CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours – Nature

Patient biopsies

Skin metastases of 20 patients with melanoma (clinical stage III–IV), obtained during routine histopathological diagnostic procedures at the Department of Dermatology of the University Medical Centre Magdeburg, were analysed by immunohistochemistry for the expression of MHC-I (1:100), MHC-II (1:200) and CD8 (undiluted), in addition to the melanoma markers MART-1 (undiluted), gp100 (undiluted), S-100 (undiluted) and Sox10 (undiluted) using the automated Ventana BenchMark platform and standard protocols. These studies were performed in the context of routine clinical workup and were approved by the ethics committee of the Otto-von-Guericke University Hospital Magdeburg (approval number 162/20).

Cell suspensions derived from immunotherapy naive tumour biopsies of 20 melanoma metastases in skin (n = 5), subcutis (n = 4) and lymph nodes (n = 11) from 19 patients (clinical stage IIIB–IV) were analysed for their messenger RNA expression profile in single cells. Methods for tumour dissociation, library construction, scRNA-seq data acquisition and analysis were described previously²⁰. This study was approved by the UZ Leuven Medical Ethical Committee and written consent obtained from all patients. The immune cells were distinguished from other tumour microenvironment cells by high immune signature score and low copy number variation score. Next, the cells were reclustered and annotated using SingleR. The MHC-I and MHC-II gene signature scores were measured using the AUCell R package⁴⁹.

MILAN (mIHC)

Multiplex immunofluorescent images were generated by sequential immunostaining and antibody removal according to the published MILAN protocol⁵⁰ from human melanoma biopsies as described previously⁵⁰. From the complete 41 protein markers included in the published panel, a reduced panel including panCK (1 µg ml⁻¹), CD3 (1 µg ml⁻¹), CD4 (1:200), CD8, (1 µg ml⁻¹), FOXP3 (1 µg ml⁻¹), MHC-II (1 µg ml⁻¹), CD11c (1 µg ml⁻¹), CD68 (1:200), MLANA (1:500), MITF (1 µg ml⁻¹) and CD31 (1 µg ml⁻¹) for staining keratinocytes, effector T cells, MHC-II expressing myeloid cell subsets, melanoma cells and vessels respectively, is shown. Image analysis was performed as described previously⁵¹. Briefly, stains were visually evaluated for quality by an experienced pathologist. Flat field correction was performed using a custom implementation of the methodology⁵². Consecutive staining rounds were registered using a previously described algorithm⁵³. Tissue autofluorescence was subtracted using a baseline image stained only with a secondary antibody.

Mice

Mice were housed in an ambient temperature- and humidity-controlled environment on a 12-h light/dark cycle to mimic natural conditions. Wild type C57BL/6J mice were purchased from Janvier or Charles River. The T cell receptor-transgenic Pmel-1 (B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J), TRP-1 (B6.Cg-Rag1tm1Mom Tyrp1B-w Tg (Tcra,Tcrb) 9Rest/J), OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J) and OT-II (B6.Cg-Tg(TcraTcrb)425Cbn/J) mice, and the fluorescent B6-eGFP (C57BL/6-Tg (UBC-GFP) 30Scha/J) and CD11c-eYFP (B6.Cg-Tg (Itgax-Venus) 1Mnz) mice were purchased from Jackson Laboratories. Pmel-1-Venus mice were generated by crossing CAG-Venus mice with Pmel-1 mice. TRP-1-eGFP mice were generated by crossing B6-eGFP mice into the TRP-1-deficient Rag1-KO background of TRP-1 mice. OT-I-Venus mice were generated by crossing CAG-Venus mice with OT-I mice. OT-II-dsRed were generated by crossing OT-II mice with hCD2-dsRed mice (kindly provided by C. Halin). All transgenic mice were bred in house. Age matched cohorts of tumour developing mice were randomly allocated to the different experimental groups. All animal experiments were conducted with male mice on the C57BL/6 background under specific pathogen-free conditions in individually ventilated cages according to the institutional and national guidelines for the care and use of laboratory animals with approval by the Ethics Committee of the Office for Veterinary Affairs of the State of Saxony-Anhalt, Germany (permit licence numbers 42502-2-1393 Uni MD, 42502-2-1586 Uni MD, 42502-2-1615 Uni MD, 42502-2-1672 Uni MD) in accordance with legislation of both the European Union (Council Directive 499 2010/63/EU) and the Federal Republic of Germany (according to §8, section 1 TierSchG and TierSchVersV).

