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Intra-tumoral production of IL18, but not IL12, by TCR-engineered T cells is non-toxic and counteracts immune evasion of solid tumors

ABSTRACT Adoptive therapy with engineered T cells shows promising results in treating patients with malignant disease, but is challenged by incomplete responses and tumor recurrences. Here, we aimed to direct the tumor microenvironment in favor of a successful immune response by local secretion of interleukin (IL-) 12 and IL-18 by sadministered T cells. To this end, we engineered T cells with a melanoma-specific T cell receptor (TCR) and murine IL-12 and/or IL-18 under the control of a nuclear-factor of activated T-cell (NFAT)-sensitive promoter. These T cells produced IL-12 or IL-18, and consequently enhanced levels of IFNγ, following exposure to antigen-positive but not negative tumor cells. Adoptive transfer of T cells with a TCR and inducible (i)IL-12 to melanoma-bearing mice resulted in severe, edema-like toxicity that was accompanied by enhanced levels of IFNγ and TNFα in blood, and reduced numbers of peripheral TCR transgene-positive T cells. In contrast, transfer of T cells expressing a TCR and iIL-18 was without side effects, enhanced the presence of therapeutic CD8+ T cells within tumors, reduced tumor burden and prolonged survival. Notably, treatment with TCR+iIL-12 but not iIL-18 T cells resulted in enhanced intra-tumoral accumulation of macrophages, which was accompanied by a decreased frequency of therapeutic T cells, in particular of the CD8 subset. In addition, when administered to mice, iIL-18 but not iIL-12 demonstrated a favorable profile of T cell co-stimulatory and inhibitory receptors. In conclusion, we observed that treatment with T cells engineered with a TCR and iIL18 T cells is safe and able to skew the tumor microenvironment in favor of an improved anti-tumor T cell response.


Introduction
Up to now, dozens of clinical trials have demonstrated the effectiveness of adoptive therapy with T cells that are geneengineered with T cell receptors (TCRs) or chimeric antigen receptors (CARs). Therapeutic successes are most prominent for the treatment of hematological malignancies, [1][2][3][4][5][6] whereas in the majority of patients with solid tumors, initial regressions are challenged by rapid tumor recurrences.
Patient responsiveness to immune therapies appears to be related to numbers and location of tumor infiltrating lymphocytes (TILs). 7,8 Tumors that have low or negligible numbers of TILs may demonstrate intrinsic changes that result in limited T cell infiltration, movement and activation, and as such pose barriers to T cell treatments when compared to tumors with high numbers of TILs. 9,10 In fact, predictive value towards response to clinical T cell therapies has recently been attributed to determinants of immunogenicity, such as extent of antigen presentation, composition of immune infiltrates, and presence of inhibitors of T cell activity. 11 Expectedly, intervention with such determinants has the ability to enhance the sensitivity of tumors for T cell treatment. 12 Here, we investigated to what extent local depositions of IL-12 and IL-18 in the tumor tissue would enhance the efficacy of adoptive T cell therapy. IL-12 is a heterodimer that consists of the subunits p35 and p40 and, although primarily produced by antigen-presenting cells, it is also a product of T cells. 13,14 Initial studies showed IL-12's ability to enhance the cytolytic activity of T cells, 15 aid in the recruitment of macrophages, 16 and induce a TNFa-mediated shift towards M1-type macrophages. 17,18 IL-18 is a pro-inflammatory IL-1-like cytokine that is primarily produced by macrophages and is able to drive T and NK cell maturation (reviewed in 19 ). Both cytokines enable an immunological shift towards Th1-type T cell responses. Notably, IL-12 increases the proportion of T cells expressing the IL-18 receptor 1 subunit (IL-18R1), resulting in further enhancement of IFNg production by IL-12 plus IL-18-stimulated T cells. 20,21 Despite IL-12's beneficial impact on antitumor immune responses in preclinical models, which also holds in early-phase trials, systemic administration of recombinant IL-12 has been reported to result in hepatotoxicity and high systemic levels of IFNg. 22,23 In contrast, administration of IL-18 revealed no such toxicities in studies with melanoma and renal cell cancer patients, however, did not reveal clinical responses either. [24][25][26] We argue that delivery of these two cytokines directly at the site of the tumor, either alone or together, will minimize toxic effects and at the same time maximize immune-stimulatory effects. In earlier studies, we have shown that the use of CAR-engineered T cells with inducible IL-12 (iIL-12) enhanced tumor killing without toxicity by increasing the numbers and activation of tumor infiltrating macrophages. 18 In the current study, we have extended these findings and exploited TCR-engineered T cells to provide antigen-specific and intra-tumoral release of IL-12 and IL-18, and investigated the impact of these enhanced T cells on tumor growth, toxicity, T cell phenotype and function.

