61
Lecture: T Cell Activation and Regulation Mark Anderson, MD,PhD UCSF Diabetes Center [email protected] 415-502-8052
Lecture: T Cell Activation and Regulation
Mark Anderson, MD,PhD UCSF Diabetes Center
[email protected] 415-502-8052
Lecture Overview • Anatomical concerns
• “The rules of engagement” – T cell activation requires more than the generation of
foreign peptide-self MHC complexes on APC’s…..
• T cell signaling
• Two signal model and co-stimulation (bulk of the lecture)
• Putting it all together
Functional responses of T lymphocytes
From: Abbas & Lichtman, Cellular & Molecular Immunology, W. B. Saunders, 2003
Kinetics of a T cell response
Signals for T cell activation
• Antigen recognition – Regulated movement of signaling receptors
and adhesion molecules at (immune synapse)
• Costimulators (second signals)
• Cytokines – Produced by APCs or T cells – Stimulate T cell expansion and differentiation
into effector cells
Antigen recognition by T cells
• Each T cell sees an MHC molecule and bound peptide – Dual recognition determines specificity and MHC restriction
• Each T cell sees very few (1-3) residues of the peptide antigen – T cells distinguish between diverse microbes based on
recognition of few amino-acids • The affinity of TCR-antigen interactions is low
– Kd on the order of 10-5 to 10-6
– Because T cells are selected by recognition of self MHC in the thymus (the only MHC they can encounter during their lives)
– T cell-APC contacts need to be stabilized by other molecules
• The activation of T cells may require multiple or prolonged TCR-antigen interactions – T cell receptors and signaling proteins assemble in the
synapse
T cells first “stick” to APC’s using cell adhesion molecules
T cells use co-receptors for antigen recognition
Formation of the immunological synapse
Regulated way of bringing together key signaling molecules
Functions of the immune synapse
• Promote signaling • Terminate signaling: recruitment of
phosphatases, ubiquitin ligases, inhibitory receptors to the site of the TCR complex
• Direct effector molecules to the relevant target: cytokines, CD40L, perforin, etc
Menu F B
TCR signalling is dynamically regulated
• Csk and CD45 are continually phosphorylating and dephosphorylating Lck
• Phosphorylation of Lck inhibits its activation acitivity
• When TCR stimulation occurs PAG1 is dephosphorylated and Csk is released thus removing the inhibitory phosphorylation of Lck
TCR signalling is dynamically regulated (cont).
• Cbl family proteins are Ubiquitin Ligases that tag phosphorylated adaptors for destruction in the lysosome
• When Cbl-b is knocked out, mice develop a severe autoimmune syndrome highlighting the importance of the termination of signaling
Initial responses to activation • #1 rule- key cytokine the T cell needs
to make is IL-2
• Proliferation. Mostly dependent on IL-2 through an autocrine pathway.
• Other cytokines, cytokine receptors will also get produced and lead to effector T cell development (lecture upcoming…)
Figure 8-20
The Two-Signal Model of T-cell Activation
TCRMHC
CD4 or CD8
1
2
DC T cell
COSTIMULATION
Two signal requirement for lymphocyte activation
• Naïve lymphocytes need two signals to initiate responses
• Signal 1: antigen recognition – Ensures that the response is antigen-specific
• Signal 2: microbes or substances produced during innate immune responses to microbes – Ensures that the immune system responds to microbes
and not to harmless antigenic substances – Second signals for T cells are “costimulators” on APCs
and cytokines produced by APCs
Marc Jenkins and Ronald Schwartz in the late 80’s :The first definitive experimental demonstration that TCR engagement alone was insufficient for T cell activation.
Proliferative response of T cell clones (pigeon cytochrome c peptide 81-104 presented by I-Ek)to normal or ECDI(chemical crosslinker)-fixed peptide-pulsed APCs
ECDI-treated APCs fail to stimulate proliferation by normal T cell clones :Not the result of extensive modification of the MHC class II moleculeECDI treatment inactivated an accessory (costimulatory) function of the APC
Jenkins M.K., and Schwartz R.H. J. Exp. Med. 165:302-319, 1987.