Cell lines and cell culture

The mouse melanoma cell line HCmel12 was established from a primary melanoma in the Hgf-Cd4k^R24C mouse model by serial transplantation in our laboratory as described previously⁵⁴. The mouse melanoma cell line B16 and the human melanoma cell lines A375 and SKmel28 were purchased from ATCC. The human melanoma cell lines MaMel04 and MaMel102 were kindly provided by D. Schadendorf. All cell lines were cultured in complete Roswell Park Memorial Institute (RPMI) medium consisting of RPMI 1640 medium (Life Technologies) supplemented with 10% foetal calf serum (Biochrome), 2 mM l-glutamine, 10 mM non-essential amino acids, 1 mM HEPES (all form Life Technologies), 20 µM 2-mercoptoethanol (Sigma), 100 IU ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin (Invitrogen) in a humidified incubator with 5% CO₂. The cell lines were routinely screened for mycoplasma contamination and were authenticated by the commercial provider or by short tandem repeat fingerprinting.

In vitro cell death assays

For the measurements of cell death in mouse and human melanoma cell lines, cells were first seeded in 96-well plates in complete RPMI medium. Inflammatory mediators were added after 24 h (10 U ml⁻¹ recombinant mouse IFNγ (Peprotech); 1,000 U ml⁻¹ recombinant mouse TNF (Peprotech); 100 U ml⁻¹ animal-free recombinant human IFNγ (Peprotech); 1,000 U ml⁻¹ recombinant human TNF (Peprotech) and 100 µM SNAP (Cayman Chemicals)). After 24 h, floating and adherent cells were gathered and stained using the fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Pharmingen) and analysed using the Attune NxT acoustic focusing flow cytometer (ThermoFisher).

Adenovirus generation and expansion

To generate the adenoviral vaccine Ad-PT, a fusion construct was generated consisting of the first 150 base pairs of the human PMEL complementary DNA (coding for amino acids 1–50 of the human PMEL/gp100 protein including the CD8⁺ T cell epitope KVPRNQDWL) and 1,404 base pairs of the mouse Trp1 cDNA (coding for amino acids 51–518 including the CD4⁺ T cell epitope SGHNCGTCRPGWRGAACNQKILTVR) followed by sequences coding for a T2A viral self-cleaving peptide and the yellow fluorescent marker protein eYFP. This vaccine construct was cloned into the pShuttle vector (termed pShuttle-PT-YFP). A recombinant adenovirus vector with this sequence was then generated by a recombineering technique in Escherichia coli strain SW102 using bacmid pAdZ5-CV5-E3⁺. The E1 region of this bacmid is replaced by a selection/counter-selection cassette called ampicillin, LacZ, SacB or the ALS cassette. Next, E. coli with this bacmid were electroporated with the PT-YFP transgene with homology arms flanking the ALS cassette obtained by PCR amplification using pShuttle-PT-YFP as a template. Positive colonies were isolated after antibiotic selection on LB-sucrose plates. Ad-PT and Ad-OVA were expanded using the 911 human embryonic retinoblast cell line. A confluent monolayer of the cells in T175 cell culture flasks was infected with Ad-PT or Ad-OVA at MOI 1. The cytopathic effects were observed at around 36 h of incubation at 37 °C. Next, cells were scraped, freeze–thawed three times and the lysates were cleared by centrifuging at the speed of 7,000g for 45 min. The crude virus was then titrated by the TCID₅₀ method according to standard protocols.

