Adoptive T-cell therapy (ATT) efficacy is limited when targeting large solid tumors. The evaluation of ATT outcomes using accessory treatment would greatly benefit from an in vivo monitoring tool, allowing the detection of functional parameters of transferred T cells. Here, we generated transgenic bioluminescence imaging of T cells (BLITC) mice expressing an NFAT-dependent click-beetle luciferase and a constitutive Renilla luciferase, which supports concomitant in vivo analysis of migration and activation of T cells. Rapid transferability of our system to preestablished tumor models was demonstrated in the SV40-large T antigen model via both crossbreeding of BLITC mice into a T-cell receptor (TCR)-transgenic background and TCR transduction of BLITC T cells. We observed rapid tumor infiltration of BLITC CD8+ T cells followed by a burst-like activation that mirrored rejection kinetics. Using the BLITC reporter in the clinically relevant H-Y model, we performed female to male transfers and detected H-Y-specific alloreactivity (graft-versus-host disease) in vivo. In an H-Y solid tumor model, we found migration of adoptively transferred H-Y TCR-transgenic CD4+ T cells into the tumor, marked by transient activation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expression of inhibitory receptors. In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT, is rapidly transferable to models of interest not restricted to tumor research, and is suitable for rapid screening of TCR clones for tumor rejection kinetics, as well as off-target effects. Cancer Immunol Res; 6(1); 110–20. ©2018 AACR.
Adoptive T-cell transfer (ATT) has emerged as a promising option in cancer therapy, although first clinical trials have shown mixed results. With CD19-CAR T cells moving into successful clinical trials (1), the picture is changing for many types of cancer. However, ATT for treatment of large solid tumors has limited efficacy due to insufficient cell migration and infiltration into tumor tissues caused by inadequate inflammatory signals, the presence of metabolic factors like lactic acid, and T-cell inhibitory circuits being highjacked by the tumor microenvironment (2–4). Consequently, it has become evident that even an ideal T cell warrants accessory therapeutic interventions in order to overcome large solid tumors. By using antibodies targeting checkpoint inhibitory molecules, the therapeutic tools available for combination with ATT have gained potency surpassing conventional approaches like chemotherapy and irradiation (5, 6). However, the exact impact and optimal therapeutic window of these approaches is difficult to determine without a bulk of experimental animals or advanced noninvasive tools to monitor T-cell fate upon transfer.
Bioluminescent imaging (BLI; ref. 7) offers an optimal combination between ease-of-use and high sensitivity unrivalled by any other in vivo method, including positron emission tomography (8). Among the few reporter systems utilizing reporter T cells, most focus on migration and proliferation (9, 10). However, T-cell activation and effector function, targeted by virtually all tumor defense mechanisms, remain elusive. The few existing reporter systems that allow the detection of activated T cells are either not suited for in vivo BLI (11–14) or specifically monitor granzyme expression, an effector function primarily found on cytotoxic CD8+ T cells (15).
Here, we present a transgenic T cell–reporter mouse BLITC (BLI of T cells), that allows the concomitant analysis of T-cell migration and NFAT-dependent activation using dual-luciferase transgenic T cells upon transfer. We implemented this reporter for the study of two independent tumor models and gained important insights into the kinetics of T-cell activation. We propose that our reporter system enables in-depth analysis of T-cell biology in a spectrum of applications not limited to tumor immunology.
Materials and Methods
Mice and constructs
T-cell transfers were performed using albino B6 mice (C57BL/6(Cg)-Tyrc-2J) and albino Rag knockout mice (albino RagKO; B6.129S6-Rag2tm1Fwa N12-Tyrc-2J), both lacking skin and fur pigmentation for increased BLI sensitivity. Balb/c RagKO mice (C.129S6-Rag2tm1Fwa N12) were used as ATT recipients (bred at the MDC Berlin and originally obtained from Taconic Biosciences).
The NFAT-click beetle luciferase (NFAT-CBR) construct was adopted from a previously described construct (16). Briefly, the polyA signal in the original lentiviral vector pNFAT was PCR-amplified (Phusion polymerase; New England BioLabs), and the enclosing restriction sites StuI and XbaI were added via adapter primers (Eurofins Genomics). This 240 base-pair (bp) fragment was inserted downstream of the redshifted click beetle luciferase (CBR; Promega) sequence after cutting out a 1,390-bp XbaI-StuI fragment (all restriction enzymes from New England BioLabs) containing lentiviral elements and included the 3′-LTR. A 2.3 kb fragment containing the insulator sequence upstream of the complete NFAT-responsive CBR fragment, including the added 3′-polyA signal, was purified after digestion with StuI and HindIII. It contained the redshifted CBR (Promega) under control of an artificial NFAT-responsive promoter (ref. 17; Fig. 1A) and was microinjected into fertilized C57BL/6NCrl oocytes at the Max Planck Institute of Molecular Cell Biology and Genetics and derived founder mice were selected for proper germ-line transmission and expression of the transgene cassette. The annotated NFAT-CBR sequence is accessible online via GenBank (accession number MF462285).
