Optimized workflow improves the characterization of tumor-infiltrating T cells

César Evaristo, Ramona Siemer, David Agorku, Janina Brauner, Olaf Hardt, Christian Dose, and Anne RichterMiltenyi Biotec GmbH, Bergisch Gladbach, Germany

IntroductionImmunotherapy has proven clinical efficacy and tremendous potential in multiple cancers. Syngeneic mouse tumor models represent the gold standard to analyze effects of immunotherapy, as they possess a fully competent immune repertoire. However, the amount and composition of tumor-infiltrating leukocytes (TILs) is highly variable, complicating the analysis of individual subpopulations. In particular, small subpopulations

might escape analysis as they could get lost in the background noise. When working with large cohort sizes, even immunophenotyping of TILs by flow cytometry is time consuming and data processing highly work intensive. Therefore, pre-enrichment of TILs is highly desirable to increase the sensitivity of analysis and save time and effort during flow cytometry.

Reliable and fast isolation of T cells from syngeneic mouse tumors1Results

We have developed new CD4+, CD8+, and pan T cell specific enrichment reagents for magnetic cell iso lation (MACS® Technology) directly from dissociated tumor tissue (fig. 1A). Tumors derived from B16-F10 (left and center columns) or B16-OVA (right column) melanoma cells were inocu-lated subcutaneously into the right flank of C57BL / 6 mice. After 11–15 days, tumors were collected and dissociated using the gentleMACS™ Octo Dissociator and the mouse Tumor Dissociation Kit (TDK) optimized for epitope pre servation. CD4+, CD8+, and pan T cells were isolated using the new mouse CD4 (TIL) MicroBeads, CD8 (TIL) MicroBeads, and CD4 /CD8 (TIL) MicroBeads, respectively (fig. 1B). Frequencies of tumor-infil-trating T cells among living cells before or after isolation are shown for different tumor models, as assessed by flow cytometry. Magnetic cell isolation resulted in purities above 80%, which represents an up to 500-fold enrichment of the target cell population (fig. 1C), dramatically de-creasing time of analysis (table 1).

Using recombinantly engineered REAfinity™ Antibodies decreases background in flow cytometry analysis of tumor-derived samples2

REAfinity™ Recombinant Antibodies have specifically mutated human IgG1 Fc regions, which virtually eliminate unspecific binding to Fcγ recep-tors. Sple nocytes and tumor cells were isolated from BALB / c mice har-boring H8N8 breast cancer tumors. Res pective cells were subsequently labeled with Viobility™ 405 / 520 Fixable Dye, REA clones CD45-FITC, and CD3-APC, and either REA clone CD8α-PE or hybridoma clone CD8α-PE. Staining was performed in either presence or absence of FcR blocking rea gent and viable CD45+CD3– cells are shown.

Magnetic isolation improves the quality of flow cytometry analysis of tumor-infiltrating CD8+ T cells3

We used optimized panels of fluorochrome-con jugated REAfinity Anti-bodies to phenotypically characterize tumor-infiltrating CD8+ T cells (table 2). Total (fig. 3A) or magnetically isolated CD8+ T cells (fig. 3B) from B16-OVA tumors were labeled with the reagents described in table 2 and analyzed by flow cytometry using the MACSQuant® Analyzer 10. Gated popula tions show viable cells.

Enzymatic tumor dissociation is essential for efficient recovery of key populations of tumor-infiltrating CD8+ T cells4

B16-F10 tumors were collected and dissociated using the gentleMACS™ Octo Dissociator in the presence (fig. 4A) or absence (fig. 4B) of Tumor Dis-sociation Kit enzymes. Cells were subsequently labeled with REAfinity™

Antibodies (table 1). Gating of cell popu lations was performed as previ-ously mentioned (fig. 3).

Conclusion

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. gentleMACS, MACS, the MACS logo, MACSQuant, REAfinity, Vio, Viobility, VioBlue, and VioBright are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Copyright © 2017 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.

• Standardized processing of tumor samples using TDK enhanced reproducibility and was essential for recovery of key populations of TILs.

• Enrichment of tumor-infiltrating T cells by magnetic cell separation greatly reduced time and costs of downstream analysis while the quality of data obtained from TIL analysis was significantly increased.

• In comparison to hybridoma-derived antibodies, recombinantly engineered REAfinity Antibodies virtually eliminated unspecific binding to Fcγ receptors leading to improved quality of flow cytometry analysis.

• Complete workflows for TIL analysis, validated in different mouse tumor models, significantly reduced the processing and analysis time while data quality was greatly improved.

