IMMUNOLOGY · MOLECULAR BIOLOGY

T Cell Activation
& Immune Checkpoints

From Molecular Mechanisms to Cancer Immunotherapy

Based on Peer-Reviewed Literature 2013–2024

Key References Chen & Flies, Nat Rev Immunol 2013 · Frontiers Immunol 2024

TCR

PD-1

CTLA-4

CD28

MHC

01

Overview & Learning Objectives

🔬

T Cell Activation

Two-signal model, TCR signaling cascade, immunological synapse formation

⚡

Signal Transduction

Lck → ZAP-70 → LAT → PLC-γ1 → Ca²⁺/NFAT, PKCθ/NF-κB, RAS/ERK pathways

🛑

Immune Checkpoints

CTLA-4 and PD-1/PD-L1 as brakes on T cell activation — mechanisms and biology

💊

Clinical Translation

FDA-approved checkpoint inhibitors, clinical trial data, combination strategies

KEY CONCEPT The balance between co-stimulatory and co-inhibitory signals determines T cell fate: activation, anergy, or exhaustion

02

The Two-Signal Model of T Cell Activation

APC / Dendritic Cell

MHC-II
+ peptide

CD80/
CD86

Signal 1

Signal 2

Naïve T Cell

TCR/
CD3

CD28

✓ Signal 1 + Signal 2 → Full Activation

✗ Signal 1 alone → Anergy / Deletion

⚠ No Signal → No Response

Signal 1 — Antigen Recognition

TCR engages peptide-MHC complex on APC. CD4 or CD8 co-receptors stabilize interaction. Lck (associated with CD4/CD8) phosphorylates ITAMs on CD3ζ chain.

Chen & Flies, Nat Rev Immunol, 2013

Signal 2 — Co-stimulation

CD28 binds CD80/CD86 on APC. Recruits PI3K via YMNM motif and Lck/GRB2 via PYAP motif. Amplifies TCR signaling, induces IL-2 production, prevents anergy.

Frontiers Immunol, 2024 · PMC3786574

Signal 3 — Cytokine Context

Cytokines (IL-12, IL-4, TGF-β) from the APC direct T cell differentiation into Th1, Th2, Th17, or Treg lineages.

BSI Immunology Review, 2024

03

TCR Signaling Cascade

🔗

TCR–pMHC Engagement Kinetic segregation model; CD45 exclusion from synapse

▼

⚡

Lck & Fyn Activation Src-family kinases phosphorylate ITAMs on CD3ζ (Y83, Y94, Y111, Y123, Y142, Y153)

▼

🎯

ZAP-70 Recruitment & Activation SH2 domains bind phospho-ITAMs; Lck phosphorylates ZAP-70 at Y315, Y319, Y492, Y493

▼

🔀

LAT Phosphorylation → Signalosome ZAP-70 phosphorylates LAT; recruits PLCγ1, Grb2-SOS, Gads-SLP-76

▼

Ca²⁺/NFAT

PLCγ1 → IP3 → Ca²⁺ release → Calcineurin → NFAT dephosphorylation → Nuclear translocation

IL-2, IL-4 gene expression

PKCθ/NF-κB

DAG → PKCθ → CARMA1-BCL10-MALT1 → IKK → IκB degradation → NF-κB

Survival, proliferation genes

RAS/ERK

RASGRP1 → RAS-GTP → RAF → MEK → ERK1/2 → AP-1 (Fos/Jun)

Cytokine production, differentiation

04

The Immunological Synapse

cSMAC

TCR CD3 Lck ZAP-70 CTLA-4

pSMAC

LFA-1 ICAM-1 Talin

dSMAC

CD45 CD43

🎯 cSMAC (Central)

TCR microclusters concentrate here. Site of sustained signaling. CTLA-4 competes with CD28 in cSMAC, physically excluding CD28 to attenuate activation.

🔗 pSMAC (Peripheral)

Adhesion ring formed by LFA-1/ICAM-1 interactions. Stabilizes T cell–APC contact. Required for sustained signaling duration.

🚫 dSMAC (Distal)

CD45 phosphatase excluded from cSMAC — critical for maintaining Lck in active state. CD43 also segregated here.

⚡ CD28 Costimulation at Synapse

CD28 YMNM motif recruits PI3K; PYAP motif recruits Lck and GRB2. Enhances Lck dynamics and sustains ZAP-70 phosphorylation even with low-affinity antigens.

