DOI: 10.5281/zenodo.15678770
title: Exhaustion-resistant T cell clones form functional memory after chronic infection and immunotherapy [TCR-seq]

description: Bulk TCR-seq Data Associated with the manuscript: Exhaustion-resistant T cell clones form functional memory after chronic infection and immunotherapy 

Manuscript Abstract: 

Chronic antigen exposure promotes T cell exhaustion and results in permanent impairment of T cell function after antigen clearance. However, the differentiation patterns of individual exhausted T cell (Tex) clones are heterogeneous, and whether some clones escape this trajectory and preserve memory potential remains unresolved. Here, we used single cell RNA/T cell receptor sequencing of antigen specific CD8+ T cells during chronic viral infection and human cancer to determine the clonal differentiation patterns of stem-like Tex. These data revealed that while most clones enter the Tex differentiation trajectory during chronic antigen stimulation, a subset of clones resist terminal exhaustion and maintain a stem-like progenitor (Tex-Prog) cell state. These Tex-Prog biased clones persist long-term and preferentially form central memory T cells (Tcm) after the clearance of chronic antigen exposure. Upon in vivo rechallenge, these Tex-Prog derived Tcm robustly expand and differentiate conferring protective immunity. Underlying their functional recall capacity, Tcm derived from Tex-Prog biased clones show minimal epigenetic imprinting of chronic antigen exposure demonstrating the capacity of these clones to resist the Tex differentiation pathway. Finally, we identified an analogous differentiation pattern of long-lived Tex-Prog biased clones among neoantigen-specific T cells in tumor samples and that these clones support checkpoint blockade responses in vivo. Together, these data reveal a novel differentiation trajectory of chronically stimulated CD8+ T cells, in which select clones remain in a stem-like state during chronic infection and maintain the capacity to form bona fide memory and respond to PD-1  blockade.

Bulk TCR-seq data are from four (4) datasets associated with the manuscript:



TCR-seq of GP33+ and GP276+ T cell subsets from LCMV-Cl13 infection 100+ dpi

TCR-seq of PD1+ Tex subsets from the blood and spleen 28 dpi with LCMV-Cl13

TCR-seq of CD8+ T cell subsets from LCMV-Cl13 or LCMV-Arm infection 100+ dpi prior to transfer of cells and LCMV-Arm rechallenge 

Longitudinal TCR-seq of CD8+ T cell subsets from LCMV-Cl13 infection 21, 35, and 100+ dpi +/- αPD-L1 treatment


Methods Details

(1) TCR-seq of GP33+ and GP276+ CD8+ T cell subsets from LCMV-Cl13 infection 100+ dpi

OVERVIEW: TCR sequencing was performed on sorted populations of GP33+ and GP276+ T cell subsets at 126 days post-infection. GP33+ cells were also sorted from blood of the same at 28 days post-infection.

DETAILS: Peripheral blood was collected at 28 days post-infection by retro-orbital bleed, and spleen  was collected at 126 days post-infection.  CD8+ T cells were enriched by magnetic separation, and peripheral blood was treated with chloride-potassium bicarbonate for red blood cell lysis. Cells were stained with viability dye, GP33 or GP276 tetramer (separate mice per specificity), and surface antibodies. Live CD8+ tetramer+ T cells were sorted based on surface protein expression using a BD FACSAria into RPMI supplemented with 10% FBS. The cells were centrifuged, the supernatant was removed, and dry pellets were snap-frozen. Genomic DNA extraction was performed by Adaptive Biotech (Seattle, WA).  TCR-seq was performed by Adaptive via their immunoseq platform at the survey depth. Adaptive performed intial data alignment and processing. 

(2) TCR-seq of PD1+ Tex subsets from the blood and spleen 28 dpi with LCMV-Cl13

OVERVIEW: CD8+ T cells populations from the spleen and blood of the same mouse were sorted 28 days post-infection with LCMV-Cl13.

DETAILS: Mice were infected with LMCV-Arm or LCMV-Cl13 and sacraficed at 28 dpi. Spleen and peripheral blood (collected via cardiac puncture) were collected and enriched for CD8+ T cells using magnetic separation. Cells were stained with viability dye, GP33 tetramer, and surface antibodies. Live CD8+ GP33+ T cells were sorted based on surface marker expression using a BD FACSAria into RPMI supplemented with 10% FBS. The cells were centrifuged, the supernatant was removed, and dry pellets were snap-frozen. Genomic DNA extraction was performed by Adaptive Biotech (Seattle, WA).  TCR-seq was performed by Adaptive via their immunoseq platform at the survey depth. Adaptive performed intial data alignment and processing. 