TCR and inducible IL-12 and IL-18 constructs
TCRa and b genes specific for the human gp100 280-288 epitope presented by HLA-A2 (gp100/A2) were derived from CTL-296 clone, 28 murinized and codon-optimized as described earlier. 29 Subsequently, TCR genes were cloned into the pMP71 vector (kindly provided by prof. Wolfgang Uckert, Max-Delbr€ uck Center, Berlin, Germany) with TCRa and b genes separated by an optimized T2 A ribosome skipping sequence. Sequences of murine IL-12 (a linked dimer consisting of p35 (NM_000882) and p40 (NM_002187)) and the processed form of IL-18 (NM_001562) were ordered from Geneart (not codon-optimized) and cloned into a pSIN vector, which contains 6 repeats of the NFAT response element and a minimal mIL-2 promotor, as described in. 18 The plasmids with iIL-12 and iIL-18 contain a neomycin resistance gene to allow for selection of transduced T cells.

T cell transduction
Total mouse splenocytes were isolated, activated with Concanavalin A (0.5 mg/ml) and rhIL-2 in mouse T cell medium, and transduced with retroviral supernatant from a co-culture of single construct (either TCR, iIL-12 or iIL-18) transfected 293 T and Phoenix-Amp cell lines as described by Pouw and colleagues. 29 To generate T cells that express TCR and either iIL-12 or iIL-18, T cells were simultaneously incubated with two retroviral supernatants in a 1:1 ratio (yielding TCRCiIL-12 or TCRCiIL-18 T cells). To obtain T cell populations that provide an inducible source for both cytokines, used in some experiments, TCRCiIL-12 and TCRCiIL-18 T cells were mixed in a 1:1 ratio (yielding TCRCiIL-12CiIL-18 T cells). Control (mock) T cells were transduced with empty retroviral vector. TCR and mock T cells were cultured as described above, whereas T cells harboring (in addition to the TCR) iIL-12 or iIL-18 underwent selection with 1 mg/ml of G418 starting at 24 h after transduction and lasting for 72 h (for a detailed flowchart of the T cell activation, transduction and selection procedure, please see Fig. 1A and supplementary figure 1 A, B).

Adoptive T cell therapy
Experiments with mice were approved by the Experimental Animal Committee of the Erasmus MC Cancer Institute and carried out in accordance with institutional and national guidelines. For adoptive T cell therapy studies we used HLA-A2 transgenic (tg) mice that express a chimeric HLA-A Ã 0201 transgene (HHD, referred to as HLA-A2). 30 Adoptive transfer of T cells was done as described before. 27 In short, at day -15, HLA-A2 tg mice were injected subcutaneously with 0.5 £ 10 6 of a B16:A2-YLEP clone and at days -4 and -3 mice received a total of two Busulfan injections intraperitoneally (16.5 mg/kg ea.), followed a day later by a single Cyclophosphamide injection intraperitoneally (200 mg/kg). In some experiments, the mice were not transplanted with tumor cells, but followed the same conditioning and T cell treatment protocol. Mice were treated with T cells at day 0 and grouped according to one of the following treatments: mock T cells (number of T cells equal to TCR T cell group); TCR T cells (total number corrected for 7.5 £ 10 6 transduced T cells); TCRCiIL-12 T cells; TCRCiIL-18 T cells; and TCRCiIL12C18 T cells). Tumor growth was measured by caliper 3 times a week and tumor volumes were estimated with the formula 0.4 x (AxB2) where A represents the largest diameter and B the diameter perpendicular to A. Tumor regression or response to treatment was defined as a >30% reduction in size compared to day 0, while relapse was defined as a size-increase of the tumor over the course of at least 3 days preceded by response to treatment. Survival was monitored daily for up to 45 days after administration of T cells. Body weight and edema like symptoms were recorded starting at the day of tumor inoculation and every third day thereafter. Peripheral blood was collected at day 6 after T cell transfer and at weekly intervals thereafter and used to determine numbers of administered T cells, whereas plasma was collected at days 16 and day 21 and used to measure cytokine levels. Tumor tissues of regressing tumors were collected at day 5 after T cell transfer and snap-frozen in liquid nitrogen and stored at -80 C for downstream assessment of cytokines in tumor lysates and performance of in situ immune fluorescence. Another part of tumor tissue was directly processed into TIL suspensions. To this end, tissue was cut into smaller fragments, incubated with collagenase (1 mg/ml; Sigma, St. Louis, MI) for 45 minutes at 37 C, checked microscopically and washed prior to flow cytometry.

