alone did not affect the ability of T to inhibit melanoma. The addition of anti-IL-2 blocking antibody blunted the ability of eff T +T to inhibit melanoma, but it could not be determined if this effect was exerted on T or T (data not shown). This experiment was eff mem eff mem repeated twice with similar results, 3 samples per group. (p-values as shown on horizontal bars) Conclusions complementary differences in temporal killing of Traditional approaches to adoptive immunotherapy tumor cells by effector and memory CD8+ T cells, as have used effector CD8+ T cells for their rapid prolif- well as local paracrine potentiation of effector CD8+ erative capacity and ability to engage and clear tumor T cell function by memory T cell-secreted IL-2. These cells. Experimental evidence suggests that memory findings suggest that combinatorial approaches to CD8+ T cells may have physiological advantages over cell-based immunotherapy harnessing diverse states of effector CD8+ T cells. Our observations indicate that T cell differentiation may open the door to even a combinatorial approach to adoptive immunotherapy greater therapeutic benefit. using effector and memory CD8+ T cells concurrently Abbreviations results in superior tumor control associated with ACT: Adoptive cell transfer; LCMV: Lymphocytic choriomeningitis virus; maximal induction of endogenous T cell responses to T : Effector CD8+ T cell; TIL: Tumor infiltrating lymphocyte; T : Memory eff mem tumor. This strategy appears to take advantage of CD8+ T cell Contreras et al. Journal for ImmunoTherapy of Cancer (2018) 6:41 Page 10 of 10 Funding memory T cell-based adoptive immunotherapy in mice. Cancer Immunol This work was supported by grant support from the Department of Veterans Immunother. 2016;65:601–11. Affairs CDA-2 and Merit Review Award (1I01BX001619-01A1) to CSC, National 8. Wentworth L, Meyers JV, Alam S, Russ AJ, Suresh M, Cho CS. Memory T cells Institutes of Health T32 (CA090217) to MVB, National Institutes of Health T32 are uniquely resistant to melanoma-induced suppression. Cancer Immunol (GM081061 and AI55397) to AC, and National Institutes of Health AI48785 to Immunother. 2013;62:149–59. MS. The content is solely the responsibility of the authors and does not 9. Russ AJ, Wentworth L, Xu K, Seroogy CM, Rakhmilevich A, Sondel PM, et al. represent the views of the Department of Veterans Affairs or the United Suppression of T-cell expansion by melanoma is exerted on resting cells. States Government or the National Institutes of Health. Ann Surg Oncol. 2011;18:3848–57. 10. Russ AJ, Xu K, Wentworth L, Alam S, Meyers JV, Macklin MD, et al. Availability of data and materials Melanoma-induced suppression of tumor antigen-specific T cell expansion The datasets used and analyzed during the current study are available from is comparable to suppression of global T cell expansion. Cell Immunol. the corresponding author on reasonable request. 2011;271:104–9. 11. Klebanoff CA, Scott CD, Leonardi AJ, Yamamoto TN, Cruz AC, Ouyang C, Authors’ contributions et al. Memory T cell-driven differentiation of naïve cells impairs adoptive AC and MVB performed the mouse and lymphocyte experiments and immunotherapy. J Clin Invest. 2016;126:318–34. analyzed and interpreted the data and contributed equally to the writing of 12. Mahvi DA, Meyers JV, Tatar AJ, Contreras A, Suresh M, Leverson GE, et al. this manuscript. AJT, SS and PS performed the mouse and lymphocytes CTLA-4 blockade plus adoptive T cell transfer promotes optimal melanoma experiments. MS interpreted the data. TKL performed the co-culture experiments immunity in mice. J Immunother. 2015;38(2):54–61. and contributed to the writing of this manuscript. CSC analyzed and interpreted 13. Schiavoni G, Gabriele L, Mattei F. The tumor microenvironment: a pitch for the data and contributed to the writing of this manuscript. All authors read and multiple players. Front Oncol. 2013;3:1–15. approved the final manuscript. 14. Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol. 2014;192:5451–8. Ethics approval and consent to participate 15. Heemskerk B, Liu K, Dudley ME, Johnson LA, Kaiser A, Downey S, et al. All mouse work was reviewed and approved by the University of Wisconsin Adoptive cell transfer for patients with melanoma, using tumor-infiltrating and William S. Middleton Memorial VA Hospital and VA Ann Arbor lymphocytes genetically engineered to secrete interleukin-2. Hum Gene Healthcare Animal Care and Use Committees. Ther. 2008;19:496–510. 16. Tahtinen S, Kaikkonen S, Merisalo-Soikkeli M, Gronerg-Vaha-Koskela S, Competing interests Kanerva A, Parviainen S, et al. 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Le HK, Graham L, Miller CH, Kmieciak M, Manjili MH, Bear HD. Incubation of Department of Surgery, University of Wisconsin School of Medicine and antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin Public Health, 600 Highland Avenue, Madison, WI 53792, USA. Department in IL-7 + IL-15 increases yield of cells capable of inducing regression of of Pathobiological Sciences, University of Wisconsin School of Veterinary melanoma metastases compared to culture in IL-2. Cancer Immunol Medicine, 2015 Linden Drive, Madison, WI 53706, USA. VA Ann Arbor Immunother. 2009;58:1565–76. Healthcare System, 2215 Fuller Road, Ann Arbor, MI 48105, USA. 20. Sukumar M, Liu J, Ji Y, Subramanian M, Crompton JG, Yu Z, et al. Inhibiting Department of Surgery, University of Michigan Medical School, 1500 East glycolytic metabolism enhances CD8+ T cell memory and antitumor Medical Center Drive, Ann Arbor, MI 48109, USA. function. J Clin Invest. 2013;123:4479–88. 21. 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Published: May 29, 2018

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