MATERIALS AND METHODS
The aim of this study was to investigate mechanisms by which CD8+ TSL cells are maintained in tumors over the course of cancer progression. We utilized an autochthonous model, as well as an orthotopic transplant mouse model, of lung adenocarcinoma in which tumor cells express the neoantigen GP33 from LCMV. We evaluated the presence of tumor-specific CD8+ TSL cells in various lymphoid and non-lymphoid tissues by FACs using tetramer-specific cell staining. We analyzed single-cell RNA-sequencing to assess the differentiation state and trajectory of the cell subsets present after FACs sorting on endogenous tetramer-specific CD8+ T cells from tumors and draining lymph nodes of tumor-bearing mice, comparing them to tetramer-specific CD8+ T cells from spleens of mice infected with acute or chronic LCMV. In order to determine the clonal relationship of these cells from tumors and draining lymph nodes, we analyzed single-cell TCR sequencing. We tested the effect of blocking lymphocyte migration into tumors after three weeks of FTY720 treatment in autochthonous mice by FACs. Lastly, to assess whether similar phenomena occur in humans, we analyzed a publicly available single cell RNA-sequencing dataset from lymph nodes and lungs of non-small cell lung cancer patients.
Lung Tumor Initiation
Autochthonous tumor generation: KP-NINJA mice were infected intratracheally with 2.5 × 107 PFU Ad5mSPC-Cre (Dr. Anton Berns, Netherlands Cancer Institute), after precipitation with 10mM CaCl2 for 20-60 min, or 5 × 104 PFU Lenti-cre. To induce expression of NINJA neoantigen in infected cells, mice were given doxycycline hyclate chow (625mg/kg; Envigo cat. TD.09628) days 7-11 post infection (p.i.) and concomitantly treated with 4.4mg tamoxifen (MP Biomedicals cat. MP215673894) in corn oil (ThermoFisher Scientific cat. S25271) by gavage on days 8-10 p.i. To induce neoantigen expression via lentivirus, KP mice were infected with 2.5 × 104 PFU mClover-GP33-80-Cre lentivirus and assessed at 8 weeks p.i. Orthotopic KPN1 tumor transplants: Established KPN1 cells were maintained in complete DMEM (10% HI-FBS, 55μM beta-mercaptoethanol, 1x Pen/Strep and 1x L-Glut). Prior to injection, cells were washed 3x with 1xPBS and 200,000 cells were injected intravenously via tail vein injection. Subcutaneous KPN1 transplants: Established KPN1 cells, sorted for GFP+ (NINJA-expressing) cells, were maintained in complete DMEM (10% HI-FBS, 55μM beta-mercaptoethanol, 1x Pen/Strep and 1x L-Glut). Prior to injection, cells were washed 3x with 1xPBS and 500,000 cells were injected s.c. and measured using standard caliper measurements. Tumor volume = (LxW2)/2.
Tissue processing for flow cytometry
Ex Vivo IFNγ expression
Single cell suspensions were obtained as described above. The number of cells from draining lymph nodes and tumors was determined using hemocytometer. Samples were then plated in 96-well flat bottom plates at a ratio of 25:75 with CD45.1 splenocytes and stimulated in 10% HI-FBS RPMI-1640 (Thermo Fisher Scientific cat. 11875085) containing Brefeldin A (eBioscience cat. 00-4506-51), and LCMV GP33-41 peptide (AnaSpec cat. AS-61296), or left unstimulated in 10% HI-FBS RPMI-1640 (ThermoFisher Scientific cat. 11875085) containing Brefeldin A (eBioscience cat. 00-4506-51). Plates were incubated for 4-6 hours at 37°C, and samples were then transferred to 96-well round bottom plates. Samples were stained for extracellular markers (see Flow cytometry section), fixed with BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences cat. 554714), and stained with anti-IFNγ for intracellular cytokine assessment (see Flow cytometry section) in BD Perm/wash Buffer (BD Biosciences cat. 554714) as per manufacturer’s protocol.