Flow cytometry
T cells or TILs (5 £ 10 5 ) were washed with PBS and incubated with antibodies at 4 C or with pMHC multimers at 37 C for 30 min. Following staining, T cells were washed again and fixed with 1% paraformaldehyde. Absolute T cell counts in mouse blood samples were determined using Flow-Count Fluorospheres. Events were acquired on a FACS Canto flow cytometer and analysed using FCS Express 4 software (BD Biosciences).

In situ immune fluorescence
Tissue sections were cut at 5 mm and fixed with acetone for 10 min, dried, washed, and blocked with PBS/10% donkey serum/0.3% Triton for 30 min prior to immune staining. Next, sections were incubated with the primary antibody overnight at 4 C, washed and incubated with the donkey anti-rat IgG Alexa Fluor 488 for 2 h at room temperature in the dark. Sections were covered with Vectashield, and kept subsequently at room temperature for 2 h and at 4 C overnight. Sections were examined microscopically (Leica, DM IL, 200x magnification) and photographed (Leica DFC 3000G camera and LAS4 software). Recorded photographs were analyzed using Fiji software. 31 The number of pictures from each tumor ranged between 12 and 16, and using an in-house developed algorithm, the mean number of positively stained cells was determined, and normalized for percentages of nucleated cells (DAPI staining).

Statistical analyses
The different treatment groups were compared with the Student's t-tests, or the Mantel-Cox test in case of survival data, using GraphPad Prism5 (GraphPad Software, La Jolla, CA). P values < 0.05 were considered statistically significant.

TCRCiIL-12 T cells, and to a lesser extent TCRCiIL-18 T cells, produce enhanced levels of IFNg
We developed a protocol that yielded high numbers of T cells transduced with both the gp100/HLA-A2-specific TCR and iIL-12 or IL-18. Such inducible T cells showed maximal responsiveness towards antigen-positive tumor cells (see Fig. 1A and supplementary figure 1 ACB for details). Surface expression of TCR genes and binding to cognate pMHC ranged between 65-75%, irrespective of the presence of iIL-12 or iIL-18 (Fig. 1B). TCRCiIL-12 T cells produced >5.5 ng/10 6 cells of IL-12, and TCRCiIL18 T cells produced >75 pg/10 6 cells of IL-18 upon co-culture with antigen-positive cells (see Fig. 2A). Co-culturing TCRCiIL-12 T cells and TCRCiIL-18 T cells at a 1:1 ratio (TCRCiIL-12CiIL-18) demonstrated an expected drop in the antigen-specific production of IL-12 and IL-18. Notably, T cells that harbor iIL-12 produced minor amounts of IL-12 independent of TCR engagement, which was not the case for T cells harbouring iIL-18 (see Fig. 2A). When testing antigen-specific production of IFNg by these T cell populations, we observed a significant increase in case T cells harbored iIL-12, iIL-18 or both (see Fig. 2B) when compared to TCR T cells. TCRCiIL-12 and TCRCiIL-12CiIL-18 T cells produced more than twice as much IFNg than TCRCiIL-18 T cells. Furthermore, testing for antigen-specific production of IL-10 revealed that both TCRCiIL-12 and TCRCiIL-12CiIL-18 T cells produced significantly increased levels of this cytokine. While the various T cell populations also produced IL-2 as well as TNFa upon co-culture with antigen-positive B16 cells, their levels of were not affected by either iIL-12, IL-18 or both (see Fig. 2B).

