Gfi1 controls the formation of effector-like CD8+ T cells during chronic infection and cancer

Abstract

During chronic infection and tumor progression, CD8⁺ T cells lose their effector functions and become exhausted. These exhausted CD8⁺ T cells are heterogeneous and comprised of progenitors that give rise to effector-like or terminally-exhausted cells. The precise cues and mechanisms directing subset formation are incompletely understood. Here, we show that growth factor independent-1 (Gfi1) is dynamically regulated in exhausted CD8⁺ T cells. During chronic LCMV Clone 13 infection, a previously under-described Ly108⁺CX₃CR1⁺ subset expresses low levels of Gfi1 while other established subsets have high expression. Ly108⁺CX₃CR1⁺ cells possess distinct chromatin profiles and represent a transitory subset that develops to effector-like and terminally-exhausted cells, a process dependent on Gfi1. Similarly, Gfi1 in tumor-infiltrating CD8⁺ T cells is required for the formation of terminally differentiated cells and endogenous as well as anti-CTLA-induced anti-tumor responses. Taken together, Gfi1 is a key regulator of the subset formation of exhausted CD8⁺ T cells.

Fig. 1. Gfi1 is dynamically regulated in exhausted CD8⁺ T cell subsets during chronic viral infection.

A Gfi1tdTomato reporter mice were infected with LCMV Cl-13, followed by the assessment of Gfi1tdTomato expression on Days 5 (n = 6), 8 (n = 6), 15 (n = 6) and 30 (n = 8) post-infection. B–E Identification of Day 30 exhausted CD8⁺ T cell subsets based on CD44, PD-1, TCF1, Ly108 (B), Ki67, and CD69 expression (C). D Quantification of Ki67 and CD69 expressions as in (C) (n = 9). E. Expression and quantification of Ly108 and CX₃CR1 on the 4 exhausted subsets (n = 9). F GMFIs of PD-1, 2B4, LAG-3, Tim-3, CX₃CR1, CD44, TCF1, T-bet, Eomes, and TOX in the 4 exhausted subsets (progenitor, Ly108⁺CX₃CR1⁺, effector-like, and Term-Exh) (n = 9–10). G Expression and quantification of CXCR6 in the 4 exhausted subsets (n = 5). H Gfi1tdTomato expression on the 4 exhausted CD8⁺ T cell subsets (n = 8). Data were pooled from 2 independent experiments and depicted with mean ± s.e.m. Data in A were analyzed by one-way ANOVA; data in (D–H) were analyzed by repeated one-way ANOVA. Holm-Sidak’s post hoc test was used for all analysis. Source data are provided as a Source Data file.

Fig. 2. Ly108⁺CX₃CR1⁺ cells possess unique chromatin accessibility and transcriptional features, some of which are shared with effector-like cells.

Day 30 exhausted GP33⁺ CD8⁺ T cell subsets were sorted based on Ly108 and CX₃CR1 expression for ATAC-Seq (A–G) and RNA-Seq (H). A Statistically significant and differentially accessible chromatin regions (DACRs) with > 1.2 log2 fold change from pairwise comparisons were shown. Prog = Progenitor, Eff-like = Effector-like, and Term-Exh = Terminally-exhausted. B Promoter and Enhancer distribution of more and less accessible DACRs in the Ly108⁺CX₃CR1⁺ cells versus progenitors. C Accessibility profiles of DACRS from (B) were visualized in effector-like and Term-Exh cells. D Venn diagram depicting the intersection of DACRs in Ly108⁺CX₃CR1⁺ cells versus progenitors with those in effector-like cells versus progenitors (Left), with the bar graph showing the unique DACRs in effector-like cells (Middle), and heatmap visualization of these regions in progenitors, Ly108⁺CX₃CR1⁺, and effector-like cells (Right). E Bar graph depicting distribution of DACRs in Term-Exh versus Ly108⁺CX₃CR1⁺ cells. F^. Genomic track of accessibility at the Mef2c locus in the 4 exhausted subsets. G De novo HOMER motif analysis of DACRs from Ly108⁺CX₃CR1⁺ versus progenitor cells. H Heatmap visualization of select transcription factors among the 4 exhausted subsets. ATAC- and RNA-seq data were from 2 and 3-4 independent replicates, respectively. Each replicate was a pooled sample of 3-5 mice.

