Flow Cytometric Applications for

Immunotherapy of Cancer

Bone Marrow Transplantation and Cellular Therapy

Program and Divisions of Neuro-oncology and Neurosurgery

University of Alabama at Birmingham

Flow cytometry has evolved beyond immunophenotypinginto a powerful tool for immunotherapy design and monitoring.

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are needed to see this picture.

©2006 Wall Street Journal

Flow Cytometric Immune

Monitoring

• Preparation: Pre-treatment immune status - immunophenotyping, immune function, cytotoxicity

• Weapons: Graft qualification -composition and potency.

• Surveillance: immunophenotyping, activation and cytotoxicity and minimal residual disease assessment (in hematopoietic malignancies).

MALIGNANT GLIOMA

Basic concepts

• Innate immune activation as cancer cells are stressed - a new hope?

• Chemotherapy, radiation, and tumor-derived immunosuppressive cytokines suppression - the empire strikes back.

• Immune reconstitution and immunotherapy strategies - return of the cancer-killing Jedi?

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Gliomas express stress-associated antigens

that are targets for NK and γδγδγδγδ T cells

From Friese MA et al

Cancer Res. 2003; 63: 8996-9006

Tumor-derived Immunosuppresion

and Immune Escape

• Down-regulation of MHC Class I

• Secretion of immunosuppressive proteins

• Induction of regulatory T cells

(CD3+CD4+CD25+FoxP3+).

• MSC population of tumor stroma

• Interference with costimulatory factors

such as down regulation of CD40L

• NK inhibitory receptors on MHC Class I-

cells

Test

CD3 CD3+CD8+ CD3+CD4+

Akso

lute

Lym

ph

ocyte

Co

un

t x

10

6

0

500

1000

1500

2000

Control <45

Control >45

Pre-Resection

Early Postop

Late PostOP

VL Postop

Test

γδ+γδ+γδ+γδ+

Akso

lute

Lym

ph

ocyte

Co

un

t x

10

6

0

20

40

60

80

100

120

140

Control <45

Control >45

Pre-resection

Early Postop

Late Postop

VL Postop

αβαβαβαβ and γδγδγδγδ T cell phenotypes in GBM

patients (Six-color

immunophenotyping)

NK and γδγδγδγδ T cell phenotypes in GBM

patients

Test

γδ+γδ+γδ+γδ+

Akso

lute

Lym

ph

ocyte

Co

un

t x

10

6

0

20

40

60

80

100

120

140

Control <45

Control >45

Pre-resection

Early Postop

Late Postop

VL Postop

Test

CD3-CD16/56+

Akso

lute

Lym

ph

ocyte

Co

un

t x

10

6

0

100

200

300

400

500

Control <45

Control >45

Pre-Resection

Early PostOP

Late PostOP

VL Postop

Circulating Regulatory T cells in

Patients with Gliomas

Flow Cytometric Immune

Monitoring

• Preparation: Pre-treatment immune status - immunophenotyping, immune function, cytotoxicity

• Weapons: Graft qualification -composition and potency.

• Surveillance: immunophenotyping, activation and cytotoxicity and minimal residual disease assessment (in hematopoietic malignancies).

Mitogen-stimulated γδγδγδγδ T cell

proliferation in GBM patients

γδγδγδγδ T Cell Source

1 2 3 4 5

Fo

ld γ

δγδ γδγδ E

xp

an

sio

n

0

200

400

600

800

1000

C<45 C>45 Pre-op Post-op Post-therapy

Pre-selection Post selection

Flow Cytometric Tracking of Graft

Manipulation and Purity

γδγδγδγδ T cells and αβαβαβαβ T cells vs. U251

Suspension cytotoxicity assay (4

hr)

γδγδγδγδ T cell cytotoxicity vs. primary

GBM and GBM cell lines (PKH26/ToPro

Iodide)

AstrocytesD54MG U251MG U373 GBM 1 GBM 2

Cyto

toxic

ity a

t E:T

Ratio

16:1

0

10

20

30

40

50

60

Cytotoxicity of GBM patient-derived

γδγδγδγδ T cells vs. U251 (PKH26/ToPro

Iodide)

E:T Ratio

1 2.5 5 10

% K

illing

0

20

40

60

80

100

E:T Ratio vs Preoperative

E:T Ratio vs Postoperative

U251luc GBM Treated with

allogeneic expanded/activated

γδγδγδγδ T cells

Saline-injected control mice

γδγδγδγδ-injected mice (5:1)Week 2 post-injection

Effect of γδγδγδγδT cells on Induction and

Growth of U251ffLuc Intracranial Gliomas

Days After Tumor Induction

0 7 14 21 28 35 42 49 56 63 70 77 84

Pro

port

ion

Surv

ivin

g

0.0

0.2

0.4

0.6

0.8

1.0 U251ffLuc Cells (2.4x105)

MST = 21 daysU251 Cells + γδγδγδγδT cells

(1.2x106)

MST = 48 days

U251ffLuc cells injected alone or with γδγδγδγδT cells

p < 0.001

Flow Cytometric Immune

Monitoring

• Preparation: Pre-treatment immune status - immunophenotyping, immune function, cytotoxicity

• Weapons: Graft qualification -composition and potency.