Cells were prepared from various tissues and stained with extracellular antibodies in FACs Buffer (0.5% FBS, 20% sodium azide in water, PBS 1X without Mg2+/Ca2+). Staining reagents included PECF594 anti-CD45 (30-F11) and FITC anti-IFNγ (XMG1.2) from BD Biosciences; PERCP anti-CD90.2 (30-H12), BV421 anti-CD279 (PD-1; 29F.1A12), APCFire750 anti-CD90.1 (THY1.2; OX-7), BV605 anti-CD90.2 (30-H12), PECY7 anti-CD366 (TIM3; RMT3-23), BV421 anti-CD279 (29F.1A12), PE anti-SLAMF6 (330-AJ), BV421 anti-CD8α (53-6.7), BV711 anti-CD44 (IM7), APC/Fire750 anti-CD90.1 (THY1.1; IM7), and APC-Cy7 anti-CD45.1 (A20) from Biolegend, FITC anti-CD8α (CT-CD8α), and PeCy5 anti-CD8α (CT-CD8α) from Thermo Fischer Scientific; PE TCF1/7 (C63D9) from Cell Signaling Technologies. H-2D(b) LCMV GP 33-41 tetramer-KAVYNFATM-APC was provided by the NIH tetramer core. Cells were stained at 4°C for 30 min followed by fixation and permeabilization with apropriate intracellular staining kit. For intracellular staining of cytokines, the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit from BD Biosciences was used as per manufacturer’s protocol. For intracellular staining, FoxP3/Transcription Factor Staining Buffer set (eBioscience cat# 00-5523-00) was used as per manufacturer’s protocol. Data were collected on LSRII cytometer (BD Biosciences). For sorting, indicated populations were sorted to >90% purity with FACSAria III cytometer (BD Biosciences).
Histology and IHC staining
Tumor-bearing lungs of KP-NINJA mice were fixed in 1x Formalin solutions in PBS (Millipore-Sigma) for 24 hours at 4°C, switched into 70% ETOH, and submitted to Yale histology core for paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining. Unstained slides of KP-NINJA autochthonous lung tumors were stained with anti-CD3 (ab5690) using the ImmPACT DAB Peroxidase kit (Vector Labs) for immunohistochemistry. H&E and anti-CD3 IHC stained sections were imaged on a Nikon TE2000 microscope (Micro Video Instruments, Inc. Avon, MA) using a 20x objective.
KP-NINJA mice were infected intratracheally with 2.5 × 107 PFU Ad5mSPC-Cre (Dr. Anton Berns, Netherlands Cancer Institute) and treated with tamoxifen and doxycycline as previously described. From 6 to 9 weeks following intratracheal infection, mice were treated with 0.3 mg/kg FTY720 or vehicle (saline) i.p. every other day.
Cell line generation
LCMV-Clone 13 and -Armstrong infections
Sorting and single cell RNA- and TCR- sequencing of GP33-specific CD8+ T cells
KP-NINJA mice were infected with Ad5mSPC-Cre, treated with doxycycline and tamoxifen, and lungs and dLN were harvested 8 and 17 weeks p.i. after i.v. injection of anti-CD45-PECF594 antibody (clone 30-F11, BD Biosciences), as described. Spleens were harvested from C57BL/6 mice 28 days following infection with LCMV-Clone 13 (or LCMV-Armstrong – Figure S3I-K). Tissues were dissociated as previously described and GP33-specific CD8+ T cells were sorted (i.v.CD45–CD8+GP33-loaded MHC I tetramer+) and submitted to the Yale Center for Genome Analysis for single-cell RNA and TCR sequencing. Single cell RNA-sequencing data was demultiplexed using Cell Ranger 3.0 Software and then further analyzed using Python. Data represents cells from n=3 pooled at each time point. Pooled GP33-specific endogenous cells from tumors and matched dLNs, as well as from spleens, were submitted for 10X single cell RNA-and TCR-sequencing to Yale Center for Genome Analysis (YCGA).
Bioinformatics analysis of GP33-specific CD8+ T cells
Single group TCR sequence diversity was calculated using Simpson’s index based on number of clones and number of cells with clonal sequences (shared by 2 or more cells). Number of clonal sequences and number of cells with clonal sequences, respectfully: Early tumor (448 and 767), Early dLN (1098 and 1734), Late tumor (216 and 675), Late dLN (346 and 886). Morisita-Horn index between samples was calculated to compare overlap between samples. These calculations were conducted using R program.
Human CD8+ T cell single cell RNA-sequencing analysis
All statistical analyses were performed using Prism V8.3.0 software.