ESMO PRECEPTORSHIP ON

Title: From basic to tumor immunologyfor oncologists

Name: Pedro Romero

Date: May 10th 2019

From basic to tumorimmunology for oncologists

ESMO Preceptorship Immuno Oncology, May 10 th 2019, Zurich

Pedro RomeroLudwig Cancer Research Center,

Department of Fundamental Oncology,FBM,University of Lausanne

DISCLOSURES

Pedro Romero

Personal financial interests

Speaker honoraria: BMS, Astra Zeneca, Roche

Member Scientific Advisory Board: Immatics biotechnologies, Enterome

Grant for research: Roche pRED, Zurich

Non-financial interests

Transgene - Member Scientific Advisory Board

NexImmune - Member Scientific Advisory Board

Editor-in-chief - Journal for Immunotherapy of Cancer

Major steps to the deciphering of the biology of T cells

IL-2 discovered in early 70s (TCGF)

First T cell subsets (Lyt-1, Lyt-2) in mid 70s (Shiku, Old, Boyse)

TCR cloned in 1982 (Davis, Mak)

Antigenic peptides in 1989 (Townsend)

Tregs in 1995 (Sakaguchi)

CTLA-4 as negative regulator in 1995 (Allison, Mak)

PD-1 cloning in 1992 (Honjo)

CYTOLYTIC CD8 + T LYMPHOCYTES(CTL) ,

the major effectorsof anti-tumoradaptive immunity

- Direct lysis (perforin, GZB)- IFN-γ, TNF-α, GM-CSF …- Fas L- CD40L

The tumor

microenvironment:

multiple cellular and

molecular circuits

mediating

immunosuppression

Joyce J & Fearon D, Science 2015

1. The normal T cell repertoire

By recombination, random insertion, deletion and substitution, the small set of genes that encode the T-cell receptor has the potential to create between 1015 and 1020 TCR clonotypes (a clonotype is a population of T cells that carry an identical TCR)

There are only an estimated 10 13 T cells in the human body , and many clonotypes are of high abundance due to strong selection forces

High-throughput sequencing (HTS) allows greater sequencing depth and significantly more accurate quantification of TCR clonotype abundance. However, HTS is still subject to PCR bias and sequencing error

~160 TCR genes Laydon et al. Philos Trans R Soc Lond B Biol Sci 2014

Thymic anatomy and compartmentalized selection,or the I-O preceptorship for T cells

Cortex

Medulla

+ selection

Negative

(95%, RIP)

Palmer, Nat Rev Immunol 2003

Thymic selection depends on T-cell receptor affinity for self peptide–MHC complexes

Palmer, Nat Rev Immunol 2009

Aire: from transcriptional regulation to tolerance induction

Mathis & Benoist, Nat Rev Immunol 2007

mTECs(medullary

thymic epithelial cells

A new generation oftumor specific antigens:the « neoantigens »

Schumacher & Schreiber, Science 2015

Somatic mutations

No central tolerance

Highly individual

NGS – Bioinformatic pipelinefor identification

2. Antigen recognition by T cells

105 MHC-I molecules on the surface of target cells

Diversity of peptide ligands – 10 4, according to MS (M Bassani)

1 – 10 pMHC-I are enough to activate an effector CD8 T cell!

But it is literally like finding a needle in a haystack

Zoete, Irving, Michielin, J Mol Recognit 2010

Affinity range: 10 -4 – 10-6 M

CD8 coreceptor: 10-fold increase

Kinetic parameters also important

Serial engagement allows exquisiteantigen sensitivity

Measuring «functional avidity» of specific TCRs

- Lysis (4h, kinetically)

- IFN-γ (16h)

- Other cytokines, chemokines

Functions measured

Measuring «structural» avidity» of specific TCRs

Schmid et al. J Immunol 2010

Measuring «structural» avidity» of specific TCRs

Schmid et al. J Immunol 2010

Tetramer association rateKon

Tetramer dissociation rateKoff

Measuring «structural» avidity» of specific TCRs

Schmid et al. J Immunol 2010

Measuring 2D affinity of specific TCRs

Hong et al. Nat Immunol 2018

The TCR is (also) a mechanosensor

TCR specificity and TCR degeneracy

Mason Immunol Today 1998

CTL clone LAU 203/1.5: HLA-A2/EAAGIGILTV (Mela-A/MART-1)

