Human CD1c-autoreactive T-cells recognise Mycobacterium tuberculosis-infected antigen-presenting cells and display cytotoxic effector programmes
- School of Clinical and Experimental Sciences, NIHR Southampton Biomedical Research Centre, Faculty of Medicine, University of Southampton, United Kingdom
- Research and Evaluation, UK Health Security Agency, United Kingdom
- Immunocore Limited, United Kingdom
- Africa Health Research Institute, South Africa
- School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, South Africa
- Division of Infection and Immunity, University College London, United Kingdom
- Institute for Life Sciences, University of Southampton, United Kingdom
Figures
Figure 1 with 1 supplement
Autoreactive T-cells expand upon CD1c stimulation.
(A) Representative histogram overlays showing CD1c, major histocompatibility complex (MHC) -I, MHC-II, β2-microglobulin (β2m), CD1b, and HLA-E expression on wild-type THP1 cells, and engineered THP1-KO and THP1-CD1c antigen-presenting cells (APCs). (B) Representative flow cytometry analysis of T-cells from a healthy donor expanded with THP1-CD1c cells in the absence of exogenous lipid antigen and then cultured overnight with THP1 cells. Cells were gated on live CD3+CTV- T-cells, and T-cell activation was measured with anti-CD69, anti-CD25, and anti-CD137. Plots show expression of CD69 and CD137 (top), CD25 and CD137 (bottom). Significant numbers of CD1c-autoreactive T-cells were present in expanded cultures. (C) Cumulative data from 14 healthy donors showing frequency of CD3+CTV-CD69+CD137+ T-cells (top), and CD3+CTV-CD25+CD137+ T-cells (bottom) from lines first expanded with THP1-CD1c and then overnight culture with THP1-KO or THP1-CD1c APCs. CD1c-autoreactive T-cells were present in the majority of donors. **p<0.01; ***p<0.001 (C, Wilcoxon matched pairs signed-rank test).
Figure 1—figure supplement 1
CD1c-dependent activation and cytokine production by CD1c-autoreactive T-cells.
(A) Flow cytometry gating strategy analysing the expression of the T-cell activation markers CD69, CD25, and CD137 on proliferated CD3+CTV- T-cells. (B) Cytokine release by T-cells first expanded with THP1-CD1c antigen-presenting cells (APCs) and then stimulated overnight with THP1-KO or THP1-CD1c APCs. Cytokine secretion was measured by Luminex array. ns, not significant; *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA with Tukey’s multiple comparison test). Mean and SD of triplicate measurements are shown and are representative of three individual donors.
Figure 2 with 1 supplement
CD1c is expressed in human tuberculosis (TB) and downregulated in antigen-presenting cells (APCs) by M. tuberculosis (Mtb) infection.
(A) Lung biopsies from patients with active TB were stained with anti-CD1c antibodies. (i–iii) Representative granulomatous regions, including central granuloma areas where CD1c immunoreactivity is infrequent. (iv) Control staining of the same granuloma with secondary antibody only and avidin biotin-peroxidase complex (ABC) detection shows no immunoreactivity. (v) CD1c staining is more apparent in inflammatory tissue remote from the TB granuloma centre. (vi) CD1c staining in B-cell follicles adjacent to the granuloma. (B) RNA-seq heatmap showing significant reduction in CD1A, CD1B, and CD1C expression in monocyte-derived dendritic cells (MoDCs) from five donors at 48 hr after Mtb infection (MOI = 1). Differential gene expression was performed on filtered normalised counts using the voom-limma pipeline in RStudio, with p-value adjustment performed using the Benjamini-Hochberg method. Differentially expressed genes were identified as having log2FC >+1 (upregulated) or <–1 (downregulated) with adjusted p-values<0.05. (C) MoDC histograms of normalised counts demonstrating a reduction in CD1a, CD1b, and CD1c expression on differentiated MoDCs following live Mtb infection (MOI = 1). Data representative of an experiment conducted in two donors performed in triplicate. (D) Flow cytometry histograms showing CD1c expression on THP1-CD1c cells at 72 hr following infection with live Mtb (MOI = 1). Mtb infection does not change CD1c expression on THP1-CD1c cells. Data representative of two experiments performed in triplicate.