Transgenic BLITC (bioluminescence imaging of T cells) mice were obtained by crossing NFAT-CBR mice with ChRLuc mice (10) expressing Renilla luciferase (Rluc) under the previously described CAG composite promoter (18, 19) to obtain dual-reporter BLITC mice. In order to rapidly establish transgene homozygosity, we determined the genomic position of both reporter cassettes using an adapter ligation-mediated PCR method (20). Briefly, we designed long-strand oligos and 3′-modified short strand oligos (supplied by Eurofins Genomics) that, after ligation to genomic DNA, were digested using specific restriction enzymes, which allowed for PCR amplification from the respective transgene sequence into the immediately adjacent genomic sequence followed by Sanger sequencing. Upon determination of genomic location of transgene insertion, the following primer sets were designed and used for mouse screening: NFAT-CBR (Chromosome 4qC7): 5′–CCG GCG GTT AGT AAT CTA GAC GAC C–3′, 5′–CCC CCC TCC CTC TTA AGC AGA G–3′, and 5′–CCA AGT CCA GAG GGC TTT GTA TGC–3′; Rluc (Chr. 10qA4): 5′–CGA ATA ATC TAT TGA CGT CAA TGG GC–3′, 5′–CCT CTT CCA CTG ACC AAT CCA TCT TTG C–3′, and 5′–CCT GCT CTC TTA CAC GTC AGA TTG ACC–3′ (all oligos from Eurofins Genomics).
The following TCR-transgenic mice were used for ATT and for crossbreeding with BLITC mice: Marilyn-Rag1KO [TCR-I mice transgenic for an H-Y TCR targeting SV40 large T antigen (TAg) epitope I] and MataHari-Rag2KO mice (Olivier Lantz; Institute Curie, Paris, France; refs. 21–23). After crossing of BLITC transgenic mice to Marilyn-Rag1KO mice and MataHari-Rag2KO mice, we obtained ML-BLITC (Marilyn-TCR+/+ Rag1−/− NFAT-CBR+/+ Rluc+/+) and MH-BLITC (MataHari-TCR+/+Rag2−/−NFAT-CBR+/+Rluc+/+) mice, which were used exclusively for in vitro experiments. All mouse studies were performed in adherence to institutional, state, and federal (LAGeSo) laws and guidelines.
Bone marrow transfer
Bone marrow (BM) cells were harvested from femora and tibiae of donor mice by standard procedure and suspended in PBS plus 0.5% bovine serum albumin (BSA; both from Sigma). Erythrocytes were removed from single-cell BM suspensions via incubation with a self-made erythrocyte lysis buffer containing 155 mmol/L NH4Cl, 10 mmol/L KHCO3 and 100 μmol/L EDTA (all from Sigma) in water (pH 7.3). T cells and other lineage cells were then removed via a customized MACS enrichment protocol. Briefly, BM cell suspensions were incubated with an antibody cocktail containing biotinylated CD4 and CD8 (BD Biosciences), as well as biotinylated Gr-1, Ter119, B220, CD11b, and NK1.1 (all from BioLegend). After a washing step in PBS/BSA plus 1 mmol/L EDTA (Sigma), suspensions were incubated with anti-biotin MACS beads, and the unlabeled flow-through was recovered after passing cells through LS magnetic separation columns (negative selection; beads and columns from Miltenyi Biotec). 5 × 106 lineage-negative cells (T-cell contamination was routinely below 2% as determined via flow cytometry), were i.v. injected into lethally irradiated (two consecutive doses of 4 Gy within 4 hours) female albino B6 recipients.
T-cell preparation and stimulation
T cells harvested from spleens and inguinal lymph nodes of donor mice were enriched by labeling with the biotinylated antibodies Ter119, NK1.1, B220, Ly6A/E, CD11b, and CD11c (BioLegend), followed by negative selection using anti-biotin MACS beads and magnetic columns (both from Miltenyi Biotec; purity above 95%). T cells were cultured in T-cell medium (TCM) consisting of RPMI and 10% fetal bovine serum (FBS; both from Life Technologies) supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), 1 mmol/L sodium pyruvate, 100 mmol/L MEM, nonessential amino acids, 50 μmol/L β-mercaptoethanol, and 2 mmol/L L-glutamine (all from Sigma). For some experiments, media only containing RPMI, FBS, penicillin, and streptomycin (RPMI/FBS) were used.