The Experimental Evidence of Co-stimulation
The B7:CD28 families
From Abbas, Lichtman and Pillai. Cellular and Molecular Immunology 6th edition, Elsevier, 2007
Activation of T cells by peptide-pulsed DCs in vivo: requirement for B7
DO11 T cells (Ova-specific TCR transgenic) labeled with CFSE and transferred into normal or B7-knockout recipients ----> immunized with Ova peptide-pulsed cultured dendritic cells from normal or B7-knockout recipients ---> response of DO11 cells assayed
0
100000
200000
300000
400000
500000
0 0.001 0.01 0.1 10
100000
200000
300000
400000
500000
0 0.001 0.01 0.1 1
Naïve CD4 T cells Memory CD4 T cells
Prol
ifer
atio
n (C
PM)
Antigen (µg/ml)
Memory cells are less dependent on B7 costimulation than are naïve T cells
wild type (normal; positive control)
B7.1/2-/- None (negative control)
APCs
B7:CD28 dependence of T cells
• Initiation of T cell responses requires B7:CD28
• Dependence on B7-CD28: – Naïve > Th1 > Th2 > memory – CD4 > CD8 – Regulatory T cells
CD28 Signals through its cytoplasmic tail SH2-binding sites. A major downstream signaling
enzyme is PI3 kinase.
TCR and CD28 Signaling cooperate to help promote IL-2 production
Proliferation IL-2 (transcription, mRNA stabilization) IL-2R up-regulation ↑G1 cell cycle kinases ↓Cell cycle inhibitor p27Kip Survival Bcl-xL Effector function ↑ CD40-L, OX-40, 41BB, ICOS ↑ cytokines expression ↑ cytotoxic molecules
The major effects of CD28-mediated costimulation are to augment and sustain T cell responses initiated by antigen receptor signal by promoting T-cell survival and enabling cytokines to initiate T cell clonal expansion and differentiation.
Major effects of CD28-mediated costimulation in T cells
Major effects of CD28-mediated costimulation in T cells Article
Mitochondrial Priming by CD28Ramon I. Klein Geltink,1 David O’Sullivan,1 Mauro Corrado,1 Anna Bremser,2,3 Michael D. Buck,1 Joerg M. Buescher,1
Elke Firat,4 Xuekai Zhu,5 Gabriele Niedermann,4,6 George Caputa,1 Beth Kelly,1 Ursula Warthorst,2 Anne Rensing-Ehl,2
Ryan L. Kyle,1 Lana Vandersarren,7,8 Jonathan D. Curtis,1 Annette E. Patterson,1 Simon Lawless,1 Katarzyna Grzes,1
Jing Qiu,1 David E. Sanin,1 Oliver Kretz,9,10 Tobias B. Huber,10,11,12 Sophie Janssens,7,8 Bart N. Lambrecht,7,8
Angelika S. Rambold,2,3 Edward J. Pearce,1,13 and Erika L. Pearce1,14,*1Department of Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany2Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, 79106 Freiburg, Germany3Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany4Department of Radiation Oncology, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany5Shanghai Institute for Advanced Immunochemical Studies (SIAIS), Shanghai Tech University, 201210 Shanghai, People’s Republic of China6German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany7Laboratory of Immunoregulation and Mucosal Immunology, VIB Center for Inflammation Research, 9052 Ghent, Belgium8Department of Internal Medicine, Ghent University, 9000 Ghent, Belgium9Department of Neuroanatomy, University of Freiburg, 79104 Freiburg, Germany10III. Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany11Department of Medicine IV, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany12BIOSS Center for Biological Signaling Studies and Center for Systems Biology (ZBSA), Albert-Ludwigs-University, 79104 Freiburg,Germany13Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany14Lead Contact*Correspondence: [email protected]://dx.doi.org/10.1016/j.cell.2017.08.018
SUMMARY
T cell receptor (TCR) signaling without CD28 canelicit primary effector T cells, but memory T cellsgenerated during this process are anergic, failing torespond to secondary antigen exposure. We showthat, upon T cell activation, CD28 transiently pro-motes expression of carnitine palmitoyltransferase1a (Cpt1a), an enzyme that facilitates mitochondrialfatty acid oxidation (FAO), before the first cell divi-sion, coinciding with mitochondrial elongation andenhanced spare respiratory capacity (SRC). micro-RNA-33 (miR33), a target of thioredoxin-interactingprotein (TXNIP), attenuates Cpt1a expression in theabsence of CD28, resulting in cells that thereafterare metabolically compromised during reactivationor periods of increased bioenergetic demand. EarlyCD28-dependent mitochondrial engagement isneeded for T cells to remodel cristae, develop SRC,and rapidly produce cytokines upon restimulation—cardinal features of protective memory T cells.Our data show that initial CD28 signals during T cellactivation prime mitochondria with latent metaboliccapacity that is essential for future T cell responses.