Figure 2—figure supplement 1
Mtb infection alters CD1 molecule expression on antigen-presenting cells.
(A) Flow cytometry gating strategy of live HLA-DR+/CD11c+ monocyte-derived dendritic cells (MoDCs) (top), and line graphs showing the effect of M. tuberculosis (Mtb) infection on the expression of CD1a, CD1b, and CD1c on MoDCs (bottom). (B) Flow cytometry gating strategy depicting live CD14+ THP1-CD1c cells stained with anti-CD1c antibody. *p<0.05; **p<0.01; ***p<0.001 (one-way ANOVA with Tukey’s multiple comparison test).
Figure 3 with 1 supplement
CD1c-autoreactive T-cells are cytotoxic to M. tuberculosis (Mtb)-stimulated cells.
(A–D) CD1c-endo tetramer staining and phenotypic characterisation of T-cell lines generated from two healthy donors. (A) T-cells generated from peripheral blood mononuclear cells (PBMCs) after CD1c-endo streptamer enrichment, CD1c-endo dextramer flow cytometric sorting, and subsequent in vitro expansion (Figure 3—figure supplement 1). (B) Phenotypic analysis demonstrating that the cells in (A) are αβTCR+ and CD4+. (C) T-cells generated following expansion with THP1-CD1c cells, CD1c-endo tetramer-guided sorting, and subsequent in vitro expansion. (D) Phenotypic analysis demonstrating that the cells in (C) are αβTCR+ and CD4+. (E) T-cell lines are activated in response to THP1-CD1c antigen-presenting cells (APCs). Rested T-cells and T-cells cultured with THP1-KO APCs served as control. T-cell activity was determined by measuring the upregulation of CD69 and CD25 by flow cytometry. Data is representative of three experiments from the two donor-derived lines performed in triplicate. (F) CD1c-autoreactive T-cells display significant cytotoxicity in a dose-dependent manner against THP1-CD1c APCs treated with increasing doses of UV-killed Mtb. The untreated/no-infection condition represents MOI = 0. The indicated MOI values refer only to THP1-CD1c APCs treated with UV-killed Mtb. No cytotoxicity was observed by T-cells cultured with untreated THP1-CD1c APCs or irrelevant control T-cells, lacking CD1c restriction. (G) CD1c-autoreactive T-cells displayed significant cytotoxicity in a T-cell dose-dependent manner against UV-killed Mtb-treated THP1-CD1c APCs. No cytotoxicity was observed by T-cells cultured with untreated THP1-CD1c APCs or with irrelevant control T-cells. Cytotoxicity was measured using a ToxiLight assay. Data is representative of two independent experiments, each performed in triplicate. *p<0.05; **p<0.01; ****p<0.0001 (E, one-way ANOVA with Tukey’s multiple comparison test; F–G, two-way ANOVA).
Figure 3—figure supplement 1
Flow cytometry gating strategy depicting live CD3+ T-cells comprising the negative (cells that did not bind the streptamers) or the CD1c-endo streptamer positive T-cell fraction (containing 1.14% CD1c-endo-positive T-cells) stained with CD1c-endo dextramers.
CD1c-endo dextramer positive T-cells were sorted and expanded. After expansion, T-cells were either unstained or stained with an irrelevant tetramer or with CD1c-endo tetramer. Enriched T-cells brightly stain with CD1c-endo tetramers.
Figure 4
CD1c-autoreactive T-cells lyse target cells infected with live M. tuberculosis (Mtb).