Frontiers Immunol 2024 (fimmu-15-1503018)

05

T Cell Differentiation & Fates

Naïve
CD4⁺ T Cell

→

Th1 IFN-γ, TNF-α IL-12 + T-bet

Intracellular pathogens, macrophage activation

→

Th2 IL-4, IL-5, IL-13 IL-4 + GATA3

Humoral immunity, allergy, parasites

→

Th17 IL-17A, IL-22 TGF-β + IL-6 + RORγt

Mucosal immunity, autoimmunity

→

Treg IL-10, TGF-β TGF-β + FoxP3

Immune suppression, self-tolerance

→

Exhausted PD-1⁺ TIM-3⁺ LAG-3⁺ Chronic antigen + checkpoints

Tumor microenvironment — target of ICIs

CLINICAL RELEVANCE Tumor-infiltrating T cells become exhausted via chronic PD-1/PD-L1 signaling — checkpoint blockade reverses this exhaustion program

06

CTLA-4: The First Immune Checkpoint

Mechanism of CTLA-4 Inhibition

APC

CD80/CD86 (B7)

CD28 ⟵ Activation

CTLA-4 ⟵ Outcompetes CD28 (20× higher affinity)

T Cell

CD28

CTLA-4

🔴 Recruits PP2A and SHP-2 phosphatases → dephosphorylates TCR signaling intermediates

🔴 Physically excludes CD28 from cSMAC → disrupts positive signaling

🔴 Trans-endocytosis of CD80/CD86 from APC surface → reduces ligand availability

🔴 Expressed on Tregs constitutively → suppresses effector T cells in tumor

Ipilimumab

Anti-CTLA-4 mAb

FDA Approved: 2011

Metastatic Melanoma (1st checkpoint inhibitor)

CheckMate 067: Nivolumab + Ipilimumab vs. Ipilimumab alone

5-yr OS: 52% (combo) vs. 26% (ipilimumab alone)

Grade 3-4 irAEs: ~31% with dual therapy

PMC12554671 · Nature 2024

CTLA-4 Biology

Upregulated 48–72h after T cell activation
Constitutively expressed on FoxP3⁺ Tregs
Homologous to CD28 (20–30% sequence identity)
Binds CD80/CD86 with 20× higher affinity than CD28
Nobel Prize 2018: James Allison (CTLA-4 blockade)

07

PD-1/PD-L1 Axis: Peripheral Tolerance & Tumor Escape

Tumor Cell

PD-L1
(CD274)

PD-L2
(CD273)

← IFN-γ induces PD-L1 upregulation

PD-1 ⟷ PD-L1

SHP-1/SHP-2 recruitment

↓ ZAP-70 phosphorylation

↓ PI3K/AKT signaling

↓ IL-2, IFN-γ production

Exhausted T Cell

PD-1
(CD279)

TIM-3

LAG-3

Feature	CTLA-4	PD-1
Expression	Activated T cells, Tregs	Activated T cells, B cells, NK cells
Location	Lymph nodes (priming)	Peripheral tissues, tumor
Ligands	CD80, CD86	PD-L1, PD-L2
Mechanism	Competes with CD28, phosphatase recruitment	SHP-1/2 recruitment, TCR signal dampening
irAE profile	Colitis, hypophysitis, rash	Pneumonitis, thyroid dysfunction, arthralgia

Pembrolizumab Anti-PD-1 · FDA 2014 Keytruda · Merck

Nivolumab Anti-PD-1 · FDA 2014 Opdivo · BMS

Atezolizumab Anti-PD-L1 · FDA 2016 Tecentriq · Genentech

Durvalumab Anti-PD-L1 · FDA 2017 Imfinzi · AstraZeneca

PMC12554671 · Sci Archives 2024

08

Tumor Microenvironment & T Cell Exhaustion

TUMOR

↑ PD-L1 ↑ IDO ↑ TGF-β ↓ MHC-I

Exhausted CD8⁺ T

PD-1⁺ TIM-3⁺ LAG-3⁺ TOX⁺

↓ Cytotoxicity ↓ IL-2

Treg

FoxP3⁺ CTLA-4⁺ CD25⁺

Suppresses effector T cells

MDSCs

CD11b⁺ Gr-1⁺

Arginase, ROS, NO production

TAM (M2)

CD163⁺ IL-10⁺

Pro-tumor, anti-inflammatory

T Cell Exhaustion Progression

Effector

PD-1⁻ TIM-3⁻
High cytotoxicity

→

Early Exhausted

PD-1⁺ TIM-3⁻
Partial function

→

Late Exhausted

PD-1⁺ TIM-3⁺ LAG-3⁺
TOX⁺ TCF1⁻

→

Terminal

CD38⁺ CD101⁺
Irreversible

⬆ Anti-PD-1 can rescue early/intermediate exhausted T cells

09

Clinical Evidence: Checkpoint Inhibitors

CheckMate 067

Nivolumab + Ipilimumab

Advanced Melanoma

52%5-yr OS (combo)

26%5-yr OS (ipi alone)

NCT01844505 · NEJM 2019

CheckMate 142

Nivolumab + Ipilimumab

MSI-H/dMMR Colorectal Cancer

55%ORR

85%12-mo OS rate

71%12-mo PFS rate

NCT02060188 · Overman et al. 2018

CheckMate 8HW

Nivolumab + Ipilimumab

MSI-H/dMMR CRC (Phase 3)

