Immune Netw.  2020 Feb;20(1):e2. 10.4110/in.2020.20.e2.

Re-defining T-Cell Exhaustion: Subset, Function, and Regulation

• Im SJ
• Ha SJ

• KMID: 2471042
• DOI: http://doi.org/10.4110/in.2020.20.e2

Abstract

Acute viral infection or vaccination generates highly functional memory CD8 T cells following the Ag resolution. In contrast, persistent antigenic stimulation in chronic viral infection and cancer leads to a state of T-cell dysfunction termed T-cell exhaustion. We and other have recently identified a novel subset of exhausted CD8 T cells that act as stem cells for maintaining virus-specific CD8 T cells in a mouse model of chronic lymphocytic choriomeningitis virus infection. This stem cell-like CD8 T-cell subset has been also observed in both mouse and human tumor models. Most importantly, in both chronic viral infection and tumor models, the proliferative burst of Ag-specific CD8 T cells driven by PD-1-directed immunotherapy comes exclusively from this stem cell-like CD8 T-cell subset. Therefore, a better understanding of the mechanisms how CD8 T-cell subsets are regulated during chronic viral infection and cancer is required to improve the current immunotherapies that restore the function of exhausted CD8 T cells. In this review, we discuss the differentiation of virus-specific CD8 T cells during chronic viral infection, the characteristics and function of CD8 T-cell subsets, and the therapeutic intervention of PD-1-directed immunotherapy in cancer.

Keyword

T-cell exhaustion; PD-1; Immunotherapy; Stem cell-like CD8 T-cell subset

MeSH Terms

Animals
Humans
Immunotherapy
Lymphocytic choriomeningitis virus
Memory
Mice
Stem Cells
T-Lymphocyte Subsets
T-Lymphocytes*
Vaccination

Figure

• 

  Figure 1. Differentiation pathway of Ag-specific CD8 T cells during chronic viral infection. (A) Upon acute viral infection, naïve CD8 T cells activate and differentiate into memory precursors (MP) and terminal effectors (TE). Terminal effectors die by AICD and memory precursors survive and become memory CD8 T cells (M) after the clearance of viral infection. Similarly, naïve CD8 T cells (N) are activated and differentiate into a stem cell-like subset (SL) and terminally differentiated cells (TD) upon chronic viral infection. Analogous to terminal effectors, terminally differentiated cells also die by AICD. Different from the acute infection, sustained antigenic stimulation during chronic viral infection resulted in the continual differentiation of stem cell-like CD8 T cells into terminally differentiated CD8 T cells. (B) TCF1+ CXCR5+ stem cell-like CD8 T cells maintain their population by slow self-renewal. Upon antigenic stimulation, these stem cell-like CD8 T cells differentiate into CD101− Tim-3+ transitory population. This CD101− Tim-3+ subset possesses in vivo proliferative potential after antigenic stimulation, can differentiate further into terminally differentiated CD101+ Tim-3+ CD8 T cells, and contributes to viral control with the highest cytolytic activity. With upregulation of CD101, terminally differentiated CD101+ Tim-3+ CD8 T cells lost in vivo proliferative potential and possessed impaired cytolytic function.

• 

  Figure 2. Migration of CD8 T-cell subsets during chronic viral infection. Both stem cell-like and terminally differentiated CD8 T cells are circulating after the onset of viral infection. After the establishment of T-cell exhaustion, stem cell-like cells become resident and mainly reside in the lymphoid organs, especially in the white pulp of the spleen. A fraction of the transitory CD101− Tim-3+ CD8 T cells circulate via the blood and migrate other inflamed tissues corresponding to the expression of a fractalkine receptor CX3CR1. In the red pulp of the spleen and non-lymphoid tissues, CD101− Tim-3+ CD8 T-cell subset differentiates into terminally differentiated CD101+ Tim-3+ CD8 T cells and become resident.