(A) Flow cytometry dot plots and (B) bar graphs showing CD1c-autoreactive T-cells are activated by THP1-CD1c antigen-presenting cells (APCs) but not THP1-KO APCs, with significantly greater activation when THP1-CD1c APCs are infected with live Mtb (MOI = 1). (C) Gating strategy for measuring the T-cell-mediated lysis of THP1 cells. Prior to T-cell culture, THP1 cells were stained with Tag-it Violet to allow identification of THP1 target cells in co-culture. LIVE/DEAD Fixable Near-IR Dead Cell Marker was used to measure the proportion of dead THP1 cells. Matched APC-only controls, including uninfected and Mtb-infected THP1 cells cultured without T-cells, were included in parallel to define baseline target-cell death for each condition. (D) CD1c-autoreactive T-cells lyse THP1-CD1c APCs but not THP1-KO APCs. Killing is significantly enhanced when THP1-CD1c cells are infected with live Mtb (MOI = 1). Specific lysis was calculated by subtracting baseline target-cell death observed in matched APC-only controls from target-cell death observed in the corresponding T-cell co-culture condition. (E) Lysis of THP1-CD1c is increased with Mtb infection, but not enhanced with LPS or Pam3CSK4 stimulation. Specific lysis was calculated relative to matched APC-only controls cultured in parallel. Data are representative of three independent experiments, each performed in triplicate. **p<0.01; ***p<0.001 (B, D, and E, one-way ANOVA with Tukey’s multiple comparison test).
Figure 5 with 1 supplement
TCR-CD1c interactions mediate the response to M. tuberculosis (Mtb) infection.
(A) The alpha (top) and the beta (bottom) chain sequences of EM1 and EM2 TCRs. Variable region (blue), N additions (white), and joining segment (grey) are shown. After filtering and manual curation, 11 single cells yielded productive paired αβ TCR sequences. EM1 and EM2 represented 6/11 cells and 4/11 cells, respectively. (B) CD1c-endo tetramer staining of parental Jurkats and Jurkat T-cells transduced to express EM1 and EM2 TCRs. (C) Percentage activation of Jurkat T-cells transduced with EM1 and EM2 TCRs in response to uncoated or CD1c-endo coated wells. T-cell activity was measured by flow cytometry staining with anti-CD69. (D) Jurkat T-cells transduced with EM1 and EM2 TCRs are activated by THP1-CD1c antigen-presenting cells (APCs) but not THP1-KO APCs, with significantly greater activation when THP1-CD1c APCs are infected with live Mtb (MOI = 1). Data are representative of two independent experiments, each performed in triplicate. **p<0.01; ***p<0.001; ****p<0.0001 (C, unpaired t-test; D, one-way ANOVA).
Figure 5—figure supplement 1
Validation of CD1c and TCR expression in JRT3.5 Jurkat T-cell lines.
(A) Flow cytometry dot plots showing high expression of CD1c on wild-type JRT3.5 Jurkat T-cells. (B) Flow cytometry dot plots showing the absence of CD1c and TCR expression on β2-microglobulin knockout JRT3.5 Jurkat T-cells.
Figure 6 with 2 supplements
CD1c-autoreactive T-cells secrete diverse cytokines and reduce M. tuberculosis (Mtb) luminescence.
(A) Cytokines secreted by CD1c-autoreactive T-cells cultured with UV-killed Mtb-treated THP1-CD1c antigen-presenting cells (APCs). CD1c-autoreactive T-cells produced significant amounts of the Th1 cytokines TNF-α, IFN-γ, IL-1α, and GM-CSF and the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 in a T-cell dose-dependent manner. Irrelevant control T-cells did not release cytokines. (B) Heatmap summarising cytokines released by CD1c-autoreactive T-cells or irrelevant control T-cells (CD1c unrestricted), in response to UV-killed Mtb-treated THP1-CD1c APCs. Red indicates high concentrations, and blue indicates low concentrations. Cytokine secretion was measured using a Luminex assay. Data are representative of two independent experiments, each performed in triplicate. (C) CD1c-autoreactive T-cells reduce Mtb luminescence significantly when cultured with THP1-CD1c APCs relative to when they are cultured with THP1-KO APCs. T-cells cause some reduction in THP1-KO cells, and this is greater in THP1-CD1c cells. *p<0.05; **p<0.01; ****p<0.0001 (A, two-way ANOVA; C, unpaired t-test).