79%12-mo PFS rate

72%24-mo PFS rate

NCT04008030 · André et al. 2024

NSCLC PD-L1+ Analysis

Anti-PD-1 Monotherapy

Advanced NSCLC (PD-L1 TPS ≥1%)

28.6%ORR

15.2 moMedian OS

7.4 moMedian PFS

J Hematol Oncol 2024

⚠ Immune-Related Adverse Events (irAEs)

~10% Grade 3-4 with PD-1 monotherapy

~31% Grade 3-4 with PD-1 + CTLA-4 combination

33% Any-grade irAEs in Black patients (vs 44% White) — VHA 2024

10

Next-Generation Checkpoints & Emerging Therapies

TIM-3

Ligands: Galectin-9, HMGB1, PtdSer, CEACAM1

Marks terminally exhausted T cells; co-expressed with PD-1

Sabatolimab (anti-TIM-3) — Phase 2 trials

LAG-3

Ligands: MHC-II, FGL1, LSECtin, Galectin-3

Inhibits CD4⁺ T cell activation; expressed on Tregs

Relatlimab + Nivolumab (RELATIVITY-047) — FDA 2022

TIGIT

Ligands: CD155 (PVR), CD112 (PVRL2)

Competes with CD226 (activating); suppresses NK and T cells

Tiragolumab + Atezolizumab — Phase 3 ongoing

VISTA

Ligands: VSIG-3, PSGL-1

Expressed on tumor-infiltrating myeloid cells; suppresses T cells

CA-170 (oral VISTA/PD-L1 inhibitor) — Phase 1

Bispecific Antibodies

QL1706: PD-1 × CTLA-4 · Cadonilimab: PD-1 × CTLA-4

Single molecule dual blockade; potentially reduced toxicity

Cadonilimab: FDA Breakthrough Designation 2024; APOC3 biomarker identified

CAR-T + Checkpoint

PD-1/TIGIT dual shRNA CAR-T cells

Intrinsic checkpoint resistance in engineered T cells

NCT02414269: iCasp9M28z + Pembrolizumab — Phase 1

11

Summary & Key Takeaways

T Cell Activation

1 Two-signal model: TCR (Signal 1) + CD28 (Signal 2) required for full activation; Signal 1 alone → anergy

2 Signaling cascade: Lck → ZAP-70 → LAT → three parallel pathways (Ca²⁺/NFAT, PKCθ/NF-κB, RAS/ERK)

3 Immunological synapse organizes signaling molecules; CD28 enhances Lck dynamics and ZAP-70 phosphorylation

4 T cell fate determined by cytokine milieu: Th1/Th2/Th17/Treg/Exhausted

Immune Checkpoints

5 CTLA-4 acts at lymph node priming stage; outcompetes CD28 for B7 ligands (20× higher affinity)

6 PD-1/PD-L1 axis operates in peripheral tissues/tumor; IFN-γ induces PD-L1 as adaptive resistance

7 Checkpoint inhibitors (ipilimumab, pembrolizumab, nivolumab) have transformed cancer treatment — Nobel Prize 2018

8 Next-gen: TIM-3, LAG-3, TIGIT, bispecific antibodies, CAR-T with intrinsic checkpoint resistance

"The immune system has the power to eliminate cancer — checkpoints are the brakes; immunotherapy releases them."

— James P. Allison, Nobel Laureate 2018

Key References: Chen & Flies, Nat Rev Immunol 13:227 (2013) · Frontiers Immunol 15:1503018 (2024) · PMC12554671 (2025) · J Hematol Oncol 2024 · Cancer Biol Med 2024 · CheckMate 067/142/8HW trials

T Cell Activation& Immune Checkpoints

Overview & Learning Objectives

T Cell Activation

Signal Transduction

Immune Checkpoints

Clinical Translation

The Two-Signal Model of T Cell Activation

Signal 1 — Antigen Recognition

Signal 2 — Co-stimulation

Signal 3 — Cytokine Context

TCR Signaling Cascade

The Immunological Synapse

🎯 cSMAC (Central)

🔗 pSMAC (Peripheral)

🚫 dSMAC (Distal)

⚡ CD28 Costimulation at Synapse

T Cell Differentiation & Fates

CTLA-4: The First Immune Checkpoint

CTLA-4 Biology

PD-1/PD-L1 Axis: Peripheral Tolerance & Tumor Escape

Tumor Microenvironment & T Cell Exhaustion

T Cell Exhaustion Progression

Clinical Evidence: Checkpoint Inhibitors

Next-Generation Checkpoints & Emerging Therapies

Summary & Key Takeaways

T Cell Activation

Immune Checkpoints

T Cell Activation
& Immune Checkpoints