Fig. 3. CD8⁺ T cell intrinsic Gfi1 is required for the formation of effector and Term-Exh subsets.

A, B GP33⁺ responses were evaluated in WT and Gfi1^cKO mice on Days 15 and 30 post LCMV Cl-13 infection. Frequencies (A) and cell numbers (B) of subsets (in A, n = 11 and 16 for WT mice, 12 and 13 for Gfi1^cKO mice on Day 15 and 30, respectively. For (B), n = 11 for WT mice at both time points, and n = 12 and 7 for Gfi1^cKO mice on Day 15 and 30, respectively). C, D. Frequencies (C) and cell numbers (D) of GP33 + subsets in CD4-depleted WT (n = 8 for both time points) and Gfi1^cKO mice (n = 7 and 12, respectively, for Day 15 and 30). E CD45.1⁺ mice reconstituted with WT (n = 7) or Gfi1^cKO (n = 9) CD45.2⁺ BM cells (chimeric mice) were infected with LCMV Cl-13. GP33⁺ CD8⁺ T cell responses in CD45.1⁺ and CD45.2⁺ compartments were analyzed on Day 30 post-infection. F Sorted exhausted subsets (Progenitors: n = 6; Ly108⁺CX₃CR1⁺: n = 7; effector-like: n = 6; Term-Exh: n = 6) from Day 30 LCMV Cl-123-infected Gfi1tdTomato reporter mice were adoptively transferred into naïve CD45.1⁺ recipients, followed by LCMV Cl-13 infection on the next day. Progenies of adoptively transferred cells were characterized for their expression of Ly108 and CX₃CR1 on Day 8 post-infection. Cell counts of subsets were shown by the scatter dot plot. Data (mean ± s.e.m) were pooled from 2-4 independent experiments and analyzed by two-way ANOVA with Holm-Sidak’s post-hoc test. Source data are provided as a Source Data file.

Fig. 4. Gfi1 controls the chromatin accessibility and transcriptomic programs required for the formation of exhausted effector-like and Term-Exh CD8⁺ T cells.

Exhausted GP33⁺ subsets from WT and Gfi1^cKO mice were sorted on Day 30 post-LCMV Cl-13 infection for ATAC-seq (A–E) or RNA-seq (F). A Differentially accessible chromatin regions (DACRs) between Gfi1^cKO versus WT progenitors and Ly108⁺CX₃CR1⁺ cells, with a cutoff of 1.2 log2 fold. B DACRs of progenitor versus Ly108⁺CX₃CR1⁺ subsets in WT and Gfi1^cKO cells. C Heatmap visualization of DACRs between Gfi1^cKO and WT cells were shown. k-means clusters with numbers of DACRs indicated in the parentheses for each cluster. D Genomic tracks of accessibility at Mef2c and Lef1 loci in WT (progenitor, Ly108⁺CX₃CR1⁺, effector-like) as well as Gfi1^cKO (progenitors, Ly108⁺CX₃CR1⁺) subsets. E De novo HOMER motif analysis of DACRs from Gfi1^cKO versus WT Ly108⁺CX₃CR1⁺ cells. F Heatmap visualization of select transcripts for transcription factors in WT and Gfi1^cKO GP33⁺ subsets. ATAC- and RNA-seq data were from 2 and 3-4 independent replicates. Each replicate was a pooled sample of 3-5 mice.

Fig. 5. Gfi1 is necessary for the accumulation of terminally differentiated Tim-3^hi tumor infiltrating CD8⁺ T cells in late-stage tumors.