• 

  Figure 3. Transcriptional network to regulate differentiation of CD8 T cells during chronic viral infection. TCR stimulation by persistent Ags initiates a set of transcription factors, including NFAT2 followed by BATF and IRF4. TOX is also activated by NFAT2 via separate route. TCR-driven factors generally contribute to PD-1 upregulation in both stem cell-like and terminally differentiated CD8 T cells undergoing exhaustion process. Although IRF4 together with BATF directly represses TCF1 expression and upregulates BLIMP1, highly upregulated TCF1 in stem cell-like subset increases BCL6 expression, which also accelerates E2A expression. This TCF1-BCL6-E2A axis altogether or independently induces the genes related to stem cell-like CD8 T cell signature, such as Cxcr5, Sell, and Il7r. In the other hand, terminally differentiated CD8 T cells shows a typical feature with high level of BLIMP1 and low level of TCF1. Repressed level of TCF1 are not enough to upregulate BCL6, which allows the induction of ID2 and subsequent suppression of E2A. Both BLIMP1 and ID2 contribute to the enhancement of Gzmb expression, leading to the differentiation of terminally differentiated CD8 T cells. Straight red lines with arrow symbol indicate stimulation or activation. Dotted blue lines with block symbol indicate inhibition or suppression. Thickness of lines defines a strength of signal. Straight black lines with arrow symbol and orange lines with arrow symbol exhibit a binding to upstream of the corresponding genes and their transcription, respectively.

• 

  Figure 4. Proposed mechanism of PD-1-directed immunotherapy during chronic viral infection and cancer. (A) After the establishment of T-cell exhaustion, PD-1/PD-L1 axis plays a role in 2 different ways. First, PD-1/PD-L1 interaction inhibits the proliferation and differentiation of stem cell-like CD8 T cells into the more differentiated CD8 T-cell subsets. CD28 is a major target for PD-1-mediated suppression. Next, PD-L1 on infected cells or tumor cells impairs the cytolytic function and cytokine production of PD-1+ Ag-specific differentiated CD8 T-cell subsets. (B) In the absence of PD-1-mediagted signals, the proliferation and differentiation of stem cell-like CD8 T cells into transitory and terminally differentiated CD8 T cells are accelerated and this happens in a CD28/B7-dependent manner. The increased population of transitory subset showing the highest anti-viral/tumor activity is critical for the viral clearance and tumor regression. In terms of the quality, the cytolytic function and cytokine production of transitory and terminally differentiated CD8 T-cell subsets might be increased after PD-1 blockade.

Reference

References

1. Hashimoto M, Kamphorst AO, Im SJ, Kissick HT, Pillai RN, Ramalingam SS, Araki K, Ahmed R. CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annu Rev Med. 2018; 69:301–318.

2. Sharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015; 348:56–61.

3. McLane LM, Abdel-Hakeem MS, Wherry EJ. CD8 T cell exhaustion during chronic viral infection and cancer. Annu Rev Immunol. 2019; 37:457–495.

4. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, Ahmed R. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998; 188:2205–2213.

5. Gallimore A, Glithero A, Godkin A, Tissot AC, Plückthun A, Elliott T, Hengartner H, Zinkernagel R. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J Exp Med. 1998; 187:1383–1393.

6. Velu V, Titanji K, Zhu B, Husain S, Pladevega A, Lai L, Vanderford TH, Chennareddi L, Silvestri G, Freeman GJ, et al. Enhancing SIV-specific immunity in vivo by PD-1 blockade. Nature. 2009; 458:206–210.

7. Goepfert PA, Bansal A, Edwards BH, Ritter GD Jr, Tellez I, McPherson SA, Sabbaj S, Mulligan MJ. A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon. J Virol. 2000; 74:10249–10255.

8. Shankar P, Russo M, Harnisch B, Patterson M, Skolnik P, Lieberman J. Impaired function of circulating HIV-specific CD8 (+) T cells in chronic human immunodeficiency virus infection. Blood. 2000; 96:3094–3101.

9. Kostense S, Ogg GS, Manting EH, Gillespie G, Joling J, Vandenberghe K, Veenhof EZ, van Baarle D, Jurriaans S, Klein MR, et al. High viral burden in the presence of major HIV-specific CD8⁺ T cell expansions: evidence for impaired CTL effector function. Eur J Immunol. 2001; 31:677–686.

10. Lechner F, Wong DK, Dunbar PR, Chapman R, Chung RT, Dohrenwend P, Robbins G, Phillips R, Klenerman P, Walker BD. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med. 2000; 191:1499–1512.

11. Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, Walker B, Sullivan J, Phillips R, Pape GR, et al. Sustained dysfunction of antiviral CD8⁺ T lymphocytes after infection with hepatitis C virus. J Virol. 2001; 75:5550–5558.

12. Ye B, Liu X, Li X, Kong H, Tian L, Chen Y. T-cell exhaustion in chronic hepatitis B infection: current knowledge and clinical significance. Cell Death Dis. 2015; 6:e1694.

13. Mumprecht S, Schürch C, Schwaller J, Solenthaler M, Ochsenbein AF. Programmed death 1 signaling on chronic myeloid leukemia-specific T cells results in T-cell exhaustion and disease progression. Blood. 2009; 114:1528–1536.

14. Schietinger A, Philip M, Krisnawan VE, Chiu EY, Delrow JJ, Basom RS, Lauer P, Brockstedt DG, Knoblaugh SE, Hämmerling GJ, et al. Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity. 2016; 45:389–401.

15. Lee PP, Yee C, Savage PA, Fong L, Brockstedt D, Weber JS, Johnson D, Swetter S, Thompson J, Greenberg PD, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999; 5:677–685.

16. Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, Rosenberg SA. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009; 114:1537–1544.

17. Fourcade J, Kudela P, Sun Z, Shen H, Land SR, Lenzner D, Guillaume P, Luescher IF, Sander C, Ferrone S, et al. PD-1 is a regulator of NY-ESO-1-specific CD8+ T cell expansion in melanoma patients. J Immunol. 2009; 182:5240–5249.

18. Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P, Beck A, Miller A, Tsuji T, Eppolito C, Qian F, Lele S, Shrikant P, et al. Tumor-infiltrating NY-ESO-1-specific CD8⁺ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A. 2010; 107:7875–7880.

19. Zhang Y, Huang S, Gong D, Qin Y, Shen Q. Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8⁺ T lymphocytes in human non-small cell lung cancer. Cell Mol Immunol. 2010; 7:389–395.

20. Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ. Coregulation of CD8⁺ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol. 2009; 10:29–37.

21. Gandhi MK, Lambley E, Duraiswamy J, Dua U, Smith C, Elliott S, Gill D, Marlton P, Seymour J, Khanna R. Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8⁺ T-cell function in Hodgkin lymphoma patients. Blood. 2006; 108:2280–2289.

22. Fourcade J, Sun Z, Benallaoua M, Guillaume P, Luescher IF, Sander C, Kirkwood JM, Kuchroo V, Zarour HM. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8⁺ T cell dysfunction in melanoma patients. J Exp Med. 2010; 207:2175–2186.

23. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006; 439:682–687.

24. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature. 2006; 443:350–354.

25. Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, Adams WC, Precopio ML, Schacker T, Roederer M, Douek DC, et al. PD-1 is a regulator of virus-specific CD8⁺ T cell survival in HIV infection. J Exp Med. 2006; 203:2281–2292.

26. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS, et al. Upregulation of PD-1 expression on HIV-specific CD8⁺ T cells leads to reversible immune dysfunction. Nat Med. 2006; 12:1198–1202.

27. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, Stankevich E, Pons A, Salay TM, McMiller TL, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol. 2010; 28:3167–3175.

28. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012; 366:2443–2454.

29. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012; 366:2455–2465.

30. Hargadon KM, Johnson CE, Williams CJ. Immune checkpoint blockade therapy for cancer: an overview of FDA-approved immune checkpoint inhibitors. Int Immunopharmacol. 2018; 62:29–39.

31. Page DB, Postow MA, Callahan MK, Allison JP, Wolchok JD. Immune modulation in cancer with antibodies. Annu Rev Med. 2014; 65:185–202.

32. Pauken KE, Wherry EJ. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015; 36:265–276.

33. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, Shan Q, Hale JS, Lee J, Nasti TH, et al. Defining CD8⁺ T cells that provide the proliferative burst after PD-1 therapy. Nature. 2016; 537:417–421.