(3) TCR-seq of CD8+ T cell subsets from LCMV-Cl13 or LCMV-Arm infection 100+ dpi prior to transfer of cells and LCMV-Arm rechallenge 

OVERVIEW: To track T cell clones during rechallenge, CD8+ T cells were collected from the spleens of LCMV-Cl13 immune or LCMV-Arm immune mice. Half of the cells were transferred into new host mice and challenged with LCMV-Arm. The remaining pre-rechallenge cells were subset-sorted, and bulk TCR sequencing was performed. These data are associated with scRNA/TCR-seq of the same T cell clones after rechallenge (GEO: GSE285414) 

DETAILS: Splenocytes were harvested at 151 days post-infection from mice infected with LCMV-Cl13 or LCMV-Arm, and CD8+ T cells were enriched using magnetic separation. Approximately half of the cells were transferred for rechallenge, while the remaining cells underwent TCR sequencing. CD8+ T cells that were not transferred were stained with viability dye, GP33 tetramer, and surface antibodies. Live CD8+ T cell subsets were sorted based on a combination of surface antigens using a BD FACSAria into DNA/RNA Shield (Zymo) and snap-frozen. RNA extraction was performed by iRepertoire (Huntsville, AL). iRepertoire performed TCR beta chain sequencing via Amp-RepSeq+1 Platform (Mouse TCR beta, long read). iRepertoire performed intial data alignment and processing. Reads were UMI normalized.

(4) Longitudinal TCR-seq of CD8+ T cell subsets from LCMV-Cl13 infection 21, 35, and 100+ dpi +/- αPD-L1 treatment

OVERVIEW: T cells were isolated from blood at 21 and 35 days post-infection and from the spleen of the same mice 100 days post-infection. T cell subsets were sorted at each timepoint, and bulk TCR sequencing  was performed on the sorted cells. Mice were treated six times with either aPDL1 or PBS from days 22 to 34.

DETAILS: Mice were infected with LCMV-Cl13 and dosed with 200ug aPDL1 (B7-H1) or PBS by IP injected every 2-3 days from 22-34 dpi. Peripheral blood was collected by retro-orbital bleed at 21 and 35 days post-infection, and spleen  was collected at 100 days post-infection. Splenocytes were enriched for CD8+ T cells using magnetic separation, and peripheral blood was treated with chloride-potassium bicarbonate for red blood cell lysis. Cells were then stained with viability dye, GP33 tetramer, and surface antibodies. Live CD8+ T cells were sorted based on surface protein expression using a BD FACSAria and directly into Zymo RNA lysis buffer. The cells were snap-frozen and stored at -80°C. RNA extraction was performed by iRepertoire (Huntsville, AL). iRepertoire performed TCR beta chain sequencing via Amp-RepSeq+1 Platform (Mouse TCR beta, long read). iRepertoire performed intial data alignment and processing. Reads were UMI normalized.

Samples: 

Note: For multiple experiments sorted >4 cell populations from the same animals, however our instrument only allows 4 populations to be sorted at a given time. Due to this we report the total number of cells in a subset (calculated based on absolute count beads) to normalize TCR-seq counts back to absolute frequency of a given clone.

Note: To acchieve sufficient cell numbers to perform sequencing,  each TCR-seq samples is pooled from multiple mice. 

Also availble for download in file: `TCR-seq Samples.xlsx`

 

        


Dataset
Sample Name
Total Cells (Calculated)
Organism
Organ
TCR-seq modality
File Name (Rep 1)
File Name (Rep 2)


(1) GP33+ Substs 100+ dpi
GP33_TCR_028dpi_KLRG1neg
n/a
Mus musculus
Blood
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_12.tsv
 


(1) GP33+ Substs 100+ dpi
GP33_TCR_028dpi_KLRG1pos
n/a
Mus musculus
Blood
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_11.tsv
 


(1) GP33+ Substs 100+ dpi
GP33_TCR_126dpi_Tcm
3.23E+03
Mus musculus
Spleen
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_1.tsv
 