For activation, T cells were activated in vitro for 18 hours in TCM plus IL-2 (1 ng/mL; PeproTech) in 6-well or 24-well culture plates coated over night at 4°C with anti-CD3/CD28 (anti-CD3 clone 145-2C11, 3 μg/mL; anti-CD28 clone 37.51, 2 μg/mL; BD Biosciences). Optionally, cells were expanded for up to 14 days (CD4+ T cells 7 days) on noncoated culture plates in TCM plus recombinant human IL-15 (50 ng/mL). For ATT, T cells were sorted (>97%) using a FACS Aria II or Aria III (BD Biosciences) and transplanted into anaesthetized recipient mice via retrobulbar i.v. injection using 0.3 mL insulin syringes (BD). For peptide stimulation using pulsed dendritic cells (DCs), BM cells from female albino RagKO mice were cultured in TCM containing GM-CSF (20 ng/mL). After 8 days, DCs were harvested, washed, and incubated overnight with the respective concentration of Dby or Uty peptide, including 1 μg/mL LPS. 5 × 105 T cells were cocultured with 5 × 104 DCs in 96-well plates for 24 (for cells derived from Marilyn mice) or 6 (cells derived from MataHari mice) hours.
The retroviral vector pMP71-TCR-I, bearing the genes for the alpha and beta-chain of a TCR specific for the H-2Db (MHCI)-restricted epitope I of SV40-Tag, is based on the pMP71 backbone (24, 25). T cells were harvested, sorted to high purity (routinely above 97%), and activated as described above. Retrovirus was produced by PlatE transfection (Cell Biolabs) with pMP71-TCR-I using TransIT (Mirus Bio). On days 2 and 3 after transfection, 5 × 104 T cells were spinoculated in 24-well plates for 90 minutes at 32°C in PlatE supernatant containing retroviral particles. Three days later, T cells were sorted using TCR Vß7-antibody staining (BD Biosciences).
Tumor challenge and characterization
The urothelial carcinoma cell line MB49 (ref. 26; ATCC) was grown in RPMI/FBS. For tumor challenge, MB49 cells were adjusted to 2 × 105 cells/mL in PBS, mixed 1:1 with matrigel (BD Biosciences), and kept on ice until subcutaneous injection of 100 μL (104 cells) into the hind flank of recipient mice using 1 mL insulin syringes (Omnican-Braun). For analysis of relapsed tumors, we challenged female albino RagKO mice with MB49 as mentioned above and monitored tumor remission upon treatment with 5 × 104 or 5 × 105 ML-BLITC T cells. Tumors that reemerged and grew out after a period of complete remission were harvested, T-cell depleted, and cultured in vitro as relMB49 clones for ensuing analyses.
The TAg-expressing gastric carcinoma cell line TC200.09 line, originally established in a TREloxP StoploxP TAgLuc x rtTA mouse (27), expresses a TAg-firefly luciferase fusion protein (TAgLuc) under strict dependence on doxycycline (Dox). The luciferase-deficient variant 200ΔLuc was generated by selection for a loss-of-function mutation generated by CRISPR-Cas9 mutation of the luciferase portion of TAgLuc and was passaged in RPMI/FBS plus Dox (0.5 μg/mL; ref. 28). For tumor challenge, 5 × 106 200ΔLuc cells were injected (as above described for MB49) into albino RagKO mice that continued to receive light-protected drinking water supplemented with Dox (1 mg/mL) and 5% sucrose. Mean tumor volumes were calculated by measuring tumor length (L) and width (W) with a digital caliper (Fine Science Tools) using cylindric approximation (V = W × L2/2).
Cytolytic activity of Marilyn T cells against MB49 and their relapsed counterparts relMB49 was determined in vitro via an LDH assay using a commercial kit [CytoTox96(R) NonRadioactive Cytotoxicity assay from Promega] according to the manufacturer's instructions using triplicates. Percent lysis was calculated as 100 × (experimental − effectorspontaneous − targetspontaneous)/(effectormaximum − targetspontaneous) with spontaneous and maximum values determined on medium supernatant or completely lysed cells, respectively.