Treatment of melanoma-bearing mice with TCRCiIL-18 T cells results in more cures, yet treatment with TCRCiIL-12 T cells adversely affects survival
We treated HLA-A2 transgenic mice with palpable B16:A2-YLEP tumor (»400 mm 3 ) either with 7.5 £ 10 6 TCR, TCRCiIL-12, TCRCiIL-18, TCRCiIL-12CiIL-18 or mock T cells (see Materials and Methods for details). In the TCR T cell group, 66% of mice showed tumor regression, whereas in the TCRCiIL-12, TCRCiIL-18 and TCRCiIL-12CiIL-18 T cell groups these percentages were 66, 100 and 78, respectively (see Fig. 3A; for individual tumor growth curves, see supplementary Fig. 2). Moreover, treatment with TCR T cells resulted in complete responses (i.e., no detectable tumor) until the end of the experiment (day 45 after T cell transfer) in 11% of mice, a result that was not improved by treatment with TCRCiIL-12 T cells (see Fig. 3B). In contrast, treatment with TCRCiIL-18 or TCRCiIL-12CiIL-18 T cells resulted in complete responses in 33% of mice. In addition, in the group treated with TCRCiIL-12 T cells, mice suffered from tumor recurrence at earlier time points than mice in the group treated with TCRCiIL-18 T cells (see supplementary Fig. 2). Fig. 3B provides an overview of the fractions of complete, partial and no responses among the different treatment groups. When assessing survival, 29% and 14% of mice treated with TCR or TCRCiIL-12 T cells were alive at the end of experiment, respectively, whereas 57% and 43% of mice treated with TCRCiIL-18 or TCRCiIL-12CiIL-18 T cells were alive at the end of experiment (see Fig. 3C). Treatment-related mortality, defined as death of mice after T cell treatment not coinciding with tumor outgrowth, occurred in TCRCiIL-12 and TCRCiIL-12CiIL-18 T cell-treated mice, with percentages of mice that died within 14 days after T cell transfer being 33 and 22, respectively. Treatment-related mortality was absent in the TCR and TCRCiIL-18 T cell-treated mice (see Fig. 3B). Along these lines, we observed enhanced loss of weight in 56-78% (see supplementary Fig. 3) as well as edema-like toxicities in 29% of the treatment groups with iIL-12. Table I provides details on observed therapy-related effects.

Administration of TCRCiIL-12 T cells results in compromised T cell persistence and enhanced plasma levels of inflammatory cytokines
As peripheral T cell persistence has been directly linked to clinical anti-tumor efficacy, 32 blood samples were taken at various time points after T cell transfer and analyzed by flow cytometry for the presence of TCR-engineered T cells. We observed increased numbers of pMHC-binding T cells in blood of TCRCiIL-18 and the TCRCiIL-IL-12CiIL-18 T cell-treated mice at day 6 after T cell transfer when compared to TCR T cell-treated mice (see Fig. 4A). Numbers of pMHC-binding T cells were decreased in the blood at day 6 after treatment with TCRCiIL-12 T cells. It is noteworthy, that the ratio of CD8 to CD4 positive T cells was significantly enhanced in the treatment groups that received treatment with iIL-18 when compared to the TCRCiIL-12 group (see Fig. 4B). These findings are in line with our in vitro observation that cultures of Figure 2. Inducible T cells produce IL-12 and IL-18 upon recognition of cognate antigen, which results in enhanced production of IFNg. T cells were transduced with one or a combination of the following constructs: empty retroviral vector (mock); gp100 TCR (TCR); gp100 TCR and iIL-12 (TCRCiIL-12); gp100 TCR and iIL-18 (TCRCiIL-18). In an additional set of experiments, TCRCiIL-12 and TCRCiIL-18 T cells were mixed at a 1:1 ratio (TCRCiIL-12CiIL-18). These T cell populations were co-cultured with B16 melanoma cells that were either positive or negative for the gp100 target antigen at an effector:target (E:T) ratio of 3:1 for 24 h. As a control, T cells were cultured in medium only. (A) Levels of IL-12 and IL-18 in culture supernatants were measured via ELISA, and displayed as mean cytokine production per 10 6 T cells §SEM (n D 5). (B) Levels of IFNg, IL-10, IL-2 and TNFa in culture supernatants were measured via ELISA, and displayed as mean cytokine production per 10 6 T cells §SEM (n D 5). Statistically significant differences between T cell populations that were co-cultured with antigen-positive B16 cells were calculated with Student's t-test: Ã p<0.05; ÃÃ p<0.01; ÃÃÃ p<0.005. figure 1 A, lower panel). In addition to whole blood, we also collected plasma samples on day 16 after T cell transfer, which were analyzed via multiplex assay for the presence of cytokines (see Fig. 5). While plasma levels of IL-12 were low in all treatment groups (<7 pg/ml), plasma levels of IL-18 were considerably high in all groups (>700 pg/ml) and highest in mice treated with TCRCiIL-12CiIL-18 T cells (>1300 pg/ml). Of interest, in particular in relation to the abovementioned toxicities, is the observation that plasma levels of IFNg and TNFa were significantly higher in the TCRCiIL-12 and TCRCiIL-12CiIL-18 T cell groups (and negligible in the TCR and TCRCiIL-18 T cell groups). IL-2 plasma levels showed no significant difference among the tested groups. Complete response is defined as absence of a palpable tumor at the end of the experiment (day 45); partial response is defined as 30% tumor regression followed by relapse; no-response is defined as < 30% tumor regression; and therapy-related death is defined as death not coinciding with tumor progression (n D 9 per group). (C) Survival curves of treatment groups. Two mice of each group were sacrificed at day 5 after T cell transfer to collect tumors and are omitted from this figure (n D 7 per group). Plasma levels of IL-10 were increased in mice treated with TCRCiIL-12 T cells compared with those mice treated with TCRCiIL-18 and TCRCiIL-12CiIL-18 T cells.

