CD8+ T cell-derived Fgl2 regulates immunity in a cell-autonomous manner via ligation of FcγRIIB

The regulatory circuits dictating CD8+ T cell responsiveness versus exhaustion during anti-tumor immunity are incompletely understood. Here we report that tumor-infiltrating antigen-specific PD-1+ TCF-1− CD8+ T cells express the immunosuppressive cytokine Fgl2. Conditional deletion of Fgl2 specifically in mouse antigen-specific CD8+ T cells prolongs CD8+ T cell persistence, suppresses phenotypic and transcriptomic signatures of T cell exhaustion, and improves control of the tumor. In a mouse model of chronic viral infection, PD-1+ CD8+ T cell-derived Fgl2 also negatively regulates virus-specific T cell responses. In humans, CD8+ T cell-derived Fgl2 is associated with poorer survival in patients with melanoma. Mechanistically, the dampened responsiveness of WT Fgl2-expressing CD8+ T cells, when compared to Fgl2-deficient CD8+ T cells, is underpinned by the cell-intrinsic interaction of Fgl2 with CD8+ T cell-expressed FcγRIIB and concomitant caspase 3/7-mediated apoptosis. Our results thus illuminate a cell-autonomous regulatory axis by which PD-1+ CD8+ T cells both express the receptor and secrete its ligand in order to mediate suppression of anti-tumor and anti-viral immunity.

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Mandy L. L. Ford May 13, 2024 Libraries were validated by by capillary electrophoresis on on a TapeStation 4200 (Agilent), pooled at at equimolar concentrations, and sequenced with PE100 reads on on an an Illumina NovaSeq 6000, yielding ~25 million reads per sample on on average.Alignment was performed using STAR version 2.9.7a and transcripts were annotated using GRCm38_102.Transcript abundance estimates were calculated internal to to the 20 20 STAR aligner using the algorithm of of htseq-count.
Raw counts were analyzed in in R using the DESeq258 version 1.41.12 and clusterProfiler59 4.8.2 packages from Bioconductor.Differentially expressed gene analyses were performed and differentially expressed genes were processed through GSEA.HALLMARK gene sets and C7 C7 immunological signature gene sets from the Mouse Molecular Signatures Database (MSigDB) were used.Specifically, "HALLMARK_APOPTOSIS" and "GSE41867_MEMORY_VS_EXHAUSTED_CD8_TCELL_DAY30_LCMV_UP" are shown.A p-value <0.05 adjusted with the Benjamini-Hochberg correction was considered significant.Adjusted pvalue and enrichment scores are shown.No No custom software that is is not publicly available was utilized in in this study.However, code is is available upon request.

