Sustained AhR activity programs memory fate of early effector CD8+ T cells

doi:10.1073/pnas.2317658121

. 2024 Mar 12;121(11):e2317658121.

doi: 10.1073/pnas.2317658121. Epub 2024 Mar 4.

Sustained AhR activity programs memory fate of early effector CD8⁺ T cells

Huafeng Zhang^#^{1

2}, Zhuoshun Yang^#^{3

4}, Wu Yuan^#⁴, Jincheng Liu^#⁴, Xiao Luo¹, Qian Zhang¹, Yonggang Li⁵, Jie Chen⁶, Yabo Zhou⁶, Jiadi Lv⁶, Nannan Zhou⁶, Jingwei Ma⁷, Ke Tang⁴, Bo Huang^{4

6}

Affiliations

¹ Department of Pathology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
² Institute of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
³ Institute of Biomedical Research, Department of Infectious Diseases, Regulatory Mechanism and Targeted Therapy for Liver Cancer Shiyan Key Laboratory, Hubei Provincial Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.
⁴ Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
⁵ Hubei Provincial Key Laboratory for Applied Toxicology, Hubei Provincial Center for Disease Control and Prevention, Wuhan 430079, China.
⁶ Department of Immunology and National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
⁷ Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

^# Contributed equally.

PMID: 38437537
PMCID: PMC10945852
DOI: 10.1073/pnas.2317658121

Sustained AhR activity programs memory fate of early effector CD8⁺ T cells

Huafeng Zhang et al. Proc Natl Acad Sci U S A. 2024.

. 2024 Mar 12;121(11):e2317658121.

doi: 10.1073/pnas.2317658121. Epub 2024 Mar 4.

Authors

Affiliations

¹ Department of Pathology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
² Institute of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
³ Institute of Biomedical Research, Department of Infectious Diseases, Regulatory Mechanism and Targeted Therapy for Liver Cancer Shiyan Key Laboratory, Hubei Provincial Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.
⁴ Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
⁵ Hubei Provincial Key Laboratory for Applied Toxicology, Hubei Provincial Center for Disease Control and Prevention, Wuhan 430079, China.
⁶ Department of Immunology and National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
⁷ Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

^# Contributed equally.

PMID: 38437537
PMCID: PMC10945852
DOI: 10.1073/pnas.2317658121

Abstract

Identification of mechanisms that program early effector T cells to either terminal effector T (T_eff) or memory T (T_m) cells has important implications for protective immunity against infections and cancers. Here, we show that the cytosolic transcription factor aryl hydrocarbon receptor (AhR) is used by early T_eff cells to program memory fate. Upon antigen engagement, AhR is rapidly up-regulated via reactive oxygen species signaling in early CD8⁺ T_eff cells, which does not affect the effector response, but is required for memory formation. Mechanistically, activated CD8⁺ T cells up-regulate HIF-1α to compete with AhR for HIF-1β, leading to the loss of AhR activity in HIF-1α^high short-lived effector cells, but sustained in HIF-1α^low memory precursor effector cells (MPECs) with the help of autocrine IL-2. AhR then licenses CD8⁺ MPECs in a quiescent state for memory formation. These findings partially resolve the long-standing issue of how T_eff cells are regulated to differentiate into memory cells.

Keywords: CD8+ memory T cells; HIF-1α; aryl hydrocarbon receptor; transcription factor.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