Dby expression of MB49 was determined via semiquantitative RT-PCR. Briefly, RNA was isolated from tumor cells using TRIzol (Invitrogen), and cDNA was synthesized via SuperScript II Reverse transcriptase (Thermo Fisher Scientific) using a equimolar mix of oligo-dT12-18 and dN6. Different cDNA samples were normalized using HPRT PCR prior to Dby-specific PCR (Phusion polymerase, New England BioLabs). The following primers were used (all from Eurofins Genomics): Dby fwd: 5′–CAA TAG CAG CCG AAG TAG TGG TAG T–3′, Db rev: 5′–AAC TGC CTG GGA GTT ATA ATT TCC T–3′, HPRT fwd: 5′–CAA CGT AGG AGG ACC CTT TAA TGC–3′, HPRT rev: 5′–CCA CAG GAC TAG AAC ACC TGC TAA–3′.
For in vitro bioluminescence measurement, we used a dual-luciferase reporter assay (Promega) and acquired luminescence on a Xenogen IVIS 200 (Caliper Life Sciences) in triplicates. In vivo BLI was performed using the IVIS 200 after mice were anaesthetized with isoflurane (Baxter) in an XGI-8 anesthesia system (PerkinElmer). Due to the spectral spillover from the bright Rluc signals into the 600 nm spectrum reserved for CBR-specific detection, acquisition of both luminescence parameters was performed sequentially. NFAT-CBR features considerably lower flux compared with Rluc, allowing the acquisition of Rluc signals 10 minutes after NFAT-CBR measurement.
3 minutes before Rluc acquisition, native coelenterazine (Biosynth) was dissolved in 10 μL DMSO, diluted in 100 μL PBS, and immediately injected i.v. into anaesthetized mice (100 μg native coelenterazine per mouse). For NFAT-CBR, signals were acquired 10 minutes after i.p. injection of D-Luciferin in PBS (0.3 mg/g body weight). BLI acquisition was performed for 5 minutes at small binning. Bioluminescent data was acquired, analyzed, and visualized using Living Image software (PerkinElmer). BLI quantification was performed by digitally setting regions of interest (ROI) around prospective or visible tumor areas and computing respective total flux values. For each experiment, ROIs were kept identical in size and shape for all time points. Unless indicated otherwise, flux density was visualized using the rainbow color table at the following scales: 5 to 20 × 103 for NFAT-CBR and 104 to 105 for Rluc flux. Sequential analysis was performed after sufficient blank measurements (no substrate) for control of signal carry over.
Flow data was acquired on a FACS Canto II (BD Biosciences), and all analyses were performed using FlowJo software (Treestar Corp.). For T-cell sorting and identification of tumor-infiltrating lymphocytes (TILs), the following antibodies were used: CD3, CD25, CD69, TCR Vß6, TCR Vß7, IFNy (all BioLegend); PD-1 (Miltenyi Biotec), CD4, CD8, Vß8.3 (all BD Bioscience), Tim-3, and Lag-3 (eBioscience). For better discrimination of T lymphocytes, non-T lineage cells were stained with Gr-1, Ter119, B220, CD11b, and NK1.1 (all BioLegend) and negatively gated for tumor-infiltrating lymphocyte analysis and sorting.
BLITC reporter: in vitro and in vivo features
After generating the NFAT-CBR transgenic mouse (Fig. 1A) and successfully testing its suitability for detecting in vivo T-cell activation in a classical (C57BL/6 to Balb/c) major mismatch setting (Supplementary Fig. S1), we proceeded by crossing NFAT-CBR mice with ChRluc mice (10) that constitutively express Rluc to enable detection of bulk T-cell accumulation and migration. Mice containing both reporter constructs were coined BLITC mice. No overt differences in behavior, development, or immunological features were observed between BLITC mice (Rluc+/+ NFAT-CBR+/+) and nontransgenic littermates. We further crossed BLITC mice with H-Y TCR-transgenic Marilyn and MataHari mice to generate the TCR-transgenic reporter mice ML-BLITC and MH-BLITC, respectively (21, 22).
Using sorted H-Y TCR transgenic BLITC T cells in vitro and in vivo, we proceeded to determine the sensitivity of our reporter system. In vitro, we detected Rluc signals of 10 T cells and NFAT-CBR signals of 103 activated T cells (Fig. 1B). In order to mimic in vivo conditions, we subcutaneously injected T cells into albino B6 mice lacking pigmentation. We detected 104 T cells emitting a clear signal above background for both luciferases (Fig. 1C and D). Antigen-specific stimulation, using DCs pulsed with a dilution series of H-Y peptide, revealed high specificity for the cognate antigen and sensitivity comparable to standard activation markers detected via flow cytometry but surpassed by ELISA detection of IL2 and IFNy, as shown in Supplementary Fig. S2A and S2B for MH-BLITC.