INTRODUCTION
CD28 is the receptor for B7 molecules (CD80 and CD86), whichare expressed on activated antigen presenting cells, and pro-
vide essential signals for full T cell activation. Over the years,it has become clear that CD28 signals do not act solely toamplify T cell receptor (TCR) signaling, but control a wide rangeof processes, including the cell cycle, epigenetic modifications,metabolism, and post-translational modifications (Esenstenet al., 2016). Nevertheless, a complete understanding of thebiology of CD28 is lacking. Because CD28 and its family mem-bers are targets of current and developing immunotherapies,understanding how these accessory receptors regulate T cellfunction is of broad interest and clinical importance (Esenstenet al., 2016).A prevailing model in immunology is that CD28 promotes the
glycolytic flux needed for full effector T (TE) cell activation, dif-ferentiation, and proliferation (Frauwirth et al., 2002; Jacobset al., 2008; MacIver et al., 2013). However, in vivo, T cellsdo not always require this initial CD28 costimulation to mountprimary responses or to form long-lasting antigen-specificT cells but need it specifically to develop into protective mem-ory (TM) cells, which ‘‘remember’’ past infections and respondrobustly to secondary antigen challenge (Borowski et al.,2007; Kundig et al., 1996; Mittrucker et al., 2001; Villegaset al., 1999). How initial CD28 signals contribute to the genera-tion of protective TM cells remains unclear. We have shown thatlong-lived TM cells utilize fatty acid oxidation (FAO) and main-tain fused mitochondria with tight cristae and spare respiratorycapacity (SRC) (Buck et al., 2016; van der Windt et al., 2012),the reserve energy generating capacity in the mitochondriabeyond the basal state. This metabolic phenotype facilitatestheir rapid recall function (van der Windt et al., 2013). We there-fore speculated that CD28 primes mitochondria during theinitial phase of T cell activation with the metabolic capacityimportant for future recall of TM cells.
Cell 171, 385–397, October 5, 2017 ª 2017 Elsevier Inc. 385
Article
Mitochondrial Priming by CD28Ramon I. Klein Geltink,1 David O’Sullivan,1 Mauro Corrado,1 Anna Bremser,2,3 Michael D. Buck,1 Joerg M. Buescher,1
Elke Firat,4 Xuekai Zhu,5 Gabriele Niedermann,4,6 George Caputa,1 Beth Kelly,1 Ursula Warthorst,2 Anne Rensing-Ehl,2
Ryan L. Kyle,1 Lana Vandersarren,7,8 Jonathan D. Curtis,1 Annette E. Patterson,1 Simon Lawless,1 Katarzyna Grzes,1
Jing Qiu,1 David E. Sanin,1 Oliver Kretz,9,10 Tobias B. Huber,10,11,12 Sophie Janssens,7,8 Bart N. Lambrecht,7,8
Angelika S. Rambold,2,3 Edward J. Pearce,1,13 and Erika L. Pearce1,14,*1Department of Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany2Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, 79106 Freiburg, Germany3Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany4Department of Radiation Oncology, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany5Shanghai Institute for Advanced Immunochemical Studies (SIAIS), Shanghai Tech University, 201210 Shanghai, People’s Republic of China6German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany7Laboratory of Immunoregulation and Mucosal Immunology, VIB Center for Inflammation Research, 9052 Ghent, Belgium8Department of Internal Medicine, Ghent University, 9000 Ghent, Belgium9Department of Neuroanatomy, University of Freiburg, 79104 Freiburg, Germany10III. Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany11Department of Medicine IV, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany12BIOSS Center for Biological Signaling Studies and Center for Systems Biology (ZBSA), Albert-Ludwigs-University, 79104 Freiburg,Germany13Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany14Lead Contact*Correspondence: [email protected]://dx.doi.org/10.1016/j.cell.2017.08.018
SUMMARY
T cell receptor (TCR) signaling without CD28 canelicit primary effector T cells, but memory T cellsgenerated during this process are anergic, failing torespond to secondary antigen exposure. We showthat, upon T cell activation, CD28 transiently pro-motes expression of carnitine palmitoyltransferase1a (Cpt1a), an enzyme that facilitates mitochondrialfatty acid oxidation (FAO), before the first cell divi-sion, coinciding with mitochondrial elongation andenhanced spare respiratory capacity (SRC). micro-RNA-33 (miR33), a target of thioredoxin-interactingprotein (TXNIP), attenuates Cpt1a expression in theabsence of CD28, resulting in cells that thereafterare metabolically compromised during reactivationor periods of increased bioenergetic demand. EarlyCD28-dependent mitochondrial engagement isneeded for T cells to remodel cristae, develop SRC,and rapidly produce cytokines upon restimulation—cardinal features of protective memory T cells.Our data show that initial CD28 signals during T cellactivation prime mitochondria with latent metaboliccapacity that is essential for future T cell responses.