Treatment with TCRCiIL-18 T cells results in enhanced accumulation of CD8 and TCR-transgene-positive T cells within the tumor
To better understand the in vivo behavior of TCRCiIL-12 and TCRCiIL-18 T cells, we assessed how the different treatments affect T cell infiltration into solid tumors and the phenotype of intra-tumoral T cells. To this end, we have performed flow cytometry analyses of TILs and observed an enhanced frequency of CD8 C TCR C T cells amongst CD3 C TILs upon treatment with TCRCiIL-18 T cells when compared to TCRCiIL-12 T cells (39 vs 9%) (see Fig. 6A, left graph). Looking into the ratio of CD8 C and CD4 C T cells, it became apparent that treatment with TCRCiIL-18 but not with iIL-12 T cells results in an enrichment of CD8 C TILs (» 7 vs 0.5, respectively; see Fig. 6A, right graph). Next, we studied the phenotype of TILs, in particular with respect to co-stimulatory as well as co-inhibitory receptor expression. The analysis of percentages of individual co-stimulatory or co-inhibitory receptors by CD8 C TILs, nor analysis of their co-expression by these cells, revealed no significant difference between treatment groups (see Fig. 6B and C). Also, phenotypic differences were not observed when analyzing CD4 C TILs or analyzing mean fluorescence intensities (data not shown).

Mice treated with TCRCiIL-12 T cells demonstrate extensive intra-tumoral infiltration of macrophages
In addition to numbers and phenotype of intra-tumoral T cells, we also assessed the cellular composition of the immune infiltrate in more detail. TILs from the different treatment groups were assessed by flow cytometry for the presence of T cells (CD3 C ), macrophages (CD68 C ), NK cells (CD335 C ) and neutrophils (Ly6G C ). Strikingly, we observed that frequencies of macrophages were increased in mice receiving TCRCiIL-12 T cells, an observation that went hand in hand with a decrease in frequencies of T cells (see Fig. 7). Neither NK cell nor neutrophil numbers showed significant differences between treatment groups. To substantiate these findings we assessed the presence of IL-12 and IL-18 in lysates from the same tumor, and showed that the concentrations of IL-12 were higher in tumors from the groups treated with TCRCiIL-12 or TCRCiIL-12CiIL-18 T cells, whereas the concentrations of IL-18 were higher in tumors from the groups treated with TCRCiIL-18 when compared to treatment with TCR T cells. Endogenous levels of IL-18 ranged between 400 and 1000 pg/ml, while those of IL-12 were below 100 pg/ml (see supplementary Figure 4 A). Furthermore, we also assessed the composition of the immune infiltrate using in situ immune fluorescence, and quantified the various immune cell types per area tumor tissue. The numbers of T cells (CD3 C ), macrophages (F4/80 C ), NK cells (CD335 C ) and neutrophils (Ly6G C ) that were found in the tumors from the different treatment groups confirmed the findings of the flow cytometric analysis (see supplementary Figure 4B and C). It is noteworthy that NK cells and neutrophils were readily detectable by flow cytometry, while staining for the same markers in tissues slices yielded no to negligible detection of either cell type. This discrepancy between both techniques most likely relates to the use of different antibodies in different applications.