Acknowledgements: We wish to acknowledge the support of Frauke Alves and Oliver Reinhardt from the University Medical Center Göttingen and Chrystel Flores, Charlie Norkus, Annkristin Heine and Christian Kurts from the Institute of Experimental Immunology, University Hospital Bonn.

Panel 1 Clone Fluorochrome Panel 2 Clone Fluorochrome

CD8β REA793 VioBlue CD8β REA793 VioBlue

Viobility 405/520 Viobility 405/520

Lag-3 REA776 VioBright™ 515 CD103 REA789 VioBright™ 515

TIM-3 REA602 PE CXCR3 REA724 PE

CD4 REA604 PE-Vio615 CD4 REA604 PE-Vio615

PD-1 REA802 APC CD39 REA870 APC

CD44 REA664 APC-Vio770 CD44 REA664 APC-Vio770

Table 2

Figure 1

A Bulk tumor Negative fraction Isolated TILsLias et dit

B

C

Cell type Cells to analyze Events to collect Flow cytometry time/ sample*

Total flow cytometry time**

CD4+ T cells

Bulk 5,000 7.96×10⁶ 66.3 min >10 h

Isolated*** 5,000 5.41×10⁴ 0.5 min ~11 min

CD8+ T cells

Bulk 5,000 2.80×10⁶ 23.3 min >3.5 h

Isolated*** 5,000 4.37×10⁴ 0.4 min ~10 min

T cells

Bulk 10,000 8.13×10⁵ 6.8 min >1 h

Isolated*** 10,000 3.24×10⁴ 0.3 min <10 min

10³-101

10¹ 10²0

10³

10²

10¹

-1 1

Bu

lk tu

mo

r

CD8 (TIL) MicroBeads

CD4 (TIL) MicroBeads

CD4/CD8 (TIL) MicroBeads

Iso

late

d T

ILs

CD4-PE CD8β-VioBlue CD8β-VioBlue

CD

3ε-A

PC-V

io77

0

CD

4-PE

-Vio

615

CD

4-PE

-Vio

615

10³-101

10¹ 10²0

10³

10²

10¹

-1 1 10³-101

10¹ 10²0

10³

10²

10¹

-1 1

10³-101

10¹ 10²0

10³

10²

10¹

-1 1 10³-101

10¹ 10²0

10³

10²

10¹

-1 1 10³-101

10¹ 10²0

10³

10²

10¹

-1 1

0.18%

0.96%

2.55%

3.69%

92.4%

80.5%

31.3%

53.6%

100

80

60

40

20

0

B16-F10 (n

=4)

% C

D4+

T c

ells

4T1 (n=2)

CD4 (TIL) MicroBeads

100

80

60

40

20

0

B16-F10 (n

=6)

% C

D8+

T c

ells

4T1 (n=3)

CT26.WT (n

=2)

B16-OVA (n

=4)

CD8 (TIL) MicroBeads CD4 / 8 (TIL) MicroBeads

100

80

60

40

20

0

B16-F10 (n

=3)

% C

D3+

T c

ells

CT26.WT (n

=1)

B16-OVA (n

=2)

Figure 2

10³-101

10¹ 10²0

10³

10²

10¹

-1 1

Sple

no

cyte

s

Hybridoma antibodyREAfinity Antibody

Tum

or

cells

CD

45-F

ITC

w/o FcR blocking

10³-101

10¹ 10²0

10³

10²

10¹

-1 1

10³-101

10¹ 10²0

10³

10²

10¹

-1 1 10³-101

10¹ 10²0

10³

10²

10¹

-1 1

1.34% 1.59%

2.29% 74.2%

REAfinity Antibody

with FcR blocking

Hybridoma antibody

10³-101

10¹ 10²0

10³

10²

10¹

-1 1 10³-101

10¹ 10²0

10³

10²

10¹

-1 1

10³-101

10¹ 10²0

10³

10²

10¹

-1 1

1.19% 1.21%

1.68%

10³-101

10¹ 10²0

10³

10²

10¹

-1 1

5.33%

CD8a-PE

Figure 3

A

B

Bu

lk tu

mo

r (1

50,0

00 e

ven

ts)