CRISPR–Cas9-mediated genetic cell engineering

To generate Ciita-KO, Trp1-KO, Jak1-KO and Tyr-KO HCmel12 variants, HCmel12 melanoma cells were used that can be readily genetically modified using CRISPR–Cas9-mediated gene editing⁵⁵. Cells were seeded into a 12-well plate at a density of 5 × 10⁵ cells per well and cotransfected with 1.6 μg pX330-sgRNA and 0.4 μg plasmid expressing GFP (pRp-GFP) using Fugene HD transfection reagent (Promega) according to the manufacturer’s instructions. GFP positive cells were single-cell sorted using a FACSAria III Cell Sorter (BD) to generate polyclonal and monoclonal populations per targeted gene. HCmel12 cells were mock transfected with pX330 plasmid without single-guide RNA and the polyclonal cell line was used as a CRISPR-control in all performed experiments. Genomic DNA from cultured knockout variants was extracted using the NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer’s protocol. A two-step PCR protocol was performed to generate targeted PCR amplicons for next-generation sequencing. In the first PCR, specific primers for the target gene with more adapter sequences complementary to the barcoding primers were used to amplify the genomic region of interest with Phusion HD polymerase (New England Biolabs). In a second PCR, adapter-specific universal primers containing barcode sequences and the Illumina adapter sequences P5 and P7 were used (Illumina barcodes D501-508 and D701-D712). Next-generation sequencing was performed with MiSeq Gene and Small Genome Sequencer (Illumina) according to manufacturer’s standard protocols with a single-end read and 300 cycles (MiSeq Reagent Kit v.2 300 cycle). For the detection of insertions or deletions, the web-based program Outknocker (http://www.outknocker.org/) was used as previously described⁵⁶. FASTQ files were imported, and the sequence of the target gene amplicons was used as reference sequence for alignment.

Western blot

Melanoma cells were lysed using the M-PER mammalian protein reagent (Fermentas) with protease inhibitors (Thermo Scientific). The protein concentration was spectrophotometrically measured by a Bradford-based assay using Pierce BCA protein assay kit (Thermo Scientific) according to manufacturer’s protocol. Laemmli buffer was added and lysates were boiled at 95 °C for 5 min. Then, 10 μg of protein was loaded and separated according to size by SDS–PAGE gel electrophoresis on a 3% stacking and 10% polyacrylamide gel. Proteins were transferred to polyvinyl difluoride membranes with a 0.2 μm pore size (GE Healthcare) by means of wet blotting for 1 h. Unspecific binding was blocked with 5% skimmed milk in PBS with Tween for 1 h. Blots were stained with a goat polyclonal Trp1 antibody (1:1,000, Novus Biologicals) overnight at 4 °C. Next, the blots were incubated with anti-goat IgG HRP (1:2,000, Santa Cruz) for 1 h at room temperature. Horseradish peroxidase conjugated β-actin (200 µg ml⁻¹) was used as loading control. Bound antibody was detected by SignalFire ECL reagent (Cell Signaling Technology) and chemiluminescence was visualized using an Octoplus QPLEX imager (NH DyeAgnostics).

Retroviral transduction

To generate tagBFP, mCherry and OVA-tagBFP-expressing cell lines, retroviruses were produced by transfecting human embryonic kidney 293T cells with the retroviral packaging constructs pCMV-gag-pol and pMD.2G (expressing VSVg) and the retroviral plasmids pRp-tagBFP, pRp-mCherry and pRp-OVA-tagBFP, respectively, according to standard protocols. Retrovirus-containing supernatant was used to transduce the target cell lines and antibiotic selection of transduced cells was started 48 h after transduction using 10 µg ml⁻¹ Puromycin.

Tumour transplantation experiments

For tumour inoculation, a total of 2 × 10⁵ cells were injected intracutaneously (i.c.) into the shaved flanks or hindlegs of mice with a 30G (0.3 × 13 mm) injection needle (BD). Tumour development was monitored by inspection and palpation. Tumour sizes were measured three times weekly and presented as mean diameter. Mice were euthanized when tumours exceeded 15 mm mean diameter or when mice showed signs of sickness in adherence with the local ethical regulations. All animal experiments were performed in groups of four to six mice and repeated independently at least twice.