Next, the kinetics of activation-dependent NFAT-CBR signaling were elucidated via in vitro stimulation. Two hours after CD3/CD28-stimulation in vitro, we observed a distinct NFAT-CBR increase that peaked after 6 hours and remained elevated for up to 24 hours before gradually declining along with total cell numbers, whereas accumulated IL-2 and IFNy in supernatants remained high (Fig. 2A). The NFAT-CBR peak preceded the differentiation into fully activated CD25+CD69+ T cells by several hours (Fig. 2B; ref. 29). When removed from stimulus, NFAT-CBR signals of activated T cells rapidly returned to background intensities within 6 hours of removal from stimulus (Fig. 2B), whereas the frequency of CD25+CD69+ cells and IL-2 only started to decrease after 12 hours of withdrawal. However, IFNy was fairly stable in this setting.
In order to monitor homeostatic expansion of BLITC T cells in an MHC-matched host, we performed BM transplantation experiments. Lineage-depleted BM cells from female BLITC mice (non-TCR transgenic) were transferred into lethally irradiated female albino B6 recipients (Fig. 3). Between days 17 and 28, mice developed thymic NFAT-CBR signals, indicating large-scale activation that persisted beyond day 78. Rluc kinetics displayed a distinct spatiotemporal pattern indicative of hematopoietic reconstitution. We further ruled out rejection of BLITC cells on account of potentially antigenic luciferases by transferring BLITC T cells into sublethally irradiated albino B6 mice, where reporter T cells expanded unhindered despite the presence of functional host T cells (Supplementary Fig. S3). These data clearly demonstrate high sensitivity and in vivo applicability of the BLITC system. Reporter T cells display long-term reporter expression and independent regulation of NFAT-CBR and Rluc.
Monitoring of T-cell activation in the SV40 TAg tumor model
In the following experiment, we exemplified the customization of the BLITC reporter to desired tumor models by rapidly transferring the system to a SV40 TAg-driven tumor model via two strategies widely used for TCR modifications (23, 27). In the first approach, we crossed BLITC mice with SV40 mice transgenic for a TAg-specific TCR (TCR-I) and isolated TAg-specific CD8+ T cells from the (TCR-I × BLITC)F1 offspring. In the second approach, reporter T cells were retrovirally transduced with TCR-I (tdTCR-I-BLITC T cells; Fig. 4). In order to avoid confounding BLI signals, we used the H-Y expressing and luciferase-deficient cell line 200ΔLuc, derived from the bioluminescent cell line 200.09 via CRISPR-Cas9-mediated targeted gene disruption (ref. 27; Supplementary Fig. S4). Upon subcutaneous injection of 5 × 106 200ΔLuc cells into male albino RagKO mice, tumors were established within 40 to 45 days (mean volume of 300 mm3) and were challenged with 5 × 104 (BLITC × TCR-I)F1 or TCR-I-transduced BLITC T cells. In contrast to controls, tumors started regressing 6 to 7 days after ATT and disappeared within the following two weeks (Fig. 4A). All mice remained relapse-free up to day 70 after tumor challenge.
Transferred T cells were monitored in vivo for both activation (NFAT-CBR) and migration/proliferation (Rluc). (TCR-I × BLITC)F1 T cells entered the draining inguinal lymph nodes at day 4 before migrating into the tumor tissue within the next two days (Fig. 4B and D). After reaching maximal strength, Rluc signals of TILs decreased in correlation with regressing tumors and were rarely detectable after tumor remission. In contrast, TIL activation followed a burst-like kinetic, as inferred from respective NFAT-CBR signals. While absent in draining lymph nodes, activation-dependent luminescence was observed at the tumor site within a narrow time frame of one to two days. This activation burst stringently coincided with the onset of tumor remission but rapidly disappeared independently of residual tumor burden.
The BLI kinetics of ATT using tdTCR-I-BLITC T cells roughly mirrors the one observed in the (TCR-I × BLITC)F1 group, albeit with overall reduced BLI signal intensities, as well as delayed onset of tumor remission and activation burst (Fig. 4C). Comparable BLI kinetics for both ATT approaches suggests that the NFAT-CBR signal can be highly predictive of tumor rejection in this tumor model.