Positional scanning combinatorial peptide libraries

Rubio et al. J Immunol 2002

A given TCR exhibits high peptide specificity but at the same time, when appropriately queried, itcan recognize thousands of peptides with the same or even higher functional avidity

3. Priming an anti-tumor antigenspecific CD8 T cell response

T cell priming� A rare event

� Only o ne specialized APC may initiate a T cellresponse: XP-DC

� Highly l ocalized event: lymph node (TuDLN)

� Takes a certain affinity of receptors

� Takes t ime

� At least three s ignals – highly regulated

Activation of Naïve T cells (priming)• T cells require multiple signals (minimum of THREE) to become fully activated 1

• In addition to antigen stimulation further positive co-stimulation is required 1

• Key co-stimulatory receptor is CD28 2

• Third signal: cytokines

T cell

Antigen-presenting cell

T cell becomes anergic a

Specific signal alone

1T cell

Antigen-presenting cell

No effect on T cell

Co-stimulatory signal alone

2T cell

Antigen-presenting cell

Activates T cell

Co-stimulatory signal and specific signal

MHC class I

TCR

Co-stimulator

CD8

21

aAnergy describes a state of functional inactivationMHC = major histocompatibility complex

1. Janeway CA, et al. Immunobiology 2008; 2. Pardoll DM. Nat Rev Cancer 2012;12:252–64

4. T cell Expansion, Differentiationand Migration

Two effector CD8 T cells with distinctmemory fates

Naive

SLEC

MPECMPEC

KLRG1high CD127low

KLRG low CD127high Long-lived memory T cells

Kaech et al., Cell 2002Wherry et al., Nat. Immunol., 2003Kaech et al., Nat. Immunol., 2003Sarkar et al., J. Exp. Med., 2008

SLEC: short lived effector cellMPEC: memory precursor effector cell

The morphing of the quiescent T cell precursorinto a fully differentiated effector

• The duration as well as the strength of signal 1 determines the extentof clonal expansion and functional differentiation

• The cytokine environment determines the polarity of the response

(type 1, type 2 immune responses)

• The origin of the DC imparts a tissue specific migration program (addressins, integrins, chemokine receptors)

• Clearance of antigen is followed by contraction of the T cell polyclonalcohort, to enter into the memory phase.

• Cues from the tissue of residence shape the effector phenotype and functions of the infiltrating T cells

Zajac et al. Immunology 2010

Divergent patterns ofCD8 T-cell responsesdevelop followingacute, protracted, andchronic infections.

Naïve T cell

Effector T cell

Memory T cell

Exhausted T cell

Zhang L and R

omero P,

Trend

s in M

ol. M

edicin

e, 2018

Lymphocyte trafficking,

extravasation and TILs

4. Residence in the tumormicroenvironment

Tissue resident memory T cells

Sentinels poised to immediate response

Route of vaccination matters – cDC1 required

Do not recirculate

Specific transcription factors: Runx3 +, Notch +, Hobit +, Blimp1 +, AHR, BATF +, EOMESneg, Tbet lo

CD103 (αE, β7), CD49a (VLA-1 or α1β1), CD69

Role of CD103 integrin in anti-tumor T RM

Mami-Chouaib et al. J Immunother Cancer 2018

CD103+ TILs c arry prognostic value in humancancer

In human lung cancer – Djenidi F et al. J Immunol 2015 (n= 101)

In human ovarian cancer – Webb JR et al. Clin Cancer Res 2014 (n= 497), and is co-expressed with PD-1 (Webb JR Cancer Immunol Res 2015)

In human breast cancer – Wang Z-Q et al. Clin Cancer Res 2015 (n= 424)

Bladder, colorectal, melanoma, cervical, head and nec k and pancreatic carcinomas

Melanoma TILs

Murray et al. Front Immunol 2016

VLA-1+ CD103+ TILs control tumor growth

Murray et al. Front Immunol 2016

Factors that oppose efficient T cellinfiltration and / or residence

(«cold, excluded t umors»)

β-Catenin signaling prevents migration of effector T cells into tumors

Intra-vital imaging revealed failed T cell entry into β-catenin expressing tumors

Effector T cell migration depends on the presence of CD103+ DCs producing CXCL10 (XP-DCs)

Lack of CD103+ DC-mediated effector T cell recruitment prevents immune control

Spranger S et al. Cancer Cell 2017

Understanding the cellular and molecular bases of tumor infiltration by T cells

Galunisertib (Tauriello et al

Soluble receptor (Mariathasan et al)

Traps (Ravi R et al)