ACT therapy protocol

ACT therapy was performed as previously described^23,24. In brief, when transplanted melanoma cell lines reached a mean diameter of 3–5 mm, mice were preconditioned for ACT by a single intraperitoneal (i.p.) injection of 2 mg (roughly 100 mg kg⁻¹) of cyclophosphamide in 100 µl of PBS 1 day before intravenous (i.v.) delivery of splenocytes isolated from TCR-transgenic Pmel-1 and/or TRP-1 donor mice harbouring naïve Pmel-1/gp100-specific CD8⁺ T cells and/or naïve TRP-1-specific CD4⁺ T cells (in 100 µl of PBS). Unless otherwise indicated, we transferred splenocytes containing 5 × 10⁵ antigen-specific T cells. The adoptively transferred T cells were stimulated in vivo by a single i.p. injection of 2.5 × 10⁸ PFU of the recombinant adenoviral vaccine Ad-PT in 100 µl of PBS. On day 3, 6 and 9 after T cell transfer, tumours were injected with 50 µg of CpG 1826 (MWG Biotech) and 50 µg of polyinosinic:polycytidylic acid (polyI:C, Invivogen) diluted in 100 µl of distilled water. Seven days after T cell transfer, blood was taken routinely from the Vena facialis to confirm successful expansion of transferred T cells by flow cytometry.

Supplementary in vivo treatments

NK-cell depletion was performed by a single i.p. injection of 200 µg anti-NK1.1 antibody (clone PK136, BioXCell) in 100 µl, diluted in pH 7.0 Dilution Buffer (BioXCell). CD8⁺ T cell depletion was performed by i.p. injections of initially 100 µg, followed by weekly injections of 50 µg of anti-CD8 antibody (clone 2.43, BioXCell). MHC-II blockade was performed by a single i.v. injection of 500 µg of anti-MHC-II antibody (clone Y-3P, BioXCell) directly after inducing anaesthesia for 2P-IVM and roughly 30 to 60 min before data acquisition. IFNγ blockade was performed by weekly i.p. injection of 500 µg of anti-IFNγ antibody (clone XMG1.2, BioXCell) in 100 µl, diluted in pH 8.0 buffer. Monocyte depletion was performed by i.p. injections of 20 µg of anti-CCR2 (clone MC21, provided by M. Mack) for five consecutive days. Neutrophil depletion was performed by i.p. injections of 100 µg of anti-Ly6G (clone 1A8, BioXCell) every fifth day. Inhibition of iNOS was performed by daily i.p. injection of 200 µg of L-NIL (Cayman Chemicals) diluted in 100 µl of PBS.

Flow cytometry

Immunostaining of single-cell suspensions was performed according to standard protocols. Single suspensions were incubated with anti-CD16/CD32 (1:300, Biolegend) before staining with fluorochrome-conjugated monoclonal antibodies CD45-APC Fire 750 (1:1,600), CD11c-APC (1:200), F4/80-PE (1:300), CD11b-BV711 (1:200), Ly6C-PE-Cy7 (1:2,000), CD45R-PE (1:1,000), CD3ε-BV421 (1:500), CD4-BV605 (1:500), NK1.1-APC (1:400), CD45-FITC (1:1,000), F4/80-APC (1:200), Ly6C-BV421 (1:800), iNOS-PE, (1:300), I-A/I-E-BV510 (1:800), CD45-BV711 (1:200), CD11c-APC Fire 750 (1:100), Siglec H-FITC (1:400), CD4-PE (1:1,600), CD11b-PE-Cy7 (1:2,000), Ly6G-PE (1:800), CD3ε-BV711 (1:100), CD8α-APC Fire 750 (1:1,600), H2-Kb-PE (1:500), I-A/I-E-APC (1:2,000), CD3ε-FITC (1:100), CD335-APC (1:100), CD8α-PE (1:800), Vβ14-FITC (1:2,000), T-bet-PeCy7 (1:200) and Foxp3-Alexa Fluor 647 (1:100). Intracellular staining was carried out using a Fixation/Permeabilization Solution Kit (BD or Biolegend). Single-cell suspensions from tumours were first stained with antibodies against-cell-surface antigens, then fixed and permeabilized, followed by intracellular staining. Dead cell exclusion was performed using 7-aminoactinomycin or propidium iodide. All data were acquired with an Attune NxT acoustic focusing flow cytometer (ThermoFisher). Gating and subsequent analyses were performed using FlowJo v.10.8.1 for Windows (Tree Star, Inc.). Fluorescence-activated cell sorting was performed using an Aria III (BD Biosciences).