10³-101

10¹ 10²0

10³

10²

10¹

CXCR3-PE

CD

39-A

PC

-1 1

39.7%

15.5%

CXCR3-PE

CD

39-A

PC

10³-101

10¹ 10²0

10³

10²

10¹

CD103-FITC

CD

39-A

PC

-1 1

72.4%

19.8%

CD103-FITC

CD

39-A

PC

10³-101

10¹ 10²0

10³

10²

10¹

CD8β-VioBlue

CD

4-PE

-Vio

615

-1 1

CD8β-VioBlue

10³-101

10¹ 10²0

10³

10²

10¹

Lag3-FITC

Tim

3-PE

-1 1

18.0%

23.2%

Lag3-FITC

Tim

3-PE

100000

500 750

250

500

750

1000

250

Forward scatter-A

Fo

rwar

d sc

atte

r-H

Forward scatter-A

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Tim

3-PE

-1 1

2.58%

18.6%

PD1-APC

Tim

3-PE

10³-101

10¹ 10²0

10³

10²

10¹

CD44-APC-Vio770

CD

8b-V

ioB

lue

-1 1

CD44-APC-Vio 770

CD

8b-V

ioB

lue

100000

500 750

250

500

750

1000

250

Forward scatter

Si

de s

catt

er

Forward scatter

Sid

e sc

atte

r

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Lag

3-FI

TC

-1 1

1.03%

15.5%

PD1-APC

Lag3

-FIT

C

Iso

late

d T

ILs

(50,

000

even

ts)

10³-101

10¹ 10²0

10³

10²

10¹

CD44-APC-Vio770

CD

8b-V

ioB

lue

-1 1

CD44-APC-Vio 770

CD

8b-V

ioB

lue

10³-101

10¹ 10²0

10³

10²

10¹

CXCR3-PE

CD

39-A

PC

-1 1

37.3%

13.5%

CXCR3-PE

CD

39-A

PC

10³-101

10¹ 10²0

10³

10²

10¹

CD103-FITC

CD

39-A

PC

-1 1

72.3%

19.8%

CD103-FITC

CD

39-A

PC

10³-101

10¹ 10²0

10³

10²

10¹

CD8β-VioBlue

CD

4-PE

-Vio

615

-1 1

CD8β-VioBlue

10³-101

10¹ 10²0

10³

10²

10¹

Lag3-FITC

Tim

3-PE

-1 1

14.0%

22.0%

Lag3-FITC

Tim

3-PE

100000

500 750

250

500

750

1000

250

Forward scatter-A

Fo

rwar

d sc

atte

r-H

Forward scatter-A

Forw

ard

sca

tter

-H

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Tim

3-PE

-1 1

2.25%

17.6%

PD1-APC

Tim

3-PE

100000

500 750

250

500

750

1000

250

Forward scatter

Si

de s

catt

er

Forward scatter

Sid

e sc

atte

r

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Lag

3-FI

TC

-1 1

0.47%

15.4%

PD1-APC

Lag3

-FIT

C

Figure 4

A

Wit

h tu

mo

r

dis

soci

atio

n

B

Wit

ho

ut t

um

or

dis

soci

atio

n

10³-101

10¹ 10²0

10³

10²

10¹

Lag3-FITCTi

m3-

PE-1 1

Tim

3-PE

Lag3-FITC

10³-101

10¹ 10²0

10³

10²

10¹

Lag3-FITC

Tim

3-PE

-1 1

Tim

3-PE

Lag3-FITC

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Lag3

-FIT

C

-1 1

Lag3

-FIT

C

PD1-APC10³

-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Tim

3-PE

-1 1

Tim

3-PE

PD1-APC

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Tim

3-PE

-1 1

9.3%

Tim

3-PE

PD1-APC

10³-101

10¹ 10²0

10³

10²

10¹

CXCR3-PE

CD39

-APC

-1 1

CXCR3-PE

CD

39-A

PC

10³-101

10¹ 10²0

10³

10²

10¹

CXCR3-PE

CD10

3-FI

TC

-1 1

CXCR3-PE

CD

103-

FITC

10³-101

10¹ 10²0

10³

10²

10¹

CXCR3-PE

CD10

3-FI

TC

-1 1

CXCR3-PE

CD

103-

FITC

10³-101

10¹ 10²0

10³

10²

10¹

CXCR3-PE

CD39

-APC

-1 1

CXCR3-PE

CD

39-A

PC

10³-101

10¹ 10²0

10³

10²

10¹

PD1-APC

Lag3

-FIT

C

-1 1

Lag3

-FIT

C

PD1-APC

CD

4-PE

-Vio

615

Forw

ard

sca

tter

-H

CD

4-PE

-Vio

615

Table 1

* Flow rate: 2,000 events/s** Considering 9 samples (3 experimental groups × 3 replicas/group). Includes 45 s automated mixing and rinsing between samples on the MACSQuant Instrument*** Isolation using CD8 (TIL), CD4 (TIL), or CD4/CD8 (TIL) MicroBeads, respectively

Bulk Isolated