(1) GP33+ Substs 100+ dpi
GP33_TCR_126dpi_Tem1
2.17E+04
Mus musculus
Spleen
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_5.tsv
 


(1) GP33+ Substs 100+ dpi
GP33_TCR_126dpi_Tem2
1.13E+04
Mus musculus
Spleen
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_4.tsv
 


(1) GP33+ Substs 100+ dpi
GP33_TCR_126dpi_TexProg
2.96E+03
Mus musculus
Spleen
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_2.tsv
 


(1) GP33+ Substs 100+ dpi
GP33_TCR_126dpi_TexTerm
3.88E+03
Mus musculus
Spleen
genomic DNA
TCRgp33_Processed_bulkTCRseq_GP33posCR43_3.tsv
 


(1) GP276+ Substs 100+ dpi
GP276_TCR_126dpi_Tcm
1.04E+03
Mus musculus
Spleen
genomic DNA
TCRgp276_Processed_bulkTCRseq_GP276posCR43_6.tsv
 


(1) GP276+ Substs 100+ dpi
GP276_TCR_126dpi_Tem1
1.72E+04
Mus musculus
Spleen
genomic DNA
TCRgp276_Processed_bulkTCRseq_GP276posCR43_10.tsv
 


(1) GP276+ Substs 100+ dpi
GP276_TCR_126dpi_Tem2
1.22E+04
Mus musculus
Spleen
genomic DNA
TCRgp276_Processed_bulkTCRseq_GP276posCR43_9.tsv
 


(1) GP276+ Substs 100+ dpi
GP276_TCR_126dpi_TexProg
2.70E+03
Mus musculus
Spleen
genomic DNA
TCRgp276_Processed_bulkTCRseq_GP276posCR43_7.tsv
 


(1) GP276+ Substs 100+ dpi
GP276_TCR_126dpi_TexTerm
9.97E+03
Mus musculus
Spleen
genomic DNA
TCRgp276_Processed_bulkTCRseq_GP276posCR43_8.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Blood_TexInt
5.75E+04
Mus musculus
Blood
genomic DNA
TCRspleenblood_Processed__PB_M4_PD1Pos_TexInt.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Blood_TexKLR
7.52E+04
Mus musculus
Blood
genomic DNA
TCRspleenblood_Processed__PB_M4_PD1Pos_TexKLR.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Blood_TexProg
9.82E+03
Mus musculus
Blood
genomic DNA
TCRspleenblood_Processed__PB_M4_PD1Pos_TexProg.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Spleen_TexInt
1.40E+05
Mus musculus
Spleen
genomic DNA
TCRspleenblood_Processed__SP_M4_PD1Pos_TexInt.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Spleen_TexKLR
1.40E+05
Mus musculus
Spleen
genomic DNA
TCRspleenblood_Processed__SP_M4_PD1Pos_TexKLR.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Spleen_TexProg
2.61E+04
Mus musculus
Spleen
genomic DNA
TCRspleenblood_Processed__SP_M4_PD1Pos_TexProg62LNeg.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Spleen_TexProgCD62L
3.55E+04
Mus musculus
Spleen
genomic DNA
TCRspleenblood_Processed__SP_M4_PD1Pos_TexProg62LPos.tsv
 


(2) Blood vs Spleen 28 dpi
TCR_28dpi_Spleen_TexTerm
4.74E+04
Mus musculus
Spleen
genomic DNA
TCRspleenblood_Processed__SP_M4_PD1Pos_TexTerm.tsv
 


(3) Pre-Rechallenge
PreRechal_Arm_Tcm_bulkTCR
2.04E+05
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330202_pep.csv
TCRrechalenge_Processed_330210_pep.csv


(3) Pre-Rechallenge
PreRechal_Arm_Tem_bulkTCR
2.60E+04
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330203_pep.csv
TCRrechalenge_Processed_330211_pep.csv


(3) Pre-Rechallenge
PreRechal_Arm_tTem_bulkTCR
3.69E+04
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330204_pep.csv
TCRrechalenge_Processed_330212_pep.csv


(3) Pre-Rechallenge
PreRechal_Cl13_Tcm_bulkTCR
1.85E+04
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330198_pep.csv
TCRrechalenge_Processed_330206_pep.csv