**Fig. 1.**
AhR does not affect CD8⁺ effector cells but is required for memory formation. (A–D) Experimental scheme, WT (wild-type) and AhR^−/− (AhR knockout) mice were infected with 1 × 10⁴ Lm-OVA (A). (B) frequencies of splenic CD8⁺ H-2 Kb-OVA⁺ T cells were analyzed 7 d after infection (n = 6 mice per group). (C) The number of CD8⁺ H-2 Kb-OVA⁺ T cells were analyzed 7 d after infection (n = 6 mice per group). (D) Splenocytes were stimulated with the SIINFEKL peptide for 3 h in the presence of brefeldin A. Intracellular levels of IFN-γ, TNF-α, and Granzyme B in CD8⁺ H-2 Kb-OVA⁺ T cells were measured (n = 6 mice per group). (E and F) Experimental scheme, WT and AhR^−/− mice were infected with 1 × 10⁴ Lm-OVA, followed by rechallenge of with 1 × 10⁶ Lm-OVA 30 d later (E). (E) Splenic and liver bacterial burden was measured from day 2 re-infected mice (n = 6 mice per group). (G) C57BL/6 mice were transferred with 1 × 10⁵ OT-I CD8⁺ T cells transduced with *shNC* or *shAhR* retroviruses, followed by infection of Lm-OVA. Frequencies of CD8⁺GFP⁺ OT-I T_m cells in the spleen, lymph node, blood, and lung were analyzed 30 d after infection (n = 6 mice per group). (H) The number of splenic CD8⁺GFP⁺ OT-I T_m cells was analyzed. (I) Frequencies of CD62L⁺ of CD8⁺ T_m cells in the spleen were analyzed. (J) CD62L⁺ T_m cells were enumerated 30 d after infection. (K) CD62L expression was analyzed in IL-15 differentiated WT or AhR^−/− T_m cells (n = 3). Data are representative of ≥2 independent experiments (mean ± SD). P values were calculated by two-tailed unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, no significant.

**Fig. 2.**
TCR signaling-activated ROS up-regulates AhR in early CD8⁺ T_eff cells. (A–C) Naive CD8⁺ T cells were stimulated with anti-CD3, anti-CD28, and IL-2 (n = 3). (A and B) The expressions of AhR were analyzed by western blot (A) and qPCR (B). (C) mRNA levels of *Cyp1a1* and *Cyp1b1* were analyzed. (D) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺ CD8⁺ OT-I T cells, followed by infection of Lm-OVA. Splenic CD45.1⁺ CD8⁺ OT-I T cells were isolated from infected mice. The expressions of *Cyp1a1* and *Cyp1b1* were measured (n = 3). (E) Naive CD8⁺ T cells were treated as (A), and the Nrf2 level was analyzed by western blot (n = 3). (F and G) the expression (F) and activation (G) of AhR were analyzed in ML385-treated CD8⁺ T cells (n = 3). (H) Naive CD8⁺ T cells were treated as (A), and the cellular ROS levels were analyzed by flow cytometry (n = 3). (I and J) The expression (I) and activation (J) of AhR were analyzed in NAC-treated CD8⁺ T cells (n = 3). Data are representative of ≥3 independent experiments (mean ± SD). P values were calculated using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

**Fig. 3.**
Activated AhR constitutively exists in MPECs but transiently in SLECs. (A and B) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺ CD8⁺ OT-I T cells, followed by infection of Lm-OVA. AhR expression in CD8⁺ MPEC (CD127^highKLRG-1^low) and SLEC (CD127^lowKLRG-1^high) from the spleen at day 10 after infection (n = 3). (C) Immunofluorescence microscopy images show the expression of AhR in CD8⁺ MPEC and SLEC (Scale bar: 10 μm.) (n = 3). (D) mRNA levels of *Cyp1a1* and *Cyp1b1* in splenic MPEC and SLEC were analyzed (n = 3). (E and F) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺ CD8⁺ OT-I T cells, followed by infection of Lm-OVA. Western blot and flow cytometry analysis of AhR expression in CD8⁺CD45.1⁺ OT-I T cells (n = 3). (G–I) C57BL/6 mice were transferred with 1 × 10⁵ OT-I CD8⁺ T cells transduced with *shNC* or *shAhR* retroviruses, followed by infection of Lm-OVA (n = 5 mice per group). (G) Proportions of MPEC and SLEC were analyzed at day 10 post Lm-OVA infection. (H) CD62L expression on OVA-specific MPEC was measured. (I) mRNA levels of *Bcl6*, *Tcf7,* and *Lef1* in *shNC*- and *shAhR* transduced MPEC. Data are representative of ≥3 independent experiments (mean ± SD). P values were calculated using unpaired two-tailed Student’s t test (A, D, G, H, and I) or one-way ANOVA (F). **P < 0.01, ***P < 0.001, ****P < 0.0001.