Quantification of tumour-infiltrating immune cells

To quantify the abundance of immune cell subpopulations in tumour tissues, 2,000 cells of interest per biological sample were concatenated to a single FCS file. The t-distributed stochastic neighbor embedding (t-SNE) plots were generated in FlowJo using the opt-SNE learning configuration⁵⁷. The vantage-point tree K-nearest-neighbours algorithm and the Barnes–Hut gradient algorithm were set to 1,000 iterations, 30 perplexity and 840 learning rate. Immune cell subpopulations were annotated on the basis of heatmaps for characteristic marker combinations and their percentage in the tumour was calculated.

Analysis of tumour cell MHC expression and antigen recognition by CD4⁺ T cells

To quantify the expression of MHC molecules, tumour cells were pretreated with 100 U ml⁻¹ recombinant murine IFNγ (Peprotech) for 72 h and then analysed by flow cytometry. To assess antigen recognition by CD4⁺ T cells, TRP-1 TCRtg mice were immunized with Ad-PT and subsequently injected with 50 µg of CpG and 50 µg of polyI:C i.c. 3 and 6 days after immunization. TRP-1 CD4⁺ T cells were isolated from the spleen and purified by two rounds of magnetic cell sorting (Miltenyi). Direct antigen recognition was determined by coculturing purified CD4⁺ T cells with IFNγ pretreated HCmel12 cells. Antigen recognition in proxy was assessed by initially generating bone marrow-derived dendritic cells with recombinant GM-CSF and IL-4 (Peprotech) as previously described. After 1 week, differentiated bone marrow-derived dendritic cells were then pulsed overnight with HCmel12 lysate, before coculture with purified CD4⁺ T cells. For both direct and myeloid cell-dependent antigen-recognition assays, the production of IFNγ from the CD4⁺ T cells was measured 16 h after coculture by intracellular cytokine staining using flow cytometry according to standard protocols.

Calculations of absolute immune cell counts in tumour tissues

Tumours were excised with tweezers and scissors, then weighed using the Entris 224-1S analytical balance (Sartorius). Single-cell suspensions were created mechanically using 5-ml syringe plungers (BD) and 70 µm cell strainers (Greiner). After immunostaining, cells were suspended in a defined volume and analysed on the Attune NxT acoustic focusing flow cytometer that uses a unique volumetric sample and sheath fluid delivery system allowing for accurate measurements of the number of cells analysed in a defined sample volume. The total number of viable CD45⁺ immune cells in an individual tumour can then be derived by multiplying the number of CD45⁺ immune cells counted in a defined sample volume with the total volume of the respective single-cell suspension. Division of this total number by the total weight of the tumour yields the absolute immune cell count per mg tumour tissue. The absolute count of various immune cell subpopulations was calculated from their relative percentage in viable CD45⁺ immune cells.

Immunofluorescence microscopy

Tumours were harvested on day 5 after ACT and fixed in 4% paraformaldehyde for 24 h, then dehydrated in 20% sucrose before embedding in optimal cutting temperature freezing media (Sakura Finetek). Next, 6 µm sections were cut on a CM305S cryostat (Leica), adhered to Superfrost Plus slides (VWR) and stored at −20 °C until further use. When thawed, slides were either fixed with ice-cold acetone and stained with rat anti-mouse I-A/I-E (1:50) and anti-rat IgG-Alexa Fluor 594 (1:100) or directly mounted with Vectashield Antifade Mounting Medium (Vector Laboratories). Images were acquired on an Axio Imager.M2 with a Colibri 7 LED illumination system (Zeiss) and analysed with ImageJ v.1.52i (http://imageJ.nij.gov/ij).