INTRODUCTION
CD28 is the receptor for B7 molecules (CD80 and CD86), whichare expressed on activated antigen presenting cells, and pro-
vide essential signals for full T cell activation. Over the years,it has become clear that CD28 signals do not act solely toamplify T cell receptor (TCR) signaling, but control a wide rangeof processes, including the cell cycle, epigenetic modifications,metabolism, and post-translational modifications (Esenstenet al., 2016). Nevertheless, a complete understanding of thebiology of CD28 is lacking. Because CD28 and its family mem-bers are targets of current and developing immunotherapies,understanding how these accessory receptors regulate T cellfunction is of broad interest and clinical importance (Esenstenet al., 2016).A prevailing model in immunology is that CD28 promotes the
glycolytic flux needed for full effector T (TE) cell activation, dif-ferentiation, and proliferation (Frauwirth et al., 2002; Jacobset al., 2008; MacIver et al., 2013). However, in vivo, T cellsdo not always require this initial CD28 costimulation to mountprimary responses or to form long-lasting antigen-specificT cells but need it specifically to develop into protective mem-ory (TM) cells, which ‘‘remember’’ past infections and respondrobustly to secondary antigen challenge (Borowski et al.,2007; Kundig et al., 1996; Mittrucker et al., 2001; Villegaset al., 1999). How initial CD28 signals contribute to the genera-tion of protective TM cells remains unclear. We have shown thatlong-lived TM cells utilize fatty acid oxidation (FAO) and main-tain fused mitochondria with tight cristae and spare respiratorycapacity (SRC) (Buck et al., 2016; van der Windt et al., 2012),the reserve energy generating capacity in the mitochondriabeyond the basal state. This metabolic phenotype facilitatestheir rapid recall function (van der Windt et al., 2013). We there-fore speculated that CD28 primes mitochondria during theinitial phase of T cell activation with the metabolic capacityimportant for future recall of TM cells.
Cell 171, 385–397, October 5, 2017 ª 2017 Elsevier Inc. 385
Article
Mitochondrial Priming by CD28Ramon I. Klein Geltink,1 David O’Sullivan,1 Mauro Corrado,1 Anna Bremser,2,3 Michael D. Buck,1 Joerg M. Buescher,1
Elke Firat,4 Xuekai Zhu,5 Gabriele Niedermann,4,6 George Caputa,1 Beth Kelly,1 Ursula Warthorst,2 Anne Rensing-Ehl,2
Ryan L. Kyle,1 Lana Vandersarren,7,8 Jonathan D. Curtis,1 Annette E. Patterson,1 Simon Lawless,1 Katarzyna Grzes,1
Jing Qiu,1 David E. Sanin,1 Oliver Kretz,9,10 Tobias B. Huber,10,11,12 Sophie Janssens,7,8 Bart N. Lambrecht,7,8
Angelika S. Rambold,2,3 Edward J. Pearce,1,13 and Erika L. Pearce1,14,*1Department of Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany2Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, 79106 Freiburg, Germany3Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany4Department of Radiation Oncology, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany5Shanghai Institute for Advanced Immunochemical Studies (SIAIS), Shanghai Tech University, 201210 Shanghai, People’s Republic of China6German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany7Laboratory of Immunoregulation and Mucosal Immunology, VIB Center for Inflammation Research, 9052 Ghent, Belgium8Department of Internal Medicine, Ghent University, 9000 Ghent, Belgium9Department of Neuroanatomy, University of Freiburg, 79104 Freiburg, Germany10III. Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany11Department of Medicine IV, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany12BIOSS Center for Biological Signaling Studies and Center for Systems Biology (ZBSA), Albert-Ludwigs-University, 79104 Freiburg,Germany13Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany14Lead Contact*Correspondence: [email protected]://dx.doi.org/10.1016/j.cell.2017.08.018
SUMMARY
T cell receptor (TCR) signaling without CD28 canelicit primary effector T cells, but memory T cellsgenerated during this process are anergic, failing torespond to secondary antigen exposure. We showthat, upon T cell activation, CD28 transiently pro-motes expression of carnitine palmitoyltransferase1a (Cpt1a), an enzyme that facilitates mitochondrialfatty acid oxidation (FAO), before the first cell divi-sion, coinciding with mitochondrial elongation andenhanced spare respiratory capacity (SRC). micro-RNA-33 (miR33), a target of thioredoxin-interactingprotein (TXNIP), attenuates Cpt1a expression in theabsence of CD28, resulting in cells that thereafterare metabolically compromised during reactivationor periods of increased bioenergetic demand. EarlyCD28-dependent mitochondrial engagement isneeded for T cells to remodel cristae, develop SRC,and rapidly produce cytokines upon restimulation—cardinal features of protective memory T cells.Our data show that initial CD28 signals during T cellactivation prime mitochondria with latent metaboliccapacity that is essential for future T cell responses.