Visualization of T-cell activation in a clinical model of GvHD
To engage a clinically relevant setting, we adapted our BLITC reporter to a minor histocompatibility antigen (MiHA) H-Y transplantation model that facilitates the concurrent study of the opposed effects of graft versus tumor and graft versus host disease (GvHD; ref. 30). H-Y TCR transgenic Marilyn (ML) mice that generate CD4+ T cells targeting an H-Y antigen exclusively expressed by males (H-2Ab-Dby) were crossed with BLITC mice resulting in the ML-BLITC mouse line. When determining the minimum numbers of transferable ML-BLITC T cells resulting in lethal acute GvHD, we reproducibly found the transition from sublethal to lethal acute GvHD to be in the range of 5 × 104 to 5 × 105 cells. We transferred 5 × 104 female ML-BLITC T cells into sublethally irradiated male albino RagKO recipients and scored GvHD as described (31). As a control, 5 × 105 T cells were transferred into female hosts. Male recipients developed mild acute GvHD marked by weight loss, fur roughness, and hunched posture around day 5 after transfer but regained normal health status around day 10, whereas female controls remained without symptoms (Fig. 5A and B). In contrast, male recipients demonstrated a steep increase in Rluc total body signals starting around day 6 after transfer, indicating massive T-cell expansion and migration into secondary lymphoid organs. At day 10, we still detected persistence of peripheral T cells. Concurrently, activation signals emerged in the intestinal and genital area at day 6, further increased for another two days, and were not detectable after around day 10, while Rluc signals remained elevated in these animals. In a similar experiment, we sacrificed female and male mice at GvHD peak and after its decline (days 6 and 16). At peak, excessive T-cell activation in GvHD target organs and high levels of IFNγ in blood were detected, which closely reflected CBR-NFAT kinetics (Fig. 5C). Overall, the NFAT-CBR reporter visualized GvHD progression and proved valid for the study of alloreactivity or off-target toxicity.
CD4+ T-cell activation in an H-Y tumor model
We investigated the response of reporter T cells in a compatible tumor-specific setting by challenging female donors bearing H-Y-expressing tumors with H-Y specific BLITC T cells. Subcutaneously injected MB49 cells (H-Y expressing urothelial carcinoma line; ref. 32) were palpable within 1 week and became solid tumors around day 12, with mean volumes of 100 to 150 mm3. Untreated mice had to be sacrificed around day 24 to 28 because of excessive tumor size (>1,500 mm3). Marilyn T cells elicited an efficient response against MB49 tumors. With as few as 103 T cells, tumor growth was attenuated, and TILs could be detected via Rluc signals (Supplementary Figs. S5A and S5B). However, 5 × 105 T cells were required to elicit complete remission within 2 weeks after transfer, although MB49 tumors relapsed between 3 and 8 weeks after ATT with high frequency.
For in vivo BLI, we treated tumor-bearing mice with either 5 × 104 and 5 × 105, or no ML-BLITC T cells. The control group not treated with ATT displayed rapid tumor growth, whereas both ATT groups temporarily halted tumor growth to different extent (Fig. 6B). Between days 3 and 4 after ATT, ML-BLITC cells started accumulating in the draining inguinal lymph node and around day 5, T cells migrated into the tumor area (Rluc panel in Fig. 6A). TIL Rluc signals increased until day 6 and persisted for at least two additional weeks.
TIL activation was assessed via NFAT-CBR signals and roughly coincided with the initiation of tumor regression. T-cell activation was prevalent between days 4 and 14, while demonstrating broad interindividual variability for timing and duration of peak NFAT-CBR. Frequently, two separate activation peaks were observed during this phase. After the activation period, however, NFAT-CBR signals completely disappeared, although tumors were still clearly palpable, and Rluc signals emitted by tumor-resident T cells persisted (Fig. 6B). Using flow cytometry, we confirmed the presence of T cells in Rluc-expressing tumors excised from MB49 tumors that relapsed after regression. We also investigated the expression of inhibitory receptors on isolated TILs and found increased expression of programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and lymphocyte-activation gene 3 (LAG-3) when compared with T cells isolated from respective spleens, as well as draining and nondraining lymph nodes (Supplementary Fig. S5C). We analyzed T-cell effector function at NFAT-CBR peak (day 5 after ATT) and decline (day 10) and found that TILs isolated at day 10 weakly express IFNy upon restimulation and comprise around 3% to 4% of CD25+CD69+ cells compared with a mean of 65% at NFAT-CBR peak, suggesting an inhibitory mechanism (Fig. 6C). 27 days after transfer, Rluc signals persisted in the tumor, although activation was absent (Fig. 6C). In order to test the reinducibility of T-cell activation, we systemically injected anti-CD3 into mice demonstrating T-cell persistence. After 3 hours, bright NFAT-CBR signals were emitted from the TILs, as well as peripheral T cells, indicating the potential for T-cell reactivation by a strong extrinsic stimulus.