Figure 6—figure supplement 1
Mtb enhances CD1c-dependent cytokine responses by CD1c-autoreactive T-cells.
(A) CD1c-autoreactive T-cells secrete cytokines in response to untreated THP1-CD1c antigen-presenting cells (APCs) in an autoreactive manner. Importantly, CD1c-autoreactive T-cells release significantly higher concentrations of cytokine when cultured with UV M. tuberculosis (Mtb)-treated THP1-CD1c APCs. (B) CD1c-autoreactive T-cells secrete diverse cytokines in a CD1c-dependent manner. CD1c-autoreactive T-cells release cytokines when cultured with UV Mtb-treated THP1-CD1c APCs but not when were cultured with UV Mtb-treated THP1-KO APCs. Data are representative of two independent experiments, each performed in triplicate. *p<0.05; **p<0.01; ****p<0.0001 (two-way ANOVA).
Figure 6—figure supplement 2
UV M. tuberculosis (Mtb)-treated THP1-CD1c antigen-presenting cells (APCs) secrete chemokines IL-8 and RANTES (pink bars), in comparison to media-only (black bars) and untreated THP1-CD1c APCs (blue bars).
Data are representative of two independent experiments; each performed in triplicate. ****p<0.0001 (two-way ANOVA).
Figure 7 with 3 supplements
Single-cell profiling reveals that CD1c-endo dextramer positive T-cells are enriched for cytotoxic and effector phenotypes.
(A) Schematic overview of the sample processing workflow. Peripheral blood mononuclear cells (PBMCs) were isolated from two donors and CD3+ T-cells were subsequently enriched by negative selection. Cells were stained with CD1c-endo dCODE dextramer, and CD3+CD1c-endo+ (positive) and CD3+CD1c-endo− (negative) T-cells were sorted for single-cell RNA sequencing. (B) UMAP visualisation of 11,804 single T-cells clustered by transcriptional profile. Clusters were annotated as functional T-cell subsets, including CD4+ and CD8+ naïve, central memory (TCM), effector memory (EM and TEM), cytotoxic, and stress-response populations. Additional subsets included metabolically active T-cells and tissue-resident memory-like (Trm-like) CD4+ cells. TRM: tissue-resident memory; EM: effector memory; TEM: T effector memory; TCM: T central memory. (C) UMAP projection showing CD1c-endo dextramer binding intensity, reflecting the number of bound dextramer molecules per cell. Cells with higher binding intensities are enriched within cytotoxic and effector CD4+ and CD8+ subsets. (D) Bar plots showing the proportional distribution of T-cell subtypes among CD1c-endo-positive (top) and -negative (bottom) populations. CD1c-endo-positive T-cells were enriched for cytotoxic and effector subsets. (E) Violin plots showing expression levels of the top 10 differentially expressed genes by Wilcoxon rank-sum test. CD1c-endo-positive T-cells upregulate genes associated with cytotoxicity (e.g. NKG7, GZMA, GZMK, GNLY, CTSW) and inflammation (e.g. CCL5, NFKBIA, DUSP2), consistent with a distinct effector phenotype. (F) Chord plot illustrating functional enrichment of differentially expressed genes in CD1c-endo-positive T-cells. Upregulated genes are associated with biological processes such as cell killing, antigen processing, and presentation and response to other organisms. In contrast, downregulated genes predominantly map to ribonucleoprotein complex biogenesis. The colour gradient indicates the log fold-change (logFC) in gene expression.
Figure 7—figure supplement 1
Identification and isolation of CD1c-endo-reactive T-cell populations.