INTRODUCTION
CD28 is the receptor for B7 molecules (CD80 and CD86), whichare expressed on activated antigen presenting cells, and pro-
vide essential signals for full T cell activation. Over the years,it has become clear that CD28 signals do not act solely toamplify T cell receptor (TCR) signaling, but control a wide rangeof processes, including the cell cycle, epigenetic modifications,metabolism, and post-translational modifications (Esenstenet al., 2016). Nevertheless, a complete understanding of thebiology of CD28 is lacking. Because CD28 and its family mem-bers are targets of current and developing immunotherapies,understanding how these accessory receptors regulate T cellfunction is of broad interest and clinical importance (Esenstenet al., 2016).A prevailing model in immunology is that CD28 promotes the
glycolytic flux needed for full effector T (TE) cell activation, dif-ferentiation, and proliferation (Frauwirth et al., 2002; Jacobset al., 2008; MacIver et al., 2013). However, in vivo, T cellsdo not always require this initial CD28 costimulation to mountprimary responses or to form long-lasting antigen-specificT cells but need it specifically to develop into protective mem-ory (TM) cells, which ‘‘remember’’ past infections and respondrobustly to secondary antigen challenge (Borowski et al.,2007; Kundig et al., 1996; Mittrucker et al., 2001; Villegaset al., 1999). How initial CD28 signals contribute to the genera-tion of protective TM cells remains unclear. We have shown thatlong-lived TM cells utilize fatty acid oxidation (FAO) and main-tain fused mitochondria with tight cristae and spare respiratorycapacity (SRC) (Buck et al., 2016; van der Windt et al., 2012),the reserve energy generating capacity in the mitochondriabeyond the basal state. This metabolic phenotype facilitatestheir rapid recall function (van der Windt et al., 2013). We there-fore speculated that CD28 primes mitochondria during theinitial phase of T cell activation with the metabolic capacityimportant for future recall of TM cells.
Cell 171, 385–397, October 5, 2017 ª 2017 Elsevier Inc. 385
αCD3
αCD3/2
8-40
-20
0
20
40
60
80
SR
C (%
Bas
al O
CR
)
NaiveαC
D3
αCD3/2
80
10
20
30
40
50
Gly
coly
tic R
eser
ve
(% B
asal
EC
AR
)
NaiveαC
D3
αCD3/2
80
10
20
30
40
50
SR
C (%
Bas
al O
CR
)
Basal
+ FCCP
Basal
+ FCCP
0
100
200
300
400
500
OC
R (p
mol
es/m
in)
0 50 100 150 550
50
100
150
200
250
Time (minutes)
OC
R (p
mol
es/m
in)
αCD3αCD3/28
550 600
αCD3
αCD3/2
80
20
40
60
80
SR
C (%
Bas
al O
CR
)
BA
E
Naive CD8+
T-cells
Prime with αCD3 + IL-2
± αCD28
+IL-2 3 days
+IL-15 3 days 8hr
Naive CD8+
T-cells TM
cells
TM cells (primed as in B)
αCD3/28 Restim Oligo FCCP R+A
IFN
-γ
CD8
Primed with αCD3
Primed with
αCD3/28
No Restim Restim
10 mM Glc
0.3 mM Glc AGR
+IL-2 3 days
Naive CD8+
T-cells
Prime with αCD3 IL-2
± αCD28
TE cells overnight
F
G
1.7 70
1.2
MFI
=139
8
Prime with αCD3 + IL-2
± αCD28
200
Basal
+ FCCP
Basal
+ FCCP
0
100
200
300
400
500
OC
R (p
mol
es/m
in)
53
MFI
=700
Bas
al
Bas
al0
20
40
60
80
EC
AR
(mpH
/min
)
Basal
0
20
40
60
80
EC
AR
(mpH
/min
)
Basal
αCD3αCD3/28
C
CD80/86
-/-W
T0
100
200
300
400
500
SR
C (%
Bas
al O
CR
)
Ex-vivo Memory CD8+
T-cells
D
H
0 40 80
Oligo FCCP R+A
0
200
400
600
Time (minutes)
OC
R (%
bas
elin
e)
WTCD80/86-/-
(legend on next page)
386 Cell 171, 385–397, October 5, 2017
(legend on next page)
388 Cell 171, 385–397, October 5, 2017
Are there unique pathways for CD28 signaling?