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The raw data is viewable in the NCBI SRA database under PRJNA1056506.The data generated in this study have been deposited in the GEO database and is available under the GEO database accession number GSE252556 available here, [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE252556]).Source data are provided with this paper.
This information has been collected and is outlined in Supplementary Data Table 2. Sex has been used as an identifier.
The terms "white" "unknown" and "non-white" were used to report, external variables were not controlled for.
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For TIL studies, melanoma tumor tissues were collected (IRB #00095411), deidentified, and distributed by the Cancer Tissue and Pathology shared resource of Winship Cancer Institute of Emory University.
IRB board from Emory University.the study design and conduct complied with all relevant regulations regarding the use of human study participants and was conducted in accordance with the criteria set by the Declaration of Helsinki.
sizes were determined by the resource equation model, 2-3 independent experiments were performed.Experimental repeats were successful.
Outliers were detected using the ROUT method.
In each experiment, 3-4 mice were used per group and 2-3 independent experiments were performed.
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Sample preparation shipped from the European Mutant Mouse Archive (EMMA) and re-derived at the Emory University Transgenic Mouse Core Facility.These mice were generated using embryonic stem cells from B6 mice.These Fcgr2b-/-mice made by Boross et al.52 were bred to OT-I transgenic mice at Emory University.Fgl2-/-mice were a kind gift of Dr. Gary Levy53, University of Toronto.Fgl2-/-mice were bred to OT-I or P14 Thy1.1+TCR transgenic mice to generate Fgl2-/-OT-I mice and Fgl2-/-P14 mice.Mice were used at 8-12 weeks old.All animals were housed in specific pathogen-free animal facilities at Emory University.Control animals were bred separately in the same facility.Mice were housed at a humidity of ~55% and the dark/light cycle was 12h/12h.
No wild animals were involved in this study.
Animals were sex-matched for experiments and experiments were performed in male and female mice.No sex differences observed.
The study did not involve samples collected from the field.
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The study design and conduct complied with the regulations on the use of human study participants and was conducted in accordance with the Declaration of Helsinki.This protocol was approved by Emory University's Institutional Review Board.For TIL studies, melanoma tumor tissues were collected (IRB no.00095411), deidentified, and distributed by the Cancer Tissue and Pathology shared resource of Winship Cancer Institute of Emory University [demographic data shown in Supplementary Data Table 2 (n = 4)].
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Describe the methods by which all novel plant genotypes were produced.This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization.For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed.For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.
Report on the source of all seed stocks or other plant material used.If applicable, state the seed stock centre and catalogue number.If plant specimens were collected from the field, describe the collection location, date and sampling procedures.
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For tumor-infiltrating lymphocyte (TIL) experiments, patient tumor tissue was collected and dissociated using the Human Tumor Dissociation Kit (Miltenyi) and GentleMACS Octo Dissociator (Miltenyi) according to the manufacturer's instructions.Cells were activated in vitro using 15 l/ml anti-CD3/28-coated Dynabeads (Thermofisher) in R10 for 48 hours at 37°C and 5% CO2.Flow staining was performed using the antibodies listed in Extended Data  For tumor experiments, spleen, draining lymph node (right inguinal proximal to to tumor), and tumors of of mice were processed to to cell suspensions, blood underwent red blood cell (RBC) lysis prior to to staining.For viral experiments, spleen, brachial and axillary lymph nodes were processed to to cell suspensions, blood underwent RBC lysis prior to to staining.The samples were then stained for surface markers prior to to permeabilization for transcription factor staining using the antibodies listed in in Supplementary Data Table 1. 1. Cells were permeabilized using a FoxP3/transcription factor kit (Invitrogen).For Fgl2 cytokine staining of of OT-I, splenocytes were ex ex vivo stimulated at at 37°C for 4 hours with 10 10 nM nM OVA257-264 (SIINFEKL) peptide (B16-OVA) and 10 10 g/mL GolgiPlug (BD Biosciences).For Fgl2 cytokine staining of of P14 cells, splenocytes were ex ex vivo stimulated at at 37°C for 4 hours with 0.4 g/ml LCMV-gp33-41 (KAVYNFATM) peptide and 10 10 g/mL GolgiPlug (BD Biosciences).After 4 hours, cells were processed and stained for intracellular markers using antibodies listed in in Supplementary Data Table 1. 1. Fgl2 antibody (Abnova) were conjugated to to fluorophore with Lightning Link technology (or isotype) and validated using Fgl2-/-splenocytes.
Experiments were run using Fortessa and LSR II II flow cytometers as as well as as an an Aria sorter.
Flow cytometry data was analyzed using FlowJo (Tree Star) and Prism (GraphPad Software).Absolute cell numbers were calculated using CountBright Beads (Life Technologies) according to to the manufacturer's instructions.
Purity of of >95% for post-sort populations on on Aria Cell Sorter was determined from a check of of sample post-sort while on on cell sorter.
A gating strategy is is in in the supplementary information as as Supp.Data Figure 3. 3.For most experiments, the following gating strategy was employed: FSC-H by by FSC-A to to eliminate non-singlet events, then FSC-A by by SSC-A to to gate on on lymphocytes followed by by a CD45 gate, then CD14/19 by by CD3.On On CD3 population, subsequent events were visualized on on CD4 by by CD8 axes.Afterwards, CD8 by by congenic marker (either CD45.1 or or Thy1.1) was plotted, followed by by phenotypic and functional analyses on on this population.The list of of experiments for which this gating strategy was employed is is listed in in the figure legend of of Supp.Data Fig. 3 while the subsequent appropriate gating is shown in in the appropriate figure.
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Table 1 .
Fgl2staining was assessed via staining with Fgl2-APC antibody conjugated with Lightning Link technology.