Cancer AssociatedFibroblasts (CAFs)

TGFβ

CXCL-1, 2, 5CCL3

CTL PD-L2FASL

CXCR2 antagonistKumar V et al

PD-L2 blockadeFASL inhibitorLakins MA et al

Dans les carcinomes urothéliaux et colorectaux, le TGFβ actionne l’évasion immunitaire

en contribuant à l’exclusion des cellules T

Mariathasan S et al. Nature 2018

Tauriello DVF et al. Nature 2018

Factors that dampen T cellfunctionality in the TME

(«exhausted, anergic»)

RECEPTORS

CD5

CD8

CD28

CD103

A2AR

P2X

4-1BB

PD-1

TIM-3

LAG-3

TLR-3

NKG2A

LIGANDS

CD5, CD72?

MHC-I α3

CD80, CD86

E-cadherin

Adenosine

ATP

4-1BBL

PD-L1, -L2

Gal9, PtdSer, HMGB1

CD4, others

Poly(I-C)

HLA-E

Multiple cues s ensed and output fine tunedGreen = ++ Red = ---

Infiltrating CD8 T Lymphocyte

HIF-1α/HIF-2α

VEGFA

O2

Palazon et al. Cancer Cell 2017

Glycolysis

Cytolysis

Emerging immune checkpoints: immunometabolism

• A growing number of catabolic enzymes: IDO, arginases, phenylalanine oxidase (IL4I1), glutaminase (GLS)

• Targeting the purinergic pathways in the tumormicroenvironment (CD39, CD73, adenosinereceptors)

The tumor microenvironment: struggle to acquire key nutrients

Kishton et al. Cell Metab 2017

Le phosphoénolpyruvate (PEP) : un checkpoint métabolique des réponses anti-tumorales des cellules T

La privation en glucose supprime les fonctions effectrices des cellules T anti-tumorales

Ping-Chih Ho et al. 2015

Le métabolite PEP de la glycolyse maintient les signaux Ca2+ et NFAT en bloquant SERCA

Le signal Ca2+ est un intégrateur de l’activité glycolytique et des signaux TCR

La reprogrammation métabolique des cellules T stimule les fonctions effectrices anti-tumorales (restauration métabolique des

cellules T pour générer du PEP dans des conditions

pauvres en glucose)

Acides aminés clés :

Tryptophane

Arginine

Phénylalanine

Glutamine

Enzymes cataboliques :

IDO, TDO

Arginases

Phénylalanine oxidase (IL4I1)

Glutaminase (GLS)

Ciblage de la voie des nucléotides : ATP/adénosine, CD73

A2AR

A2AR

A2AR/A2BR

d’après Beavis et al. 2012, Ohta 2016A2AR = récepteur A2a de l'adénosine

CD39 and CD73 are expressed on distinct subsets of human memory CD4 + T cells.

Gourdin N … Menetrier-Caux C, Cancer Res 2018

CD73+ CD4+ Teffsare enriched in poly-functional Th1*, Th17 cells

CD73+ CD4+ Teffs c ooperate with Tregs to degrade ATP into Ado

Isolated CD73+ CD4+ Teffs are preferential targets of

CD39+ Tregs through the generation of Ado

Breast and ovarian tumors contain highly functional CD39 + Tregs a nd CD73+ CD4+ Teff with Th1* characteristics

Gourdin N … Menetrier-Caux C, Cancer Res 2018

TILs are heterogenous

CD39 marks TA-specific CD8 T cells

CD39 as biomarker

CD39 as checkpointSimoni, … Newell E, Nature 2018

TIL heterogeneity – implications for immunotherapy

Progenitor memory-like CD8 TILs – Tcf-1+ (Siddiqui et al, Kurtulus et al. Immunity 2019)

Early and terminally exhausted CD8+ TILs (Miller BC et al, Nature 2019)

NR4A1, a key mediator of T cell dysfunction (Liu X et al. Nature 2019)

NR4A transcription factors limit CAR T cell function (Chen J et al. Nature 2019)

Defining CD8 TIL cell states in melanoma (Sade Feldman et al. Cell 2018)

CD8 TILs: a continum from transitional to dysfunctional states in melanoma (Li H et al. Cell 2019)

Sade-Feldman, … Hacohen, Cell 2018

Memory-like

Respond to a-PD-1

Good prognosis

TCF-1

Terminal effector

Exhausted

AICD

NR4A, TOX

scRNAseq – CD8 TILs upon therapy

Thank you for your kind attention