Liang et al. 2013, Nature Immunology
Are there unique pathways for CD28 signaling?
J Exp Med 2016
2421JEM Vol. 213, No. 11
lation was indistinguishable from that after CD3 stimulation alone (Fig. 5 c). In controls’ CD8+ PHA T cells, CD3 and CD2 cross-linking alone induced intermediate and weak
P65 phosphorylation, respectively, whereas CD2 and CD3 co-stimulation synergized for P65 phosphorylation (Fig. 5 d). In contrast with controls’ CD4+ T cells and in line with cy-
Figure 5. Impaired CD28 co-stimulation in patients’ CD4+ T cells. (a) Frequency of TNF +, IFN-γ+, and IL-2+ CD4+ memory T cells in healthy controls and patients after stimulation with the P815 cell line in the presence of 5 µg/ml anti-CD3 and/or 5 µg/ml anti-CD28 mAbs. 40 ng/ml PMA and 10−5 M ionomycin (Iono) stimulation was used as a positive control. One-way ANO VA and Mann–Whitney tests were used. (b) Frequency of TNF+, IFN-γ+, and CD107a+ CD8+ memory T cells in controls and patients after stimulation with the P815 cell line in the presence of 5 µg/ml anti-CD3 and/or 5 µg/ml anti-CD28 mAbs. 40 ng/ml PMA and 10−5 M ionomycin stimulation was used as a positive control. One-way ANO VA and Mann–Whitney tests were used. (a and b) Data show 20 controls and 6 patients. (c and d) Phospho-P65 (p-P65) detection by flow cytometry in CD4+ (c) and CD8+ (d) PHA blasts after cross-linking of indicated cell surface receptors. Representative FACS plot (left) and recapitulative bar graphs of eight controls (Ctl) and four patients (A3, B1, B2, and C1; right) are shown. The values represent the mean ± SEM. Wilcoxon matched-pairs signed rank test and Mann–Whitney tests were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. stim, simulated; unstim., unstimulated.
CD40L is upregulated on T cells after initial priming. This causes APC’s to further
upregulate B7 ligands.
APC TCR
CD28
NaïveT cell
B7
B7-CD28interaction
B7-CTLA-4interaction
CTLA-4
Proliferation,differentiation
Functional inactivation
(anergy)• Knockout of CTLA-4 results in autoimmune disease and loss of normal homeostasis: - multi-organ lymphocytic infiltrate, lethal by 3-4 weeks - lymphadenopathy, splenomegaly
The opposing functions of CD28 and CTLA-4
CTLA-4 – Master regulator of T cell activation
The inhibitory functions of CTLA-4
• Role in self-tolerance: – Autoimmunity and lymphoproliferation in
knockout mice – Polymorphism associated with autoimmune
diseases in humans – Blockade or deletion makes T cells resistant to
tolerance, exacerbates autoimmune diseases (EAE, type 1 diabetes)
APC
TCR
Naïve T cell
Effector and memory T cells
CD28 B7
APC
CTLA-4
APC
Cell-intrinsic: Termination of response
Responding T cell
Regulatory T cell
APC Responding T cell
Cell-extrinsic: Treg-mediated suppression of response
Immune response
Actions of CTLA-4
Expression of CTLA-4
37
How does CTLA-4 regulate T cell function
L.S.K. Walker / Immunology Letters 184 (2017) 43–50 45
Fig. 2. Cell-extrinsic function of CTLA-4 revealed by mixed bone marrow chimeric mice. Rag-/- deficient mice were reconstituted with bone marrow from wildtype mice,CTLA4-/- mice, or a 1:1 mixture of both. CTLA-4-/- cells exhibit hyperactivation and lymphoproliferation (red) when alone, but remain non-activated (green) when in thepresence of wildtype cells. Observation first reported in Ref. [21]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web versionof this article.)
in vitro produced mixed results, with some studies demonstratingreduced Treg suppression [36,37] and some not [29]. In one study,different preparations of the same antibody clone yielded differ-ing results [38]. When Treg from CTLA-4-deficient mice were used,these generally exhibited suppressive function in vitro, sometimesas efficiently as wildtype Treg [37,39,40] and sometimes slightlyless so [36]. A major confounder for many of these studies wasthat the CTLA-4-deficient Treg were being isolated from mice thatwere in the process of developing lymphoproliferative disease.Since disease in CTLA-4-deficient mice results from unimpingedengagement of the CD28 pathway, it can be abrogated by blockingCD28 signaling [41–43], theoretically removing this confounder.However the known requirement of CD28 for normal Treg genera-tion and homeostasis [44,45] poses a limitation here: to specificallytest to role of CTLA-4 in Treg, ideally one would want to interruptCTLA-4 pathway whilst leaving the CD28 pathway intact.