Discussion
In this study, we compared the therapeutic impact of IL-12 and/or IL-18 secretion at the tumor site by antigen-triggered T cells. To this end, we equipped murine T cells both with gp100-specific TCRa/b genes and an inducible cytokine construct. T cells were antigen-responsive and, besides being producers of IL-12 or IL-18, produced enhanced levels of IFNg. The production of IFNg was prominent in case of TCRCiIL-12 T cells and highest in TCRCiIL-12CiIL-18 T cells, which is in line with previous reports where T cells were exposed to a combination of recombinant IL-12 and IL-18. 33 TCR T cells harboring iIL-18, when transferred to mice bearing melanoma positive for the gp100 target antigen, resulted in enhanced and more durable anti-tumor responses when compared to T cells engineered with a TCR only or TCR and iIL-12. Furthermore, treatment with TCRCiIL-18 T cells, but not TCRCiIL-12 T cells, prolonged survival when compared to treatment with TCR T cells.
The low survival in the TCRCiIL-12 T cell treatment group is most likely a result of IL-12-mediated toxicities. Mice receiving TCRCiIL-12 or TCRC iIL-12CiIL-18 T cells demonstrated elevated plasma levels of IFNg and TNFa, clear weight loss, edema-like symptoms and in some cases death. The detection of IFNg and TNFa in plasma is considered a consequence of IL-12-mediated productions of these cytokines by tumor-infiltrating immune cells. 34 IFNg is most likely derived from administered T cells following infiltration and activation in the tumor tissue. This notion is strengthened by our observation that CD335-positive NK cells, as an alternative source of IFNg, were only present at low frequency within the tumors of treated mice. TNFa is most likely derived from macrophages since we detected an enhanced frequency of CD68 as well as F4/80-positive macrophages in tumors isolated from mice treated with TCRCiIL-12 T cells, and in vitro assays revealed no enhanced TNFa production by these T cells. Collectively, our findings extend previous clinical studies, where systemic administration of recombinant protein or cDNA encoding IL-12 was reported to result in enhanced production of IFNg and TNFa, changes in blood vessel wall permeability, edema and even sepsis-like symptoms. 34,35 In fact, a recent study demonstrated that treatment of melanoma patients with iIL-12-transduced TILs was accompanied by liver dysfunction, high fever, and hemodynamic instability. 36 To further assess the observed toxicities, we also treated mice without tumor-implant with TCRCiIL-12 T cells and still observed enhanced weight loss when compared to mice treated with TCR T cells (see supplementary figure 3). These findings extend our in vitro observations showing targetindependent release of IL-12 by these T cells ( Fig. 2A) as well as those by Zhang and colleagues, who also reported non-specific release of IL-12 and reduced fitness when culturing iIL-12expressing TILs. 36 It is noteworthy that none of the mice without a tumor implant developed edema or showed therapyrelated mortality, arguing that although toxicities are related to antigen-independent release of IL-12, they may exacerbate in the presence of antigen-positive tumor cells. Non-specific release of IL-12, in particular in the setting of TCR-engineered T cells, may be explained by the size of the inserted construct. IL-12 is a relatively large cytokine with 550 aa, while IL-18 encompasses 192 aa and, like other smaller constructs we tested (data not shown), shows no antigen non-specific release. Finally, we cannot exclude the contribution of host, co-treatment and tumor to the observed toxicities. In one of our earlier studies, where we tested iIL-12 in mouse models utilizing CAR-transduced T cells, 18 we did not observe the toxicities as reported in this study, which may suggest that mouse strain (C57 BL/6 vs NIH-III), pre-conditioning protocol (Busulfan/Cyclophosphamide vs no pre-treatment), and type of tumor (melanoma cells vs colon carcinoma cells) affect the extent of IL-12-mediated toxicities.