(A) Representative flow cytometry gating strategy used to isolate CD3+ T-cells stained with CD1c-endo dCODE dextramers. (B) Representative plots from two donors showing distinct CD3+CD1c-endo+ and CD3+CD1c-endo⁻ T-cell populations. Percentages indicate the proportion of positive cells within the CD3+ gate.
Figure 7—figure supplement 2
Dot plot showing the top 5 ranked marker genes for each annotated T-cell population, derived from all high-quality T-cells (n=11,804).
Dot size represents the proportion of cells expressing each gene within the corresponding cluster, while colour indicates the scaled average expression (z-score). This visualisation highlights the transcriptional signatures that define distinct T-cell subsets, including naïve, memory, effector, and metabolically active populations.
Figure 7—figure supplement 3
Filtering strategy for identifying CD1c-endo dextramer-bound T-cells in single-cell RNA sequencing (scRNA-seq) data from sorted populations.
A total of 14,524 cells were initially profiled. Following quality control based on gene and UMI counts, mitochondrial and ribosomal content, doublet detection, and outlier removal, 11,804 high-quality cells were retained. Dextramer signal was then assessed using kernel density estimation (KDE) and quantile-based filtering, with extreme values excluded, leaving 11,800 cells. Of these, 9518 lacked detectable CD1c-endo dextramer binding, while 2282 were classified as CD1c-endo-positive candidates. Of the positive candidates, 2117 originated from the CD1c-endo-positive sort gate and were retained as confidently positive, whereas 165 from the negative gate were excluded. Of the 9518 cells lacking dextramer signal, 1866 originated from the CD1c-endo-negative sort gate and were retained as confidently negative, while 7652 from the positive sort gate were excluded to minimise potential false-negative classification. This strategy yielded 2117 confidently CD1c-endo-positive and 1866 confidently CD1c-endo-negative T-cells for downstream analysis.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv | DOI: https://doi.org/10.7554/eLife.52668 | Live Mtb used for APC infection (MOI = 1). | |
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv lux | DOI: https://doi.org/10.7554/eLife.52668 | Live Mtb used for APC infection (MOI = 1). | |
| Cell line (Homo sapiens) | THP1 WT | ATCC | RRID:CVCL_0006 | Myelomonocytic leukaemia line. Identity/phenotype verified in the study by flow cytometry and STR profiling. |
| Cell line (Homo sapiens) | THP1-KO | This paper | THP1 derivative generated by CRISPR-Cas9 knockout of B2M and CIITA. | |
| Cell line (Homo sapiens) | THP1-CD1c | This paper | THP1-KO derivative transduced with a beta2m-CD1c single-chain construct and sorted for CD1c expression. | |
| Cell line (Homo sapiens) | J.RT3-T3.5 Jurkat; beta2m-knockout J.RT3-T3.5 Jurkat | Pierre Vantourout, King’s College London | TCR-deficient Jurkat system; beta2m-knockout cells were a kind gift from Pierre Vantourout (King’s College London). Expected phenotype verified by flow cytometry and STR profiling. | |
| Cell line (Homo sapiens) | HEK293TN | ATCC | RRID:CVCL_UL49 | Used for lentiviral particle production. |
| Biological sample (Homo sapiens) | Peripheral blood mononuclear cells (PBMCs)/primary T-cells | Healthy blood-bank donors | 13/SC/0043 | PBMCs isolated by Ficoll-Paque; used for T-cell isolation, expansion, MoDC generation, and single-cell studies. |
| Biological sample (Homo sapiens) | Paraffin-embedded lung tissue from patients with tuberculosis | University Hospital Southampton histology archive | Ethics:12/NW/0794; SRB04_14 | Surplus archived clinical tissue used for immunohistochemistry. |