Around the time that these issues were being debated, my groupwas studying double transgenic mice expressing ovalbumin (OVA)in the pancreas under the rat insulin promoter and also expressingthe OVA-specific TCR transgene, DO11.10. This model was not origi-nally intended to be used for Treg analysis but rather was designedto study the early events in type 1 diabetes pathogenesis. How-ever, as is often the case in science, unexpected findings can openthe door to new insights. Realising that the rat insulin promoterwas active in the thymus, and that by driving expression of OVAat this site it was triggering Treg differentiation [46], we beganto consider the potential advantages of having a TCR transgenicmodel of Treg differentiation. Most notable among these was theidea that by breeding these animals to a CTLA-4 deficient strain wewould be able to generate antigen-specific Treg that lacked CTLA-4but retained an intact CD28 pathway. To avoid any variation in theantigen specificity of the Treg, we bred the mice to a rag deficientbackground (precluding expression of endogenous TCR-! chains).This had the added advantage of avoiding any residual off-targetactivation of the CTLA-4 deficient T cells, since now their specificitywas strictly restricted to the ovalbumin expressed in the pancreaticislets.
For the first time we were able to test the suppressive func-tion of CTLA-4-deficient Treg bearing an identical specificity andaffinity for antigen as their wildtype counterparts (both beingDO11.10+rag-/-), that had an intact CD28 pathway, and that had notbeen isolated from mice with lymphoproliferative disease. By puri-fying OVA-specific Treg from CTLA-4-sufficient or CTLA-4-deficientmice we were able to demonstrate that CTLA-4 expression wasabsolutely required for regulation of the autoimmune responseagainst pancreatic islets in vivo [40] (Fig. 3). This contrasted withthe results obtained in vitro using the same Treg populations [40],suggesting that in vitro suppression assays do not always faithfullyrecapitulate in vivo scenarios. A similar message came from thework of Sojka et al. [39] who found that CTLA-4 deficient Treg wereincapable of suppressing lymphopenia-induced T cell expansionin vivo despite showing intact suppressive function in vitro. Theseminal experiment from this era, which put the role of CTLA-4 inTreg function beyond doubt, was the demonstration by the Sak-aguchi lab that mice selectively lacking CTLA-4 expression in Tregsuccumbed to a spontaneous lymphoproliferation with fatal T cell-dependent autoimmune disease [47]. In fact, mice lacking CTLA-4in Treg only survived a few weeks longer than mice lacking CTLA-4systemically. This marked a change in direction for the CTLA-4 fieldthat, in some ways, was as significant as the original description ofthe knockout mice. It was now clear that a major role of CTLA-4related to its ability to function in the regulatory T cell population.Indeed, transgenic expression of CTLA-4 was later shown to be oneof three core requirements to convert a conventional CD4 T cell intoa “homemade” regulatory T cell [48].
Another key message to arise from this era was the notion thatthere is a degree of redundancy in Treg suppressive mechanisms.For example it was shown that while wildtype Treg used CTLA-4to suppress colitis, Treg lacking the CTLA-4 molecule were able toutilise IL-10 to achieve the same end [24]. Likewise, the functionof CTLA-4 deficient Treg was shown to be partially TGF-" depen-dent in a separate study [37], suggesting that, like IL-10, TGF-"can also compensate to some extent for a lack of CTLA-4. Togetherthe experimental work indicated that the CTLA-4 pathway is an
APC
TCR
Naïve T cell
Effector and memory T cells
CD28 B7
Regulatory T cell
APC Responding T cell
Cell-extrinsic: Treg-mediated suppression of response
Immune response
Actions of CTLA-4 39
APC
T Cell
CD28
B7
Costimulation à T cell activation
CTLA-4 competitively inhibits B7-CD28 engagement 40
APC APC
T Cell
CD28
B7
Costimulation à T cell activation
B7 CTLA-4
CTLA-4 blocks and removes B7 à lack of costimulation
à T cell inhibition
CTLA-4 competitively inhibits B7-CD28 engagement
T cell (activated T cell or Treg)
41
APC
T Cell
CD28
B7
Unopposed costimulation à Excessive T cell activation
Consequence of mutations in the CTLA-4 pathway
Therapy?
42
The opposing actions of CD28 and CTLA-4
CD28 and CTLA-4 both recognize B7-1, 2; yet CD28 stimulates and CTLA-4 inhibits – Kinetics: CD28 is expressed constitutively and
initiates responses; CTLA-4 appears later and terminates responses
– Affinity: CD28 binds to B7 only when B7 levels are high (microbes?), CTLA-4 (high affinity) binds when B7 is low (self antigens?)