**Fig. 4.**
SLECs use HIF-1α to inactivate AhR during the early effector stage. (A) Naive CD8⁺ T cells were stimulated with anti-CD3, anti-CD28, and IL-2. HIF-1α expressions were analyzed by western blot (n = 3). (B and C) Naive CD8⁺ T cells were stimulated with anti-CD3, anti-CD28, and IL-2 for 24 h. Flow cytometric analysis of 2-NBDG staining separated cell populations (n = 3). (B) Western blot analysis of AhR and HIF-1α in 2-NBDG^high and 2-NBDG^low CD8⁺ T_eff cells (n = 3). (C) mRNA levels of *Cyp1a1* and *Cyp1b1* in 2-NBDG^high and 2-NBDG^low CD8⁺ T_eff cells (n = 3). (D) mRNA levels of *Cyp1a1* and *Cyp1b1* in CD8⁺ T_eff cells cultured in normoxic and hypoxic conditions (n = 3). (E) mRNA levels of *Cyp1a1* and *Cyp1b1* in CD8⁺ T_eff cells treated with KC7F2 (n = 3). (F) Number of IL-15-induced T_m cells cultured in normoxic and hypoxic conditions (n = 3). (G and H) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺ CD8⁺ OT-I T cells, followed by infection of Lm-OVA and treatment of KC7F2 and CoCl2. The AhR expression (G) and activation (H) of CD45.1⁺CD8⁺ OT-I T_eff cells were analyzed 10 d post infection. (I) The cell lysate of CD45.1⁺ CD8⁺ OT-I T_eff cells was immunoprecipitated with anti-HIF-1β and blotted with AhR and HIF-1α. (J) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺ CD8⁺ OT-I T cells, followed by infection of Lm-OVA. HIF-1α expression in splenic MPEC and SLEC was analyzed 10 d after infection. Data are representative of ≥3 independent experiments (mean ± SD). P values were calculated using unpaired two-tailed Student’s t test (C–F) or one-way ANOVA (H). **P < 0.01, ***P < 0.001, ****P < 0.0001.

**Fig. 5.**
MPECs maintain AhR activity via IL-2. (A and B) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺ CD8⁺ OT-I T cells, followed by infection of Lm-OVA. (A) AhR expression in IL-2^high and IL-2^low CD8⁺ OT-I T_eff cells was analyzed 7 d after infection (n = 3). (B) mRNA levels of *Cyp1a1* and *Cyp1b1* in IL-2^high and IL-2^low CD8⁺ T cells (n = 3). (C) Percentage of IL-2-producing OT-I T cells in MPEC and SLEC (n = 6 mice per group). (D) 5HTP abundance in MPEC and SLEC was analyzed by LC/MS (n = 3). (E and F) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺CD45.2⁺CD8⁺ OT-I T cells, followed by infection of Lm-OVA 24 h later. Mice were then treated with STAT5-IN-1 (STAT5 inhibitor) or CP-10188 (Tph1 inhibitor). (E) Proportions of MPEC and SLEC were measured at day 10 post Lm-OVA infection (n = 5 mice per group). (F) mRNA levels of *Cyp1a1* and *Cyp1b1* in MPEC were analyzed by qPCR. (G and H) MPEC were purified from 10-d infected mice, followed by IL-2 neutralizing antibody treatment for 24 h in vitro (n = 3). (G) 5HTP abundance was measured by LC/MS. (H) mRNA levels of *Cyp1a1* and *Cyp1b1* were analyzed. Data are representative of ≥3 independent experiments (mean ± SD). P values were calculated using unpaired two-tailed Student’s t test (A–D, G, and H) and or one-way ANOVA (E and F). *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