| Antibody | T-cell phenotyping antibodies: anti-CD3-BV510/FITC (mouse monoclonal, UCHT1); anti-alpha/beta TCR-BV421 (mouse monoclonal, IP26); anti-gamma/delta TCR-APC (mouse monoclonal, B1); anti-CD4-FITC (mouse monoclonal, RPA-T4); anti-CD8-APC (mouse monoclonal, HIT8a) | BioLegend | 5 µL per test. | |
| Antibody | MHC/beta2m antibodies: anti-MHC-I-PE (mouse monoclonal, W6/32); anti-MHC-II-FITC (mouse monoclonal, Tu39); anti-beta2m-PerCP-Cy5.5 (mouse monoclonal, A17082A); anti-HLA-DR-PE/Cy7 (mouse monoclonal, L243) | BioLegend | 5 µL per test. | |
| Antibody | CD1 antibodies: anti-CD1a-APC (mouse monoclonal, HI149); anti-CD1b-APC (mouse monoclonal, SN13(K5-1B8)); anti-CD1c-APC/PE (mouse monoclonal, L161) | BioLegend | 5 µL per test. | |
| Antibody | Activation antibodies: anti-CD69-PE (mouse monoclonal, FN50); anti-CD25-PE/Cy7 (mouse monoclonal, BC96); anti-CD137-APC (mouse monoclonal, 4B4-1) | BioLegend | 5 µL per test. | |
| Antibody | APC/myeloid markers: anti-CD14-FITC (mouse monoclonal, M5E2); anti-CD11c-BV421 (mouse monoclonal, Bu15); anti-CD36 (mouse monoclonal, 5–271) | BioLegend | 5 µL per test. | |
| Antibody | Anti-CD1c (rabbit recombinant monoclonal, EPR23189-196) | Abcam | Immunohistochemistry (1:500), overnight at 4°C. | |
| Antibody | Goat anti-rabbit secondary antibody | 2B Scientific | Immunohistochemistry (1:800). | |
| Recombinant DNA reagent | beta2m-CD1c single-chain construct | This paper | Used to generate THP1-CD1c cells; beta2m fused to CD1c. | |
| Recombinant DNA reagent | CD1c-beta2m single-chain expression cassette in pCDNA3.1 with Avi-tag/His6 and BirA | This paper | Used to express soluble human CD1c for tetramer/dextramer generation. | |
| Recombinant DNA reagent | EM1 TCR in pELNS lentivector | GenScript | Full-length TCR generated from tetramer-guided TCR sequencing and used to transduce beta2m-knockout J.RT3-T3.5 cells. | |
| Recombinant DNA reagent | EM2 TCR in pELNS lentivector | GenScript | Full-length TCR generated from tetramer-guided TCR sequencing and used to transduce beta2m-knockout J.RT3-T3.5 cells. | |
| Recombinant DNA reagent | pCMV-VSV-G; pRSV.REV; pMDL.pg.RRE | Other | Lentiviral packaging plasmids used with pELNS. Source: University of Pennsylvania | |
| Recombinant protein | CD1c-endo monomer | This paper | Soluble human CD1c-beta2m single-chain protein produced using the Expi293 expression system and purified by nickel affinity/size exclusion chromatography. | |
| Recombinant protein | CD1c-endo tetramer | This paper | CD1c-endo monomer multimerised using PE-streptavidin (BioLegend). | |
| Other | CD1c-endo dextramer/PE-dCODE dextramer | Immudex/this paper | CD1c-endo multimers generated using PE-labelled dextran backbones; dCODE dextramers used for single-cell sorting. | |
| Other | CD1c-endo streptamer | This paper | Generated from CD1c-endo containing a Strep-Tag II sequence and Strep-Tactin magnetic microbeads (IBA Lifesciences). | |
| Commercial assay or kit | EasySep Human T-cell Isolation Kit | STEMCELL Technologies | Used to purify pan T-cells from healthy donor blood. | |
| Commercial assay or kit | CD14+ monocyte negative-selection magnetic beads | STEMCELL Technologies | Used to isolate CD14+ monocytes for MoDC generation; exact product identifier not stated in the manuscript. | |
| Commercial assay or kit | Expi293 Expression System | Thermo Fisher Scientific | Used for recombinant soluble CD1c expression. | |