34. He R, Hou S, Liu C, Zhang A, Bai Q, Han M, Yang Y, Wei G, Shen T, Yang X, et al. Follicular CXCR5- expressing CD8⁺ T cells curtail chronic viral infection. Nature. 2016; 537:412–428.

35. Utzschneider DT, Charmoy M, Chennupati V, Pousse L, Ferreira DP, Calderon-Copete S, Danilo M, Alfei F, Hofmann M, Wieland D, et al. T cell factor 1-expressing memory-like CD8⁺ T cells sustain the immune response to chronic viral infections. Immunity. 2016; 45:415–427.

36. Wu T, Ji Y, Moseman EA, Xu HC, Manglani M, Kirby M, Anderson SM, Handon R, Kenyon E, Elkahloun A, et al. The TCF1-Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness. Sci Immunol. 2016; 1:eaai8593.

37. Blank CU, Haining WN, Held W, Hogan PG, Kallies A, Lugli E, Lynn RC, Philip M, Rao A, Restifo NP, et al. Defining ‘T cell exhaustion'. Nat Rev Immunol. 2019; 19:665–674.

38. Kallies A, Zehn D, Utzschneider DT. Precursor exhausted T cells: key to successful immunotherapy? Nat Rev Immunol. 2020; 20:128–136.

39. Wherry EJ, Barber DL, Kaech SM, Blattman JN, Ahmed R. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc Natl Acad Sci U S A. 2004; 101:16004–16009.

40. Shin H, Blackburn SD, Blattman JN, Wherry EJ. Viral antigen and extensive division maintain virus-specific CD8 T cells during chronic infection. J Exp Med. 2007; 204:941–949.

41. Utzschneider DT, Legat A, Fuertes Marraco SA, Carrié L, Luescher I, Speiser DE, Zehn D. T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion. Nat Immunol. 2013; 14:603–610.

42. Speiser DE, Utzschneider DT, Oberle SG, Münz C, Romero P, Zehn D. T cell differentiation in chronic infection and cancer: functional adaptation or exhaustion? Nat Rev Immunol. 2014; 14:768–774.

43. Hudson WH, Gensheimer J, Hashimoto M, Wieland A, Valanparambil RM, Li P, Lin JX, Konieczny BT, Im SJ, Freeman GJ, et al. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1⁺ stem-like CD8⁺ T cells during chronic infection. Immunity. 2019; 51:1043–1058. e4.

44. Calabro S, Liu D, Gallman A, Nascimento MS, Yu Z, Zhang TT, Chen P, Zhang B, Xu L, Gowthaman U, et al. Differential intrasplenic migration of dendritic cell subsets tailors adaptive immunity. Cell Reports. 2016; 16:2472–2485.

45. Eisenbarth SC. Dendritic cell subsets in T cell programming: location dictates function. Nat Rev Immunol. 2019; 19:89–103.

46. Cyster JG. Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs. J Exp Med. 1999; 189:447–450.

47. Yamazaki C, Sugiyama M, Ohta T, Hemmi H, Hamada E, Sasaki I, Fukuda Y, Yano T, Nobuoka M, Hirashima T, et al. Critical roles of a dendritic cell subset expressing a chemokine receptor, XCR1. J Immunol. 2013; 190:6071–6082.

48. Kretzer NM, Theisen DJ, Tussiwand R, Briseño CG, Grajales-Reyes GE, Wu X, Durai V, Albring J, Bagadia P, Murphy TL, et al. RAB43 facilitates cross-presentation of cell-associated antigens by CD8α⁺ dendritic cells. J Exp Med. 2016; 213:2871–2883.

49. Greyer M, Whitney PG, Stock AT, Davey GM, Tebartz C, Bachem A, Mintern JD, Strugnell RA, Turner SJ, Gebhardt T, et al. T cell help amplifies innate signals in CD8⁺ DCs for optimal CD8⁺ T cell priming. Cell Reports. 2016; 14:586–597.

50. Dorner BG, Dorner MB, Zhou X, Opitz C, Mora A, Güttler S, Hutloff A, Mages HW, Ranke K, Schaefer M, et al. Selective expression of the chemokine receptor XCR1 on cross-presenting dendritic cells determines cooperation with CD8⁺ T cells. Immunity. 2009; 31:823–833.