Intravital two-photon microscopy

Mice were anaesthetized with 100 mg kg⁻¹ ketamine and 10 mg kg⁻¹ xylazine i.p., complemented by 3 mg kg⁻¹ acepromazine s.c. after the onset of anaesthesia. The animals were placed and fixed to a heated stage. Transparent Vidisic carbomer gel was applied to moisten the eyes during anaesthesia. The hind leg was fixed in an elevated position and the skin covering the melanoma was detached using surgical scissors and forceps. One drop of transparent Vidisic carbomer gel was used on the exposed site as mounting medium. Two component STD putty (3M ESPE) placed on both sides of the leg was used create a level surface using a 24 × 60 mm cover slip, which was gently pressed on the putty in a way that the coverslip made slight contact with the exposed site, without exerting pressure on the tumour. After complete polymerization of the putty, the mice were transferred onto a 37 °C heating plate under the two-photon microscope.

Imaging was performed using distilled water or transparent Vidisic carbomer gel as immersion liquid with a W Plan-Apochromat ×20/1.0 DIC VIS-IR objective mounted to a Zeiss LSM 700 upright microscope with the ZEN software environment (v.2.1, Zeiss), or a LaVision TrimScope mounted to an Olympus BX50WI fluorescence microscope stand and a XLUMPlanFl ×20/0.95 objective. Excitation on the LSM700 setup was performed with Mai Tai DeepSee (tuned to 800 nm) and Insight X3 (tuned to 980 nm) Ti:Sa oscillators (both from Spectra-Physics). Fluorescence signals were read out on a long-pass dichroic mirror detector cascade as follows: dsRed, 980 nm excitation and 555 nm dichroic transmission with a 587/45 nm bandpass filter; Venus, 980 nm excitation and 520 nm dichroic transmission with a 534/30 nm bandpass filter; second-harmonic generation, 800 nm excitation and 445 nm dichroic deflection unfiltered; tagBFP, 800 nm excitation and 490 nm dichroic deflection with a 485 nm short-pass filter; and eGFP, 980 nm excitation, 520 nm dichroic deflection and 490 nm dichroic transmission with a 525/50 nm bandpass filter. Excitation on the TrimScope setup was performed with a Chamaeleon Ultra II Ti:Sa oscillator tuned to 880 nm.  Fluorescence signals were read out with a double split detector array with a 495 nm main dichroic mirror and 445 and 520 nm secondary dichroic mirrors (all long-pass) as follows: second-harmonic generation, 495 nm and 445 nm dichroic deflection unfiltered; tagBFP, 495 nm dichroic deflection and 445 nm dichroic transmission with a 494/20 nm bandpass filter; eGFP, 495 nm dichroic transmission and 520 nm dichroic deflection with a 514/30 nm bandpass filter; and Venus, 495 nm and 520 nm dichroic transmission with a 542/27 nm bandpass filter. Non-descanned photomultiplier tubes (for second-harmonic generation, dsRed and Venus in all setups, and for eGFP and tagBFP in the TrimScope setup) and high sensitivity detectors (for tagBFP and eGFP in the Zeiss setup) were used for signal collection.

Typically, three to four representative fields of view of 353 µm² size in x-, y– and a z-range of 48 to 60 µm with 4 µm step sizes were chosen for data acquisition. Z-stacks were captured in 60–90 s intervals and individual video length was 15–30 min. Data analysis was performed with the Bitplane Imaris software (v.8.3 to 9.7). T cells were identified using the Imaris spot function. Tumour area was identified using the surface function with low surface detail. CD11c-Venus cells were identified using the surface function with high detail. T cell speed was calculated using the Imaris software. Cells were considered arrested when speed was less than 2 µm min⁻¹. Contact duration was measured as the time that the distance between the centre of mass of a T cell to the closest CD11c cell surface was less than 8 µm. Snapshot images of 3D rendering and tracking were cropped, arranged and animated for time series using ImageJ v.1.52i (http://imageJ.nij.gov/ij).