Impact of relapsing tumors on TIL effector function
The reason for the failure of ML-BLITC T cells to reject relapsing tumors is unclear and could be due to inhibition of T-cell effector functions or escape from T-cell recognition. Therefore, we analyzed various clones of relapsed MB49 (relMB49) for expression of Dby, the antigen relevant for tumor recognition by ML-BLITC T cells. We found roughly 30% of relMB49 lacked Dby expression, but we could not rule out posttranscriptional inhibition of Dby in the remaining 70% (Fig. 7A). These Dby− tumors avoided killing in vitro (Fig. 7B). Compared with tumors that feature Dby downregulation, Dby-expressing tumors elicited a 4 to 12-fold higher expression of exhaustion markers on T cells in vivo (Fig. 7C). Finally, we compared the fate of in vitro–passaged Dby-negative relMB49 and primary MB49 in albino RagKO upon challenge with 5 × 104 ML-BLITC T cells (Fig. 7D). Transferred T cells failed to attenuate growth of relMB49, as is reflected by the absence of T-cell migration (Rluc BLI) and serum IFNy.
We demonstrated that the dual-luciferase reporter transgenic BLITC mouse allows for the concomitant monitoring of migration and local activation of transplanted T cells at high sensitivity in vivo. Besides its suitability for adoptive T-cell transfer, we tested its utilization for the study of supplementary aspects of T-cell biology, including alloreactivity and T-cell development. The BLITC system that uses a transgenic mouse with two separately inherited reporter cassettes (ChRLuc+/+ NFAT-CBR+/+) allows for flexibly adjusting the system to future bioluminescent requirements, as the individual reporter constructs are readily interchangeable via crossbreeding to alternative BLI reporter-transgenic mice. BLITC was designed for monitoring critical ATT parameters, to identify the principal reasons for therapeutic failure, and to provide cues in regard to T-cell efficacy and therapeutic windows for accessory treatments. For the latter, rapid on and off kinetics of the reporter are important as any detection delay would skew the actual treatment window. We found the NFAT-CBR signal emitted by BLITC T cells to reach its maximum within 4 to 5 hours upon in vitro stimulation, which is superior to the kinetics observed via surface receptors including CD25 and CD69 (29, 33). We found Rluc signals to also be affected by the activation status of T cells, possibly due to impact of activation on the CMV-driven gene expression (34). Therefore, in this model, Rluc signals should be used with caution when drawing conclusions about T-cell numbers in conditions of T-cell activation.
The BLITC reporter supports longitudinal studies, as shown by immune reconstitution of myeloablated hosts using BLITC BM cells. BLI signals were detectable for at least 11 weeks after transfer, which signifies that the reporter cassette is stably integrated, and BLITC cells display normal homeostatic expansion. Monitoring the spatiotemporal traits of transplanted BM cells, we detected significant activation almost exclusively in the thymus. The involvement of AP-1:NFAT signaling in distinct stages of thymic T-cell development has been previously reported (11, 35). Especially in the double positive stage, T cells are subject to massive expansion, which might explain our finding. In contrast, homeostatic T-cell expansion in the periphery is thought to proceed without any marked NFAT translocation.
Our data demonstrate the high versatility of the BLITC reporter, not only for ATT studies but also for any in vivo study investigating T-cell activation patterns. In this context, we were able to rapidly transfer the BLITC reporter system to a chosen tumor model by TCR transgenesis using two common methods. TCR-transduction of BLITC T cells with a SV40 TAg specific TCR (TCR-I), as well as crossbreeding of BLITC mice with TCR-I transgenic mice, yielded the TCR transgenic reporter T cells used to perform studies on trafficking and activation of adoptively transferred T cells in a relevant tumor model.
The BLITC reporter is optimally suited for the analysis of transferred T cells, whereas the monitoring of immune reactions directly in the BLITC mouse is restricted to NFAT-CBR signaling of hematopoietic cells due to broad expression of Rluc. For the analysis of ATT and GvHD in a clinically relevant setting, we opted to use a well-established H-Y model in combination with the aggressively growing urothelial carcinoma cell line MB49 for two reasons (32, 36). First, the MiHA H-Y is expressed on most male-derived tumors and is processed into two cognate immunodominant peptides (Dby and Uty) presented in the H-2b context and can be targeted by respective H-Y TCR transgenic mice (21, 22). Secondly, H-Y expressing male mice allow for the analysis of tumor therapy in alloreactive settings, reflecting the clinical situation of donor-derived lymphocytes mediating both tumor-rejection and life-threatening GvHD.
In a classical MiHA-mismatched setting (female to male), ML-BLITC T cells emitted strong activation-dependent luminescence, which tightly correlated with respective GvHD scores and parameters of T-cell activation, whereas the localization of NFAT-CBR signals mirrored expected targets, including intestinal mucosa. Activation signals did not correspond to targets of massive T-cell infiltration, as was inferred from strong Rluc signals emanated by limbs and cervical lymph nodes. We assume that these target organs equally attract alloreactive ML-BLITC T cells but are either regulated by peripheral tolerance mechanisms or lack the proper costimulatory signals required to elicit a full response. Taken together, potential targets of autoreactivity in a given ATT setting can be rapidly and noninvasively identified by the BLITC reporter.