51. Kroczek RA, Henn V. The role of XCR1 and its ligand XCL1 in antigen cross-presentation by murine and human dendritic cells. Front Immunol. 2012; 3:14.

52. Helft J, Manicassamy B, Guermonprez P, Hashimoto D, Silvin A, Agudo J, Brown BD, Schmolke M, Miller JC, Leboeuf M, et al. Cross-presenting CD103⁺ dendritic cells are protected from influenza virus infection. J Clin Invest. 2012; 122:4037–4047.

53. Borrow P, Evans CF, Oldstone MB. Virus-induced immunosuppression: immune system-mediated destruction of virus-infected dendritic cells results in generalized immune suppression. J Virol. 1995; 69:1059–1070.

54. Mueller SN, Matloubian M, Clemens DM, Sharpe AH, Freeman GJ, Gangappa S, Larsen CP, Ahmed R. Viral targeting of fibroblastic reticular cells contributes to immunosuppression and persistence during chronic infection. Proc Natl Acad Sci U S A. 2007; 104:15430–15435.

55. Mueller SN, Vanguri VK, Ha SJ, West EE, Keir ME, Glickman JN, Sharpe AH, Ahmed R. PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice. J Clin Invest. 2010; 120:2508–2515.

56. Im SJ, Konieczny BT, Hudson WH, Masopust D, Ahmed R. PD-1+ stemlike CD8 T cells are resident in lymphoid tissues during persistent LCMV infection. Proc Natl Acad Sci U S A. 2020. 201917298.

57. Hamon P, Loyher PL, Baudesson de Chanville C, Licata F, Combadière C, Boissonnas A. CX3CR1-dependent endothelial margination modulates Ly6C ^high monocyte systemic deployment upon inflammation in mice. Blood. 2017; 129:1296–1307.

58. Mionnet C, Buatois V, Kanda A, Milcent V, Fleury S, Lair D, Langelot M, Lacoeuille Y, Hessel E, Coffman R, et al. CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung. Nat Med. 2010; 16:1305–1312.

59. Zhou X, Yu S, Zhao DM, Harty JT, Badovinac VP, Xue HH. Differentiation and persistence of memory CD8⁺ T cells depend on T cell factor 1. Immunity. 2010; 33:229–240.

60. Zhou X, Xue HH. Cutting edge: generation of memory precursors and functional memory CD8⁺ T cells depends on T cell factor-1 and lymphoid enhancer-binding factor-1. J Immunol. 2012; 189:2722–2726.

61. Gullicksrud JA, Li F, Xing S, Zeng Z, Peng W, Badovinac VP, Harty JT, Xue HH. Differential requirements for Tcf1 long isoforms in CD8⁺ and CD4⁺ T cell responses to acute viral infection. J Immunol. 2017; 199:911–919.

62. Jeannet G, Boudousquié C, Gardiol N, Kang J, Huelsken J, Held W. Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory. Proc Natl Acad Sci U S A. 2010; 107:9777–9782.

63. Choi YS, Gullicksrud JA, Xing S, Zeng Z, Shan Q, Li F, Love PE, Peng W, Xue HH, Crotty S. LEF-1 and TCF-1 orchestrate T _FH differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6. Nat Immunol. 2015; 16:980–990.

64. Xu L, Cao Y, Xie Z, Huang Q, Bai Q, Yang X, He R, Hao Y, Wang H, Zhao T, et al. The transcription factor TCF-1 initiates the differentiation of T _FH cells during acute viral infection. Nat Immunol. 2015; 16:991–999.

65. Wu JQ, Seay M, Schulz VP, Hariharan M, Tuck D, Lian J, Du J, Shi M, Ye Z, Gerstein M, et al. Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line. PLoS Genet. 2012; 8:e1002565.

66. Tunyaplin C, Shaffer AL, Angelin-Duclos CD, Yu X, Staudt LM, Calame KL. Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J Immunol. 2004; 173:1158–1165.

67. Ichii H, Sakamoto A, Hatano M, Okada S, Toyama H, Taki S, Arima M, Kuroda Y, Tokuhisa T. Role for Bcl-6 in the generation and maintenance of memory CD8⁺ T cells. Nat Immunol. 2002; 3:558–563.