Treatment with TCRCiIL-18 T cells, not revealing any toxicity, resulted in clearly enhanced anti-tumor responses. To better understand the immune-enhancing effect of this treatment, we have assessed numbers and phenotype of intra-tumoral T cells as well as the presence of other immune cell types within tumors. Notably, we observed an enhanced density of TCR-transduced TILs, in particular of the CD8 subset, when mice were treated with TCRCiIL-18 but not TCRCiIL-12 T cells. Enhanced numbers of CD8 C T cells within tumors are generally considered a beneficial factor with respect to tumor evolution and therapy response. 7,37,38 The differential treatment effect with respect to numbers of TILs can be explained by two lines of arguments. First, our in vivo analysis of tumors showed that treatment with TCRCiIL-12 when compared to TCRCiIL-18 T cells results in an enhanced presence of macrophages (see Fig. 7 and supplementary Fig. 4). These results, together with enhanced TNFa levels in sera, argue that macrophages and their product TNFa may limit the infiltration of CD8 C T cells as was observed previously using various mouse models. 39,40 Second, in vitro analysis of T cells demonstrated that iIL-18 when compared to iIL-12 provides T cells with high expression of co-stimulatory receptors and a low expression of co-inhibitory receptors (see supplementary figure 5). These traits may be representative of a preferred T cell phenotype at the time of adoptive transfer for an effective tumor response. Interestingly, when studying the phenotype of TILs, we observed no significant differences in the expression of costimulatory or inhibitory receptors by CD8 C TILs. When zooming in on TCR transgene-expressing TILs, however, we did observe that treatment with TCRCiIL-18 T cells resulted in enhanced expression of the co-stimulatory receptor ICOS and the co-inhibitory receptors PD-1 and CTLA-4, whereas treatment with TCRCiIL-12 T cells resulted in enhanced expression of the co-stimulatory receptors 4-1BB and CD40 L and the coinhibitory receptors LAG3 and CTLA-4 (see supplementary figure 6). These latter data are difficult to interpret given the fact that numbers of TCR C TILs are extremely low in case of treatment with TCRCiIL-12 T cells. With respect to treatment with TCRCiIL-18 T cells, ICOS may contribute to enhanced tumor infiltration and prolonged T cell persistence as was demonstrated in adoptive T cell therapy studies ( 41 ; Kunert, manuscript in preparation). PD-1, on the other hand, may mark successful antigen-specific TCR triggering and T cell activation. 42 Notably, the fact that TCRCiIL-12 T cell treatment, when compared to TCRCiIL-18 T cells, results in the highest CTLA-4 and LAG3 expressions may provide an additional explanation for their lowered ability to mediate tumor clearance.
The above findings argue that IL-12 has potent immunestimulatory effects, which may rapidly initiate the expression of immune checkpoints. In an effort to downscale negative feedback on T cell activation, we titrated down TCRCiIL-12 T cells and observed that lowering the numbers of these cells did not change the effects this cytokine has on the production of IFNg nor the expression of PD-1 (supplementary Figure 7; same results were observed for production of IL-10 and expression of FOXP3, data not shown), arguing that it is difficult to maintain beneficial effects of IL-12 towards anti-tumor T cell activity while preventing the induction of a negative feedback. These data, together with the toxic effects we noted when using TCRCiIL-12 T cells, do not favor further therapeutic studies with T cells carrying iIL-12.
In conclusion, our findings demonstrate that the therapeutic use of TCR-engineered T cells equipped with inducible IL-12 leads to limited anti-tumor effects and severe toxicities in vivo, in part due to antigen non-specific release, whereas equipment of TCR T cells with inducible IL-18 results in enhanced anti-tumor responses without toxicities, most clearly related to increased accumulation of CD8 and TCR-transgene-positive T cells within tumors. Taken together, these findings advocate further studies towards the use of iIL-18 T cells to address therapy resistance in the setting of adoptive T cell therapy.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.