Both tumor models—H-Y and SV40 TAg—-shared the common feature of transient T-cell activation, which was in contrast to the prolonged persistence of TILs. The duration of NFAT-CBR emission differed depending on the tumor model, with the TCR-I transgenic T cells eliciting a very short activation burst of 1 day, whereas the H-Y specific response varied in duration. After the observed activation phase, however, activation signals did not reoccur throughout the duration of experiments. The presence of TILs was clearly demonstrated by the persistence of Rluc signals, which suggests local inhibition to act in the tumor microenvironment.
Although several tumor defense mechanisms are acquired in feedback with the microenvironment and act within the tumor population, additional layers of protection are potentially acquired by individual escape variants via mutation and become manifested as tumor relapse under increased evolutionary pressure. Analysis of the Dby-expression on relapsing MB49 tumors revealed downregulation of H-Y presentation on about 30% of tumors, whereas the remainder had maintained expression levels comparable to unchallenged tumors. T cells infiltrating Dby-expressing tumors featured significantly upregulated inhibitory receptors. This hints at antigen downregulation and immune checkpoint regulation as being two tumor escape mechanisms in our tumor model. We then retransferred a Dby− relapsed MB49 into immunodeficient hosts, challenged the tumors with ML-BLITC T cells, and followed their spatiotemporal migration and activation in comparison to primary MB49. Here, the relapsed tumor line showed minimal T-cell infiltration, demonstrating the lack of intratumoral activation. Downregulation of H-Y presentation potentially prevented detection by circulating T cells. Thus, BLITC reporter T cells potentially identify the type of tumor escape mechanism by revealing either a failure of infiltration or activation. The efficacy of accessory treatments aiming at increased T-cell migration to tumors could be greatly facilitated by using the BLITC reporter. In the case of tumors affecting the inhibitory receptor expression on TILs, the BLITC reporter could be a suitable tool for elucidating the optimal therapeutic window for accessory treatment with checkpoint inhibitors by observing favorable alterations in the activation kinetics upon drug administration.
Whereas the importance of cytotoxic CD8+ T cells for tumor rejection has been demonstrated, CD4+ T cells have only gradually been found potent in tumor regression, both experimentally and in the clinics. However, studies frequently consider tumor-specific CD4+ T cells to merely act as accessory to innate immunity and CD8+ T-cell cytotoxicity (3, 37). Although helper function is difficult to formally exclude in clinical settings of CD4+ ATT (38, 39), transfer experiments into T cell–deficient mice clearly ascribe complete remission to the action of CD4+ T cells (40–42). We showed tumor regression and demonstrated transient TIL activation via in vivo BLI by both CD4+ and CD8+ T cells in two separate tumor models, although complete tumor remission was only observed in the SV40-TAg model using CD8+ T cells. However, the BLITC reporter can assist in identifying the specific kinetics of tumor response in any ATT setting, whether it be CD4+, CD8+, or the combination of both.
Overall, the BLITC reporter represents a unique method for the study of T-cell behavior in vivo. Although conceived for furthering the understanding and ultimately improving the potency of ATT against solid tumors, its range of applicability extends to all areas of T-cell biology, including T-cell development and alloreactivity.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: M. Szyska, T. Blankenstein, I.-K. Na
Development of methodology: M. Szyska
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Szyska, S. Herda, S. Althoff, A. Heimann, J. Russ, D. D'Abundo, T.M. Dang
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Szyska, S. Herda, S. Althoff, A. Heimann, J. Russ, D. D'Abundo
Writing, review, and/or revision of the manuscript: M. Szyska, S. Herda, T.M. Dang, T. Blankenstein, I.-K. Na
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Szyska, T.M. Dang, B. Dörken, T. Blankenstein, I.-K. Na
Other (conduction and documentation of experiments): I. Durieux
The authors thank R. Manteufel, I. Hoeft, K. Pawletta, and B. Frenzel for taking excellent care of experimental mice and T. Daberkow-Nitzsche for assistance with rules and regulations. They also thank HP Rahn and K. Rautenberg for providing the sorting facilities and R. Naumann, Dresden, Germany, for performing oocyte injections. They are also grateful to O. Lantz for providing MataHari mice and the group of W. Uckert for assistance and valuable reagents for retroviral transduction. This work was supported by the Deutsche Forschungsgemeinschaft through the “Sonderforschungsbereich” TR36 and the Experimental and Clinical Research Center.
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