68. Ichii H, Sakamoto A, Kuroda Y, Tokuhisa T. Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8⁺ T cells. J Immunol. 2004; 173:883–891.

69. Johnston RJ, Poholek AC, DiToro D, Yusuf I, Eto D, Barnett B, Dent AL, Craft J, Crotty S. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science. 2009; 325:1006–1010.

70. Yang CY, Best JA, Knell J, Yang E, Sheridan AD, Jesionek AK, Li HS, Rivera RR, Lind KC, D'Cruz LM, et al. The transcriptional regulators Id2 and Id3 control the formation of distinct memory CD8⁺ T cell subsets. Nat Immunol. 2011; 12:1221–1229.

71. Shaw LA, Bélanger S, Omilusik KD, Cho S, Scott-Browne JP, Nance JP, Goulding J, Lasorella A, Lu LF, Crotty S, et al. Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation. Nat Immunol. 2016; 17:834–843.

72. Miyazaki M, Rivera RR, Miyazaki K, Lin YC, Agata Y, Murre C. The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells. Nat Immunol. 2011; 12:992–1001.

73. Jadhav RR, Im SJ, Hu B, Hashimoto M, Li P, Lin JX, Leonard WJ, Greenleaf WJ, Ahmed R, Goronzy JJ. Epigenetic signature of PD-1+ TCF1+ CD8 T cells that act as resource cells during chronic viral infection and respond to PD-1 blockade. Proc Natl Acad Sci U S A. 2019; 116:14113–14118.

74. Pauken KE, Sammons MA, Odorizzi PM, Manne S, Godec J, Khan O, Drake AM, Chen Z, Sen DR, Kurachi M, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science. 2016; 354:1160–1165.

75. Sen DR, Kaminski J, Barnitz RA, Kurachi M, Gerdemann U, Yates KB, Tsao HW, Godec J, LaFleur MW, Brown FD, et al. The epigenetic landscape of T cell exhaustion. Science. 2016; 354:1165–1169.

76. Ghoneim HE, Fan Y, Moustaki A, Abdelsamed HA, Dash P, Dogra P, Carter R, Awad W, Neale G, Thomas PG, et al. De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell. 2017; 170:142–157. e19.

77. Alfei F, Kanev K, Hofmann M, Wu M, Ghoneim HE, Roelli P, Utzschneider DT, von Hoesslin M, Cullen JG, Fan Y, et al. TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection. Nature. 2019; 571:265–269.

78. Khan O, Giles JR, McDonald S, Manne S, Ngiow SF, Patel KP, Werner MT, Huang AC, Alexander KA, Wu JE, et al. TOX transcriptionally and epigenetically programs CD8⁺ T cell exhaustion. Nature. 2019; 571:211–218.

79. Yao C, Sun HW, Lacey NE, Ji Y, Moseman EA, Shih HY, Heuston EF, Kirby M, Anderson S, Cheng J, et al. Single-cell RNA-seq reveals TOX as a key regulator of CD8⁺ T cell persistence in chronic infection. Nat Immunol. 2019; 20:890–901.

80. Mann TH, Kaech SM. Tick-TOX, it's time for T cell exhaustion. Nat Immunol. 2019; 20:1092–1094.

81. Nayar R, Enos M, Prince A, Shin H, Hemmers S, Jiang JK, Klein U, Thomas CJ, Berg LJ. TCR signaling via Tec kinase ITK and interferon regulatory factor 4 (IRF4) regulates CD8⁺ T-cell differentiation. Proc Natl Acad Sci U S A. 2012; 109:E2794–E2802.

82. Nayar R, Schutten E, Bautista B, Daniels K, Prince AL, Enos M, Brehm MA, Swain SL, Welsh RM, Berg LJ. Graded levels of IRF4 regulate CD8⁺ T cell differentiation and expansion, but not attrition, in response to acute virus infection. J Immunol. 2014; 192:5881–5893.

83. Man K, Gabriel SS, Liao Y, Gloury R, Preston S, Henstridge DC, Pellegrini M, Zehn D, Berberich-Siebelt F, Febbraio MA, et al. Transcription factor IRF4 promotes CD8+ T cell exhaustion and limits the development of memory-like T cells during chronic infection. Immunity. 2017; 47:1129–1141. e5.