| Commercial assay or kit | ToxiLight cytotoxicity assay | Lonza | Used to measure cytotoxicity against UV-killed Mtb-treated target cells. | |
| Commercial assay or kit | Human cytokine 35-plex panel | Thermo Fisher Scientific | Multiplex cytokine analysis; exact catalogue identifier not stated in the manuscript. | |
| Commercial assay or kit | NGS library preparation kit | New England Biolabs | Used for targeted single-cell TCR sequencing; exact kit name/catalogue identifier not stated in the manuscript. | |
| Commercial assay or kit | Chromium Single Cell V(D)J Reagent Kit v1.1; Chromium Chip G | 10x Genomics | Used for single-cell library preparation. | |
| Chemical compound, drug | Dasatinib | Axon | 50 nM during tetramer/streptamer staining. | |
| Chemical compound, drug | CellTrace Violet (CTV) | Thermo Fisher Scientific | Used to track T-cell proliferation. | |
| Chemical compound, drug | LIVE/DEAD Fixable Near-IR Dead Cell Marker | Thermo Fisher Scientific | Used for viability discrimination in flow cytometry and single-cell sorting. | |
| Chemical compound, drug | Tag-it Violet | BioLegend | Used to identify THP1 target cells in lysis assays. | |
| Chemical compound, drug | Pam3CSK4 | InvivoGen | TLR2 agonist used at 1 µg/mL in lysis assays. | |
| Chemical compound, drug | LPS | Sigma | TLR4 agonist used at 1 µg/mL in lysis assays. | |
| Peptide, recombinant protein | rhIL-2 (Proleukin) | Chiron | 10 IU/mL during expansion cultures; 100 IU/mL during post-sort expansion. | |
| Peptide, recombinant protein | rhGM-CSF; rhIL-4 | Miltenyi Biotec | Used for MoDC differentiation (25 ng/mL rhGM-CSF; 20 ng/mL rhIL-4). | |
| Software, algorithm | FlowJo | FlowJo LLC | v10.8.1 (RRID:SCR_008520) | Flow cytometry analysis. |
| Software, algorithm | GraphPad Prism | GraphPad Software, Inc | v10 (RRID:SCR_002798) | Statistical analysis. |
| Software, algorithm | Seven Bridges | Seven Bridges | Analysis of targeted TCR sequencing data; version not stated in manuscript. | |
| Software, algorithm | Cell Ranger | 10x Genomics | v7.2.0 (RRID:SCR_017344) | Demultiplexing/alignment of single-cell sequencing data to GRCh38 (2020A). |
| Software, algorithm | Scrublet; scDblFinder | Open-source software | RRID:SCR_018098 | Independent doublet-detection methods used for scRNA-seq quality control; versions not stated. |
| Software, algorithm | enaBrowserTools; kallisto; tximport; edgeR; limma | Open-source software | enaBrowserTools v1.1.0; kallisto v0.46.1; tximport v1.28.0; edgeR v3.14.0; limma v3.56.2 | RNA-seq retrieval, quantification, and differential expression analysis. |
Additional files
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Supplementary file 1
Primer sequences used for targeted single-cell TCR sequencing.
Sequences are shown for the oligo-dT primer, universal forward and reverse primers, targeted forward primer containing the 8 bp well barcode, and reverse primers specific for TRAC, TRBC, TRGC, and TRDC constant regions.
- https://cdn.elifesciences.org/articles/110341/elife-110341-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110341/elife-110341-mdarchecklist1-v1.docx
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Source data 1
Numerical source data for Figures 1—6 and associated figure supplements.
Data are organised by figure panel in separate worksheets.
- https://cdn.elifesciences.org/articles/110341/elife-110341-data1-v1.xlsx
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Human CD1c-autoreactive T-cells recognise Mycobacterium tuberculosis-infected antigen-presenting cells and display cytotoxic effector programmes
eLife 15:RP110341.
https://doi.org/10.7554/eLife.110341.3