• Surveillance: immunophenotyping, activation and cytotoxicity and minimal residual disease assessment (in hematopoietic malignancies).

P o s t - B M T In t e rv a l

1111 2222 3333 4444 5555 6666

Ab

so

lute

Lym

ph

ocyte

Co

un

t x 1

0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

E P v s C D 8 T C D

E P v s C D 8 C O N

P o s t - B M T In t e r v a l

1111 2222 3333 4444 5555 6666

Ab

so

lute

Lym

ph

ocyte

Co

un

t x 1

0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

E P v s C D 4 T C D

E P v s C D 4 C O N

P o s t - B M T In t e r v a l

1111 2222 3333 4444 5555 66660

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

E P v s A B T C D

E P v s A B C O N

P o s t- B M T In te rv a l

1111 2222 3333 4444 5555 6666

Ab

so

lute

Lym

ph

ocyte

Co

un

t x 1

0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0

E P v s C D 3 T C D

E P v s C D 3 C O N

P o s t - B M T In t e r v a l

1111 2222 3333 4444 5555 6666

Ab

so

lute

Lym

ph

ocyte

Co

un

t x 1

0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

E P v s G D T C D

E P v s C D C O N

P o s t- B M T In te r v a l

1111 2222 3333 4444 5555 6666

Ab

so

lute

Ly

mp

ho

cy

te C

ou

nt x

10

0

5

1 0

1 5

2 0

2 5

3 0

E P v s C D 4 R A T C D

E P v s C D 4 R A C O N

Post-BMT Immune Recovery

TCD vs. T Replete Matched Unrelated Grafts

Post-BMT Interval

1111 2222 3333 4444 5555 6666

Ab

so

lute

Lym

ph

ocyte

Co

un

t x 1

06

0

100

200

300

400

500

EP vs CD19TCD

EP vs CD19CON

Post-BMT Interval

1111 2222 3333 4444 5555 6666

Ab

so

lute

Lym

ph

ocyte

Co

un

t x 1

06

0

100

200

300

400

500

EP vs CD56TCD

EP vs CD56CON

Post-BMT Immune Recovery

TCD vs. T Replete Matched Unrelated Grafts

UPN 97 T CELL RECOVERY

DAYS POST BM T

0 200 400 600 800 1000 1200 1400 1600

% O

F T

OT

AL

LY

MP

HO

CY

TE

S

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

CD3

CD3+CD4+

CD3+CD8+

R7

R4

R3R6

R5R2

CD8/CD69CD4/CD69

REST

SEB

PHA

T Cell Function 100 Days Following Allog

TCD BMT

CD4+ T Cell Function in NSCLC

Patients Following Induction

Chemotherapy2D Graph 1

UPN

9622 10249

Sti

mu

late

d C

D4

+ A

TP

Rele

as

e

0

100

200

300

400

500

600

700

Pre Chemo

Post-Chemo

Flow Cytometric Immune

Monitoring

• Preparation: Pre-treatment immune status - immunophenotyping, immune function, cytotoxicity

• Weapons: Graft qualification -composition and potency.

• Surveillance: immunophenotyping, activation and cytotoxicity andminimal residual disease assessment (in hematopoietic malignancies).

MRD Monitoring - Issues

• Requires highly trained technical staff familiar with

multiparameter Boolean gating in flow cytometry analytical software.

• Best done on the same day as immune recovery testing to establish immune status and to plan for cellular therapy if warranted.

• Element of faith “evidence of things not seen”

How to find me

• Lawrence.Lamb@ccc.uab.edu

• 205-996-2335

• THT 541 1530 Third Avenue South,

Birmingham 35294

ContributorsContributorsUABUAB

•• Richard Lopez, MDRichard Lopez, MD

•• Catalina SuarezCatalina Suarez--CuervoCuervo, MD, MD

•• Nichole Bryant, MDNichole Bryant, MD

•• Yancey Gillespie, PhDYancey Gillespie, PhD

•• Burt Burt NaborsNabors, MD, MD

•• James James MarkertMarkert, MD, MD

•• Cathy LangfordCathy Langford

•• Yun Yun SuSu

South Carolina Cancer CenterSouth Carolina Cancer Center

•• Phil Phil BuckhaltsBuckhalts, PhD, PhD

•• Phil Musk, PhDPhil Musk, PhD

•• Stephanie Stephanie MugaMuga, PhD, PhD

•• Paul Paul MeehMeeh, MS, MS

•• Blair WetmoreBlair Wetmore

•• Michelle KingMichelle King

•• Justin MarshJustin Marsh

MoneyMoney

NIH/NCI SPORE Program in Brain CancerNIH/NCI SPORE Program in Brain Cancer

NIH/NINDS Translational R21 ProgramNIH/NINDS Translational R21 Program

Brain Tumor Society and the Samuel Brain Tumor Society and the Samuel Gershon Gershon EndowmentEndowment

γδγδγδγδγδγδγδγδ

γδγδγδγδ γδγδγδγδ

Photo credits

•University of Souh Carolina Golf

Club

•Alabama History Museum

•Marshall Space and Rocket Center

•Saluda Shoals Park, Columbia, SC