84. Blackburn SD, Shin H, Freeman GJ, Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by α PD-L1 blockade. Proc Natl Acad Sci U S A. 2008; 105:15016–15021.

85. Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, Sasmal DK, Huang J, Kim JM, Mellman I, et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017; 355:1428–1433.

86. Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, Konieczny BT, Daugherty CZ, Koenig L, Yu K, et al. Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017; 355:1423–1427.

87. Zinselmeyer BH, Heydari S, Sacristán C, Nayak D, Cammer M, Herz J, Cheng X, Davis SJ, Dustin ML, McGavern DB. PD-1 promotes immune exhaustion by inducing antiviral T cell motility paralysis. J Exp Med. 2013; 210:757–774.

88. Miller BC, Sen DR, Al Abosy R, Bi K, Virkud YV, LaFleur MW, Yates KB, Lako A, Felt K, Naik GS, et al. Subsets of exhausted CD8⁺ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol. 2019; 20:326–336.

89. Siddiqui I, Schaeuble K, Chennupati V, Fuertes Marraco SA, Calderon-Copete S, Pais Ferreira D, Carmona SJ, Scarpellino L, Gfeller D, Pradervand S, et al. Intratumoral Tcf1⁺ PD-1⁺ CD8⁺ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity. 2019; 50:195–211. e10.

90. Brummelman J, Mazza EM, Alvisi G, Colombo FS, Grilli A, Mikulak J, Mavilio D, Alloisio M, Ferrari F, Lopci E, et al. High-dimensional single cell analysis identifies stem-like cytotoxic CD8⁺ T cells infiltrating human tumors. J Exp Med. 2018; 215:2520–2535.

91. Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell. 2018; 175:998–1013. e20.

92. Jansen CS, Prokhnevska N, Master VA, Sanda MG, Carlisle JW, Bilen MA, Cardenas M, Wilkinson S, Lake R, Sowalsky AG, et al. An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature. 2019; 576:465–470.

93. Yost KE, Satpathy AT, Wells DK, Qi Y, Wang C, Kageyama R, McNamara KL, Granja JM, Sarin KY, Brown RA, et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med. 2019; 25:1251–1259.

94. Scott AC, Dündar F, Zumbo P, Chandran SS, Klebanoff CA, Shakiba M, Trivedi P, Menocal L, Appleby H, Camara S, et al. TOX is a critical regulator of tumour-specific T cell differentiation. Nature. 2019; 571:270–274.

95. Wang X, He Q, Shen H, Xia A, Tian W, Yu W, Sun B. TOX promotes the exhaustion of antitumor CD8⁺ T cells by preventing PD1 degradation in hepatocellular carcinoma. J Hepatol. 2019; 71:731–741.

96. Seo H, Chen J, González-Avalos E, Samaniego-Castruita D, Das A, Wang YH, López-Moyado IF, Georges RO, Zhang W, Onodera A, et al. TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8⁺ T cell exhaustion. Proc Natl Acad Sci U S A. 2019; 116:12410–12415.

97. Kim CG, Jang M, Kim Y, Leem G, Kim KH, Lee H, Kim TS, Choi SJ, Kim HD, Han JW, et al. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol. 2019; 4:eaay0555.

98. Chen J, López-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, Yoshimura A, Scott-Browne JP, Rao A. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019; 567:530–534.

99. Shin B, Kress RL, Kramer PA, Darley-Usmar VM, Bellis SL, Harrington LE. Effector CD4 T cells with progenitor potential mediate chronic intestinal inflammation. J Exp Med. 2018; 215:1803–1812.

100. Karmaus PW, Chen X, Lim SA, Herrada AA, Nguyen TM, Xu B, Dhungana Y, Rankin S, Chen W, Rosencrance C, et al. Metabolic heterogeneity underlies reciprocal fates of T _H17 cell stemness and plasticity. Nature. 2019; 565:101–105.

101. Elsaesser H, Sauer K, Brooks DG. IL-21 is required to control chronic viral infection. Science. 2009; 324:1569–1572.