A Tumor growth dynamics of Gfi1tdTomato reporter mice subcutaneously inoculated with 1 × 10⁵ MB49 cells. B Identification of TCF1^hi progenitor and Tim-3^hi effector CD8⁺ TILs with total cell counts and ratios of Tim-3^hi to TCF1^hi on Days 7 (n = 11), 14 (n = 13), and 22 (n = 11) post-inoculation. C Histograms showing the expression of 2B4, LAG-3, PD-1, and TOX in TCF1^hi (progenitor) and Tim-3^hi TILs. D Flow plots of Gfi1tdTomato expression in progenitor and Tim-3^hi CD8⁺ TILs at different time points, with the frequencies for subsets on Day 14 shown in the scatter dot plot (n = 6). E Flow plots showing expression of Gfi1tdTomato with TCF1, TOX, Eomes, and T-bet in progenitor cells with their GMFIs depicted (n = 5–7). F Tumor growth dynamics and masses on Days 7, 12, and 21 in WT (n = 10, 16, & 17) and Gfi1^cKO (n = 11, 16 & 18) mice post the inoculation of MB49 cells. G Flow plots showing the abundance of Tim-3^hi and TCF1^hi progenitor TILs in tumors from WT (n = 10, 18, & 17) and Gfi1^cKO (n = 10, 16, & 18) mice as well as their ratios on Day 7, 12, and 21 post-tumor inoculation. H Distribution of Tim-3^hiT-bet^hi and Tim-3^hiT-bet^lo effector TILs as well as Tim-3^loT-bet^int progenitor TILs (Left) and their frequencies of TOX^hi and Eomes^hi cells (Right) on Day 21 tumors from WT (n = 8) and Gfi1^cKO (n = 10) mice. I Perforin and granzyme (B) production by WT (n = 6) and Gfi1^cKO (n = 7) CD8⁺ TILs. Data (mean ± s.e.m.) were pooled results from 2–4 independent experiments, with each dot indicating an individual mouse. Data were analyzed by two-way ANOVA (B Left, D, F–H), one-way ANOVA (B, Right), repeated one-way ANOVA (E), and unpaired two-tail t test (I). Holm-Sidak’s post hoc test was used unless otherwise stated. Source data are provided as a Source Data file.

Fig. 6. Gfi1 expression in T cells is required for anti-CTLA-4 efficacy.

WT and Gfi1^cKO mice inoculated with MB49 cells were treated with anti-CTLA-4 on Days 5, 8, and 11 post-inoculation. Tumor growth dynamics was depicted in the line graph (A, n = 4). Tumors were harvested to evaluate the infiltration of CD4⁺ and CD8⁺ T cells (B, n = 7 for untreated and 8 for treated groups of both WT and Gfi1^cKO mice) and IFN-γ production by CD8⁺ TILs (C, n = 7 for untreated WT mice, = 8 for treated WT mice, = 5 for untreated Gfi1^cKO mice, and = 6 for treated Gfi1^cKO mice). D WT (n = 4 in the untreated group and = 5 in the treated group) and Gfi1^cKO (n = 7 in the untreated group and 5 in the treated group) mice bearing palpable MC38 bladder tumors were treated with or without anti-CTLA-4 on Days 5, 8, and 11 post-tumor inoculation. Tumor growth dynamics was shown in the line graph. E, F Tumors were harvested to evaluate the infiltration of CD4⁺ and CD8⁺ T cells (E n = 8 for untreated WT and 9 for treated WT; n = 11 for untreated Gfi1^cKO and 12 for treated Gfi1^cKO) and IFN-γ production by CD8⁺ TILs (F n = 9 for untreated and treated WT; n = 11 for untreated Gfi1^cKO and 12 for treated Gfi1^cKO). Data (mean ± s.e.m.) were pooled results from 2-3 independent experiments, with each dot denoting a mouse. Two-way ANOVA with Holm-Sidak post hoc test was used. Source data are provided as a Source Data file.