A functional subset of CD8+ T cells during chronic exhaustion is defined by SIRPα expression

Prolonged exposure of CD8+ T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8+ T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα+ CD8+ T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα+ cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8+ T cell-killing in vivo. SIRPα+ CD8+ T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8+ T cells during chronic infection expands the cytotoxic subset of SIRPα+ CD8+ T cells.

Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection B.D. LSRII SORP DIVA version 8.0.1. RNAseq was performed by the Stanford Functional Genomics Facility (Illumina NextSeq). Computing for this project was perfomed on the Stanford Sherlock cluster. Stanford Functional Genomics Facility extracted and generated FASTQ files for each sample, distinguished by the Nextera dual index adapters. Raw reads were trimmed for base call quality (phred >=21) and adapter sequences using Skewer 112. RNAseq was performed by the Stanford Functional Genomics Facility (Illumina NextSeq). Computing for this project was perfomed on the Stanford Sherlock cluster. Stanford Functional Genomics Facility extracted and generated FASTQ files for each sample, distinguished by the Nextera dual index adapters. Raw reads were trimmed for base call quality (phred >=21) and adapter sequences using Skewer 112. Cytof data were collected on the CyTOF2 instrument using CyTOF2 software.

Data analysis
FlowJo software, version 10.2; TreeStar, Inc., Affymetrix arrays from GSE41867 were obtained as CEL files, MAS5 normalized using the "affy" package in Bioconductor, mapped to NCBI Entrez gene identifiers using a custom chip definition file ( https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41867) and converted to MGI gene symbols. RNAseq processed reads were aligned to mm10 and read counts were generated using STAR 2.5.3a 113. The R package 'DESeq2' was used to normalize read counts, perform differential gene expression analysis, and generate the heat map. Cytof data were de-barcoded and manually analyzed on Cytobank (cytobank.org).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The RNAseq data that support the findings of this study have been deposited in Sequence Read Archive with the project accession code SRP173611. The remaining data that support the findings of this study are available from the corresponding author upon reasonable request. The remaining data that support the findings of this study are available from the corresponding author upon reasonable request.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample sizes were determined by a sample size calculator using a 95% confidence level.
Data exclusions No data were excluded.

Replication
Experimental replicates are indicated in the figure legends. All replicate experiments were successful.
Randomization Allocation into experimental groups was not randomized because the experiments were done in age and sex-matched, genetically identical mice.

Blinding
Data identification for experimental animals during collection was done by box number/animal number. Following collection, when numbers were already entered and were unalterable, box/animal number codes were associated with experimental groups so that negative or naive controls could be used to set analysis gates. For Fig. 4, RNAseq was done blinded.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.  , which is specific for F-MuLV glycosylated Gag protein (mAb34 was produced at RML/NIAID/NIH as a culture supernatant). MAb 34 binding was detected with FITC-labeled goat anti-mouse IgG2b (R12-3, BD Pharmigen 553395, lot 32885; 1/800).

Validation
Dendritic cells, macrophages and mononcytes from mice with targeted SIRPα gene disruptions completely lose reactivity with mAb p84 (anti-SIRPα) even though their SIRPβ expression is normal. These results indicate specificity of p84 for SIRPα without cross reactivity for SIRPβ

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
As stated in the methods section: For LCMV studies26, female 4-6 week old C57BL/6J mice from NCI and Thy-

Field-collected samples
For laboratory work with field-collected samples, describe all relevant parameters such as housing, maintenance, temperature, photoperiod and end-of-experiment protocol OR state that the study did not involve samples collected from the field.

Ethics oversight
As stated in the methods section: The use of all animals for LCMV studies was conducted in accordance with and approved by the Yale University IACUC guidelines. For FV studies, Mice were treated in accordance with RML IACUC-approved animal use protocols following the regulations and guidelines of the Animal Care and Use Committee of the Rocky Mountain Laboratories and the National Institute of Health Office of Laboratory Animal Welfare.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Population characteristics
The study included fifteen HCV-infected patients. Ten patients underwent at least one previous treatment with interferon, the other five were treatment naïve. The distribution of variables set as age, sex, history of previous IFN treatment, history of transplantation, HCV genotype, and HCV infection status is detailed in the methods.

Recruitment
Patients with HCV infecttion were asked during their routine visit to Stanford Liver Clinic fif they wanted to participate. The study lasted from November 2013 to May 2016.

Ethics oversight
Study protocol number 13859 approved by the Stanford University Institutional Review Board.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Splenocytes were isolated by tissue homogenization through a 100-m filter and RBCs were removed using lysis buffer ( Cell population abundance These target cell and effector cell populations were then placed in a 2 hr in vitro cytotoxic killing assay at a 1:4 target:effector