102. Yi JS, Du M, Zajac AJ. A vital role for interleukin-21 in the control of a chronic viral infection. Science. 2009; 324:1572–1576.

103. Fröhlich A, Kisielow J, Schmitz I, Freigang S, Shamshiev AT, Weber J, Marsland BJ, Oxenius A, Kopf M. IL-21R on T cells is critical for sustained functionality and control of chronic viral infection. Science. 2009; 324:1576–1580.

104. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, Boesteanu AC, Wang Y, O'Connor RS, Hwang WT, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018; 24:563–571.

105. Klebanoff CA, Crompton JG, Leonardi AJ, Yamamoto TN, Chandran SS, Eil RL, Sukumar M, Vodnala SK, Hu J, Ji Y, et al. Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy. JCI Insight. 2017; 2:e95103.

106. Klebanoff CA, Scott CD, Leonardi AJ, Yamamoto TN, Cruz AC, Ouyang C, Ramaswamy M, Roychoudhuri R, Ji Y, Eil RL, et al. Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest. 2016; 126:318–334.

107. Crompton JG, Sukumar M, Restifo NP. Uncoupling T-cell expansion from effector differentiation in cell-based immunotherapy. Immunol Rev. 2014; 257:264–276.

108. Hinrichs CS, Borman ZA, Cassard L, Gattinoni L, Spolski R, Yu Z, Sanchez-Perez L, Muranski P, Kern SJ, Logun C, et al. Adoptively transferred effector cells derived from naive rather than central memory CD8⁺ T cells mediate superior antitumor immunity. Proc Natl Acad Sci U S A. 2009; 106:17469–17474.

109. Jin HT, Anderson AC, Tan WG, West EE, Ha SJ, Araki K, Freeman GJ, Kuchroo VK, Ahmed R. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci U S A. 2010; 107:14733–14738.

110. West EE, Jin HT, Rasheed AU, Penaloza-Macmaster P, Ha SJ, Tan WG, Youngblood B, Freeman GJ, Smith KA, Ahmed R. PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells. J Clin Invest. 2013; 123:2604–2615.

111. Penaloza-MacMaster P, Kamphorst AO, Wieland A, Araki K, Iyer SS, West EE, O'Mara L, Yang S, Konieczny BT, Sharpe AH, et al. Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection. J Exp Med. 2014; 211:1905–1918.

112. Ha SJ, Mueller SN, Wherry EJ, Barber DL, Aubert RD, Sharpe AH, Freeman GJ, Ahmed R. Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection. J Exp Med. 2008; 205:543–555.

113. Peng W, Lizée G, Hwu P. Blockade of the PD-1 pathway enhances the efficacy of adoptive cell therapy against cancer. OncoImmunology. 2013; 2:e22691.

114. Duraiswamy J, Kaluza KM, Freeman GJ, Coukos G. Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors. Cancer Res. 2013; 73:3591–3603.

115. Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y, Park S, Javinal V, Chiu H, Irving B, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8⁺ T cell effector function. Cancer Cell. 2014; 26:923–937.

116. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010; 207:2187–2194.

117. Anderson AC, Joller N, Kuchroo VK. Lag-3, Tim-3, and TIGIT: co-inhibitory receptors with specialized functions in immune regulation. Immunity. 2016; 44:989–1004.

118. Brooks DG, Ha SJ, Elsaesser H, Sharpe AH, Freeman GJ, Oldstone MB. IL-10 and PD-L1 operate through distinct pathways to suppress T-cell activity during persistent viral infection. Proc Natl Acad Sci U S A. 2008; 105:20428–20433.

119. Angelosanto JM, Blackburn SD, Crawford A, Wherry EJ. Progressive loss of memory T cell potential and commitment to exhaustion during chronic viral infection. J Virol. 2012; 86:8161–8170.

120. Ribeiro SP, Milush JM, Cunha-Neto E, Kallas EG, Kalil J, Somsouk M, Hunt PW, Deeks SG, Nixon DF, SenGupta D. The CD8⁺ memory stem T cell (T _SCM) subset is associated with improved prognosis in chronic HIV-1 infection. J Virol. 2014; 88:13836–13844.