– Preferential ligands: CD28-->B7-2 (constitutive); CTLA-4-->B7-1 (inducible)
The B7:CD28 families
ICOS expression:ICOS is not constitutively expressed on naïve T cells but is induced on CD4+ and CD8+ T cellsfollowing stimulation through the TCR and is further enhanced by CD28-mediated costimulation.
A new molecule with structural characteristic similar to the B7 molecules was identify in 1999,and was named B7h (B7-related protein 1; also GL-50 or B7RP-1 or ICOS-L).
B7h does not bind to CD28 or CTLA-4, but bind to ICOS (inducible costimulatory molecule).ICOS shares 30-40% sequence similarity with CD28 and CTLA-4.
McAdam A.J. et al. J. Immunol. 165:5035, 2000.
FIGURE 2. Expression of ICOS on activated T cells. Dissociated splenocytes from wild-type or B7-1/2-/- 129/SvS4Jae mice were incubated with anti-CD3, anti-CD3 and CD28, or no Ab. The thick line shows ICOS expression on T cells from wild-type splenocyte cultures, the dotted line shows ICOS expression on T cells from B7-1/2-/- splenocyte cultures, and the thin line represents a negative staining control (rat IgG-FITC).
B7h/ICOS costimulatory pathway
Antibody response and germinal center formation in ICOS -/- mice
ICOS is required for antibody responses and GC formation.
Tafuri A. et al (2001). Nature, 409: 105-109.
ICOS +/+
ICOS +/-
ICOS -/-
The PD-1 inhibitory pathway
• PD-1 recognizes two ligands (PD-L1, PD-L2)
• Upregulated on T cells after activation • Knockout of PD-1 leads to autoimmune
disease (different manifestations in different strains)
• Role of PD-1 in T cell suppression in chronic infections?
Virus-specific T cellsViral clearance
(spleen)
Inhibitory role of PD-1 in a chronic infection
In chronic LCMV infection in mice, virus-specific T cells become paralyzed; express high levels of PD-1; function restored by blocking the PD-1 pathway. Barber et al (Ahmed lab) Nature 2006
49
Naïve CD8+ T cells
Effector T cells
Memory T cells: enhanced antiviral responses
Exhausted T cells: inability to respond to virus (expression of inhibitory receptors, e.g. PD-1, others)
Virus
Acute infection: clearance of virus
Chronic infection: persistence of virus
T cell “exhaustion” in chronic viral infections
50
Action of PD-1
Normal response PD-1 engagement
Roles of inhibitory receptors
• Maintenance of self-tolerance • Immunosuppression in chronic infections
(HCV, HIV?) • Termination of normal immune responses? • Why so many inhibitory pathways?
52
Functions of CTLA-4 and PD-1
CTLA-4 PD-1
Major site of action Lymphoid organs Peripheral tissues Stage of immune Induction Effector phase response suppressed Mechanism of action Competitive inhibitor Signaling inhibitor
of CD28 of CD28 and TCR
Cell type suppressed CD4+ and CD8+ CD8+ > CD4+
53
T cell TCR
CD28
ICOS
OX40
GITR
CD137 (4-1BB)
CD27
Activating receptors (costimulators)
Inhibitory receptors
CTLA-4
PD-1
TIM-3
TIGIT
LAG-3
BTLA
T cell activating and inhibitory receptors
Putting it back together
Figure 8-16 Context matters: APC’s upregulate
B7 upon recognition of microbes
Figure 8-14
Figure 8-15 Anatomy of naïve T cell priming-APC’s
Figure 8-4 Anatomy of naïve T cell priming (cont.)
In Vivo T cell activation Mempel et al. Nature 2004
In vivo imaging of T cells adoptively transferred into mice with antigen loaded DC’s DC’s are red and T cells are green Observed three phases of T cell behavior: Phase 1: multiple short encounters with DC’s Phase 2: long-lasting stable contacts with DC’s Phase 3: resumed short contacts and rapid migration
Phase 1 Phase 2 Phase 3
After T cell activation, differentiation into other subsets
Summary • TCR-MHC/peptide interaction is low
affinity. T cells use multiple mechanisms to overcome this (anatomy, adhesion, synapse, etc.)
• Context of MHC-antigen is critical to outcome
• Balance of positive and negative signals determine the magnitude and nature of T cell responses
• Challenges: – Which signals are dominant in vivo under
different conditions? – How do we use this knowledge to design
therapeutic strategies?
