The malate shuttle detoxifies ammonia in exhausted T cells by producing 2-ketoglutarate

The malate shuttle is traditionally understood to maintain NAD+/NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8+ T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD+/NADH ratio and limited antiviral CD8+ T cell responses to chronic infection; however, increasing the NAD+/NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8+ T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8+ T cells is not to maintain the NAD+/NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8+ T cells during chronic infection.


Statistics
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Policy information about availability of computer code Data collection We used FACS Diva Software (version 9, BD Biosciences) to collect FACS data.FACS data were analyzed using FlowJo software (10.1r1).Western blot data were collected by Fusion (FX6 Edge, Vilber).We used the ABI Prism 7500 sequence detection system (SDS Software v1.2.3, Applied Biosystems) to collect the qPCR data.We quantified the band intensities in the NIH ImageJ program (Version 1.53t).Mass isotopologue distribution (MID) was determined using the DExSI software (Version 1.11).

Data analysis
FACS data were analyzed using Flowjo software (version 10.lrl).Western blot data were analyzed using Fusion FX6 Edge.RNA sequencing reads were first subjected to adapter trimming and low-quality read filtering with flexbar (version 2.5) with the following parameters: -u 6 -m 36 -ae RIGHT -at 2 -ao 2. Reads that were mapped to the reference sequences of rRNA, tRNA, snRNA, snoRNA, and miscRNA (available from Ensembl and RepeatMasker annotation) with Bowtie 2 (version 2.4.2) with default parameters (in --end-to-end &-sensitive mode) were excluded.The remaining reads were then mapped to the mouse reference genome (mml0) with STAR (version 2.7.7a) with key parameters --outFilterMismatchNmax 8--outFilterMismatchNoverlmax 0.1--alignlntronMin 20--alignlntronMax 1000000--outFilterType BySJout --outFilterlntronMotifs RemoveNoncanonicalUnannotated.Reads that mapped to multiple genomic sites were discarded in the following analysis.HTSeq-count (version 2.0.1) was used to count reads mapped to annotated genes, with parameters -f barn -r pos -s no -a 10.Differentially expressed gene analysis was performed with the R package DESeq2 (version 1.30.1).In brief, size factor estimation was first conducted to normalize the data across samples, and this was followed by dispersion estimation to account for the negative binomial distributed count data in RNA sequencing.Finally, gene expression fold changes were calculated, and the significance of the gene expression difference was estimated with the Wald test.To control for the false discovery rate in multiple testing, the raw p-values were adjusted with the Benjamini-Hochberg procedure.

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April 2023 ATAC sequencing data were first subjected to adapter trimming and low-quality read filtering with flexbar (version 2.5) with the following parameters: -u 5 -m 26 -ae RIGHT -at 2 -ao 1.The trimmed reads were mapped to the mouse reference genome (mmlO) with Bowtie 2 (version 2.4.2) with parameters -X 2000 --mm.Reads that mapped to mitochondrial DNA or those with low mapping quality(< 30) were excluded from downstream analysis.Duplicate reads due to PCR amplification of single DNA fragments during library preparation were identified with Picard (version 2.17.3; available at http://broadinstitute.github.io/picard)and thus were removed from the downstream analysis.MACS2 (version 2.2.7.1) was used for calling open chromatin regions.To identify peaks with differential accessibility, we counted the deduplicated reads overlapping with peaks.DESeq2 (version 1.30.1),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 and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.

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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 description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The GEO accession number for the RNA sequencing data and ATAC sequencing data is GSE220876.The dataset will become public from September 1, 2023.All data needed to evaluate the conclusions in the paper are present in the manuscript and the Supplementary Information.There are no data restrictions.
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Reporting on sex and gender
For the in vitro culture studies: buffy coat human peripheral blood mononuclear cell (PBMC) samples from healthy donors were provided by the blood bank of Mannheim.There were 4 male donors and 4 female donors.We did not observe different phenotypes between the male and female donor T cells.
For the HIV patient tissue section studies: lymph node sections were provided by the tissue bank of the German Center for Infection Research.HIV-positive samples were from 2 male donors (59-year-old, 62-year-old) and 1 female donor (34-yearold).HIV-negative samples were also from 2 male donors (65-year-old, 74-year-old) and 1 female donor (51-year-old).

Reporting on race, ethnicity, or other socially relevant groupings
We did not consider the information of race, ethnicity, or other socially relevant groupings when we requested samples from the blood bank of Mannheim or from the tissue bank of the German Center for Infection Research (DZIF, Heidelberg, Germany).We also do not have such information.

Population characteristics
For the in vitro culture studies: we used PBMC from healthy volunteers from 24 to 69 years of age.
For the HIV patient tissue section studies: lymph node sections were provided by the tissue bank of the German Center for Infection Research.Donors were 34 to 74 years of age.

Recruitment
For the in vitro culture studies: Buffy coat PBMC samples from healthy donors were provided by the blood bank of Mannheim, Germany.When choosing samples, we have considered both age (from 18 to 70 years of age) and gender (both male and female), but we did not consider other factors, such as race, ethnicity, or other socially relevant groupings.
For the HIV patient tissue section studies: lymph node sections were provided by the tissue bank of the German Center for Infection Research (DZIF, Heidelberg, Germany).Samples were chosen based on HIV positive or negative irrespective of age, gender, ethnicity or any other bias that could influence study outcomes.

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This study was performed in accordance with the approval of the ethics committee of Heidelberg University.
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Life sciences study design
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Sample size
No formal statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications nature portfolio | reporting summary

April 2023
Data exclusions We did not exclude data.

Replication
Experiments were repeated twice or three times, as indicated in the figure legends.
Randomization For the comparison between wildtype and knockout mice, littermate mice were allocated into 2 groups based on genotypes (namely wildtype and knockout mice).
For the P14 T cell adoptive transfer experiments, C57Bl/6N mice were randomly allocated into different groups.
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Flow Cytometry
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