**Fig. 6.**
AhR licenses CD8⁺ MPECs in a quiescent state for memory formation. (A and B) C57BL/6 mice (CD45.2⁺) were transferred with 1 × 10⁵ CD45.1⁺CD8⁺ OT-I T cells, followed by infection of Lm-OVA 24 h later. Mice were then treated with CH223191 every day. MPECs were purified from 10-d infected mice, and ECAR (A) and OCR (B) of MPEC were analyzed (n = 3). (C) MPECs were purified from 10-d infected mice, and then cultured in ¹³C-glucose medium for 24 h. ¹³C-labeled lactate and pyruvate were measured by LC/MS (n = 3). (D) Mice were treated as (A), the expressions of HK2 in MPECs were analyzed by western blot (n = 3). (E) ChIP-qPCR analysis was performed with an antibody to AhR and HIF-1α-promotor-specific primer (n = 3). (F) NIH3T3 cells were cotransfected with a HIF-1α promoter-luciferase reporter PGL4.10 and Flag-AhR plasmid for 24 h, followed by analysis of luciferase activity (n = 3). (G) mRNA levels of *HIF-1*α in CD8⁺ T_eff cells in the presence or absence of FICZ were analyzed (n = 3). (H) Western blot analysis of AhR expression in CD8⁺ T_eff cells treated with CHX (10 μg/mL) in the presence or absence of FICZ (10 μM) (n = 3). (I) Activated CD8⁺ T_eff cells were treated with FICZ or Kyn for 48 h. The cell lysates were immunoprecipitated with an anti-HIF-1α antibody and immunoblotted with anti-ubiquitination antibody (n = 3). (J) HIF-1α in AhR^−/− Tm cells was knocked out through the electroporation of Cas9-sgRNA ribonucleoproteins (RNPs). The expressions of AhR and HK2 in WT, AhR^−/− or AhR^−/− HIF-1α-KO T_m cells were analyzed by western blot (n = 3). (K) MPECs purified from 10-d infected mice were assayed for cell cycle analysis. Data are representative of ≥3 independent experiments (mean ± SD). P values were calculated using unpaired two-tailed Student’s t test (A–E, G, and K) and or one-way ANOVA (F). *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

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References

1. Bousso P., T-cell activation by dendritic cells in the lymph node: Lessons from the movies. Nat. Rev. Immunol. 8, 675–684 (2008). - PubMed
1. Smith-Garvin J. E., Koretzky G. A., Jordan M. S., T cell activation. Annu. Rev. Immunol. 27, 591–619 (2009). - PMC - PubMed
1. Mueller S. N., Gebhardt T., Carbone F. R., Heath W. R., Memory T cell subsets, migration patterns, and tissue residence. Annu. Rev. Immunol. 31, 137–161 (2013). - PubMed
1. Kaech S. M., Wherry E. J., Ahmed R., Effector and memory T-cell differentiation: Implications for vaccine development. Nat. Rev. Immunol. 2, 251–262 (2002). - PubMed
1. Sallusto F., Geginat J., Lanzavecchia A., Central memory and effector memory T cell subsets: Function, generation, and maintenance. Annu. Rev. Immunol. 22, 745–763 (2004). - PubMed

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[1] Bousso P., T-cell activation by dendritic cells in the lymph node: Lessons from the movies. Nat. Rev. Immunol. 8, 675–684 (2008). - PubMed

[2] Bousso P., T-cell activation by dendritic cells in the lymph node: Lessons from the movies. Nat. Rev. Immunol. 8, 675–684 (2008). - PubMed

[3] Smith-Garvin J. E., Koretzky G. A., Jordan M. S., T cell activation. Annu. Rev. Immunol. 27, 591–619 (2009). - PMC - PubMed

[4] Smith-Garvin J. E., Koretzky G. A., Jordan M. S., T cell activation. Annu. Rev. Immunol. 27, 591–619 (2009). - PMC - PubMed

[5] Mueller S. N., Gebhardt T., Carbone F. R., Heath W. R., Memory T cell subsets, migration patterns, and tissue residence. Annu. Rev. Immunol. 31, 137–161 (2013). - PubMed

[6] Mueller S. N., Gebhardt T., Carbone F. R., Heath W. R., Memory T cell subsets, migration patterns, and tissue residence. Annu. Rev. Immunol. 31, 137–161 (2013). - PubMed

[7] Kaech S. M., Wherry E. J., Ahmed R., Effector and memory T-cell differentiation: Implications for vaccine development. Nat. Rev. Immunol. 2, 251–262 (2002). - PubMed

[8] Kaech S. M., Wherry E. J., Ahmed R., Effector and memory T-cell differentiation: Implications for vaccine development. Nat. Rev. Immunol. 2, 251–262 (2002). - PubMed

[9] Sallusto F., Geginat J., Lanzavecchia A., Central memory and effector memory T cell subsets: Function, generation, and maintenance. Annu. Rev. Immunol. 22, 745–763 (2004). - PubMed

[10] Sallusto F., Geginat J., Lanzavecchia A., Central memory and effector memory T cell subsets: Function, generation, and maintenance. Annu. Rev. Immunol. 22, 745–763 (2004). - PubMed

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Sustained AhR activity programs memory fate of early effector CD8⁺ T cells

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Sustained AhR activity programs memory fate of early effector CD8⁺ T cells

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