(3) Pre-Rechallenge
PreRechal_Cl13_Tem_bulkTCR
1.36E+05
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330201_pep.csv
TCRrechalenge_Processed_330209_pep.csv


(3) Pre-Rechallenge
PreRechal_Cl13_TexProg_bulkTCR
7.50E+03
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330199_pep.csv
TCRrechalenge_Processed_330207_pep.csv


(3) Pre-Rechallenge
PreRechal_Cl13_TexTerm_bulkTCR
4.80E+03
Mus musculus
Spleen
RNA
TCRrechalenge_Processed_330200_pep.csv
TCRrechalenge_Processed_330208_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_Tcm_aPDL1
2.99E+06
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341724_pep.csv
TCRlongtiduinal_Processed_341725_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_Tem2_aPDL1
1.01E+06
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341736_pep.csv
TCRlongtiduinal_Processed_341752_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_Tem1_aPDL1
2.57E+06
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341738_pep.csv
TCRlongtiduinal_Processed_341754_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_TexProg_aPDL1
2.68E+05
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341729_pep.csv
TCRlongtiduinal_Processed_341745_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_TexTerm_aPDL1
3.23E+05
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341740_pep.csv
TCRlongtiduinal_Processed_341756_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_Tcm_PBS
3.15E+06
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341731_pep.csv
TCRlongtiduinal_Processed_341747_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_Tem2_PBS
8.44E+05
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341760_pep.csv
TCRlongtiduinal_Processed_341766_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_Tem1_PBS
2.08E+06
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341734_pep.csv
TCRlongtiduinal_Processed_341750_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_TexProg_PBS
1.70E+05
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341758_pep.csv
TCRlongtiduinal_Processed_341764_pep.csv


(4) Longtudinal
LongitudinalTCR_100dpi_TexTerm_PBS
1.28E+05
Mus musculus
Spleen
RNA
TCRlongtiduinal_Processed_341732_pep.csv
TCRlongtiduinal_Processed_341748_pep.csv


(4) Longtudinal
LongitudinalTCR_21dpi_TexInt_aPDL1
9.49E+04
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341741_pep.csv
TCRlongtiduinal_Processed_341757_pep.csv


(4) Longtudinal
LongitudinalTCR_21dpi_TexKLR_aPDL1
1.59E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341735_pep.csv
TCRlongtiduinal_Processed_341751_pep.csv


(4) Longtudinal
LongitudinalTCR_21dpi_TexProg_aPDL1
2.73E+04
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341759_pep.csv
TCRlongtiduinal_Processed_341769_pep.csv


(4) Longtudinal
LongitudinalTCR_21dpi_TexInt_PBS
9.69E+04
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341737_pep.csv
TCRlongtiduinal_Processed_341753_pep.csv


(4) Longtudinal
LongitudinalTCR_21dpi_TexKLR_PBS
1.11E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341739_pep.csv
TCRlongtiduinal_Processed_341755_pep.csv


(4) Longtudinal
LongitudinalTCR_21dpi_TexProg_PBS
2.49E+04
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341733_pep.csv
TCRlongtiduinal_Processed_341749_pep.csv


(4) Longtudinal
LongitudinalTCR_35dpi_TexInt_aPDL1
6.27E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341767_pep.csv
TCRlongtiduinal_Processed_341774_pep.csv


(4) Longtudinal
LongitudinalTCR_35dpi_TexKLR_aPDL1
4.15E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341762_pep.csv
TCRlongtiduinal_Processed_341771_pep.csv


(4) Longtudinal
LongitudinalTCR_35dpi_TexProg_aPDL1
1.21E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341768_pep.csv
TCRlongtiduinal_Processed_341775_pep.csv


(4) Longtudinal
LongitudinalTCR_35dpi_TexInt_PBS
3.61E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341763_pep.csv
TCRlongtiduinal_Processed_341772_pep.csv


(4) Longtudinal
LongitudinalTCR_35dpi_TexKLR_PBS
3.56E+05
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341765_pep.csv
TCRlongtiduinal_Processed_341773_pep.csv


(4) Longtudinal
LongitudinalTCR_35dpi_TexProg_PBS
7.63E+04
Mus musculus
Blood
RNA
TCRlongtiduinal_Processed_341761_pep.csv
TCRlongtiduinal_Processed_341770_pep.csv