Cell preparation for scRNA-seq

Three individual tumours per group were harvested and processed into single suspensions. CD45⁺ cells were enriched using a positive selection kit (Miltenyi). Next, individual samples were hashtagged with unique TotalSeq-B hashtag antibodies B0301-B0310 (1:300, Biolegend) and subsequently stained with fluorescently labelled antibodies. CD45⁺CD11b⁺Ly6G^– cells were sorted with an Aria III fluorescence-activated cell sorter (BD). Isolated cells were loaded onto one lane of a 10X Chromium microfluidics controller. cDNA of hashtag and gene expression libraries were amplified, and indices added by means of PCR. Sequencing was performed on an Illumina Novaseq on two lanes of a S1 cartridge with 150 bp read length in paired end mode. Reading depth was calculated to obtain roughly 50,000 reads per cell for the gene expression library and 5,000 reads per cell for the hashtag library.

scRNA-seq data processing and hashtag-demultiplexing

The scRNA-seq data generated using 10X Genomics Chromium technology were aligned and quantified using the Cell Ranger Single-Cell Software Suite against the mm10 mouse reference genome. The raw, unfiltered data generated from Cell Ranger were used for downstream analyses. Quality control was performed on cells on the basis of the three metrics: total unique molecular identifier (UMI) count, number of detected genes and proportion of mitochondrial gene count per cell. Specifically, cells with less than 1,000 UMIs, 1,000 detected genes and more than 25% mitochondrial UMIs were filtered out. To remove potential doublets, cells with UMI count above 40,000 were removed. Subsequently, we demultiplexed the samples tagged with distinct hashtag-oligonucleotides using Solo⁵⁸. After quality control, we normalized raw counts by their size factors using scran⁵⁹ and subsequently performed log₂ transformation. The logarithmized and normalized count matrix was used for the downstream analyses.

Dimensionality reduction, unsupervised clustering and differential gene expression analyses

Analysis of normalized data was performed using the scanpy Python package⁶⁰. Initially, the 4,000 most highly variable genes were selected for subsequent analysis using scanpy.pp.highly_variable_genes with the parameter ‘n_top_genes=4000’. Next, a principal component analysis was performed with 50 components using scanpy.tl.pca with the parameters ‘n_comps=50, use_highly_variable=True, svd_solver=‘arpack’’. Subsequently, dimensionality reduction was performed using UMAP with scanpy.tl.umap. Single cells were automatically assigned using R package SingleR⁶¹, with transcriptomes from the Immunological Genome Project as a reference. Clustering of single cells by their expression profiles was conducted by using the Leiden algorithm running scanpy.tl.leiden with the parameter ‘resolution=1.0’. Clusters with fewer than 20 cells were removed from further analysis. Differential gene expression was performed between cells classified as macrophages and monocytes from non-treated and CD4 ACT-treated mice using a hurdle model implemented in the R package MAST. Subsequent gene set enrichment analysis was performed using gene set enrichment analysis in preranked mode using the log₂ fold change as a ranking metric. The IFN score was derived by calculating a z-score for all genes from the MSigDB gene set ‘HALLMARK_INTERFERON_GAMMA_RESPONSE’ for each cell.

RNA velocity

For RNA velocity, count matrices of spliced and unspliced RNA abundances were generated using the velocyto workflow for 10X chromium samples, with the genome annotation file supplied by 10X Genomics for the mm10 genome and a repeat annotation file retrieved from the UCSC genome browser. Subsequent analyses were performed using scVelo⁶². The count matrices were loaded into the scanpy environment, merged with the previously generated anndata objects and normalized using scvelo.pp.filter_and_normalize. Next, moments for velocity estimation were calculated, gene-specific velocities were estimated and the velocity graphs were computed. Furthermore, a partition-based graph abstraction was generated with velocity-directed edges.

Statistical methods

Statistical analyses and number of samples (n) are given in each figure legend. Mann–Whitney U-tests, unpaired two tail t-tests, analysis of variance (ANOVA) and log-rank tests were performed in Graphpad Prism (v.8).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.