#5624. Characterization of Proliferation in Multiple Lymphocyte Subsets in the CT26 Murine Colon Carcinoma Model by Multi-color Flow CytometryDavid W Draper, Alden Wong, Dan Saims, Maryland Franklin, Matthew Thayer, and Scott WiseCovance Laboratories Inc., Ann Arbor, MI

Presented at the AACR Annual Meeting 2017, April 2-5, 2017, Washington, DC

Introduction and BackgroundBackground:The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4, anti-PD-1, and anti-PD-L1, has altered the paradigm of cancer treatment. Subsequently, the growing interest in the development of new single agent and combination therapies with immune-modulatory effects has generated a need for more powerful immunophenotyping techniques capable of in-depth cell characterization and proliferation assessment.

Methods:Using the CT26 syngeneic murine colorectal cancer model we developed a flow cytometry-based proliferation assay. This 9-color application incorporates CD45, CD3, CD19, CD49b, CD335, CD4, CD8, and FoxP3 antibodies, an amine reactive dye to exclude dead cells and CFSE (carboxyfluorescein succinimidyl ester), which enables generational tracking for up to seven rounds of cell division. Proliferation was measured using the high-throughput-capable 4-laser, 14-color AttuneTM NxT Flow Cytometer. Splenocytes from tumor-naïve and CT26 tumor-bearing mice (with or without anti-CTLA-4 therapeutic antibody treatment) were pre-loaded with CFSE and proliferation was induced with anti-CD3, anti-CD28 and IL-2. Immunophenotyping and proliferation assessment was performed after four days of cell culture.

Results:Differential proliferative capacity between naïve and tumor-bearing mice was detected simultaneously in five distinct lymphoid subsets. These included B cell, CD4+ helper T cell, CD8+ T cell, regulatory T cell (Treg) and natural killer (NK) cell populations. Identification of potentially responsive immune compartments, as well as characterizing proliferation, will facilitate the development of new combination therapies by expanding the depth of our ability to provide mechanistic descriptions of drug function.

Materials & Methods▶ Female 6-7 week Balb/C mice (BALB/cAnNHsd) were purchased from Envigo and were

implanted subcutaneously in the high axilla on day 0 with CT26.WT cells.

▶ Mice were treated via intraperitoneal injection with anti-mouse CTLA-4 antibody (Bio X Cell) at 10 mg/kg 3 times/week for a total of five doses.

▶ For ex vivo proliferation, spleens were mechanically dissociated into single cell suspensions and loaded with CellTrace™ CFSE (Thermo Fisher Scientific). Splenocytes were then seeded into multi-well plates coated with anti-mouse CD3 or isotype control antibody in growth media containing anti-mouse CD28 and hIL-2 (Biolegend). Splenocyte cultures were allowed to proliferate for 4 days. Antibodies were purchased from BD Biosciences.

▶ For immunophenotyping and proliferation assessment, cells were harvested, labeled with directly conjugated fluorescent antibodies, and samples were acquired on an AttuneTM NxT Flow Cytometer (Thermo Fisher Scientific). Data was analyzed using Flowjo software (Treestar).

Figure 3. MFI Can Be Used to Measure the Extent of Proliferation. The number of rounds of proliferation each subset undergoes inversely correlates with the CFSE signal MFI. B cells undergo fewer rounds of proliferation in culture compared to T cell subsets under T cell stimulating conditions.

Figure 4. Effect of In Vitro Stimulation on the Splenocyte Immune Subset Distribution in Culture. After treatment with either isotype control or anti-CD3 antibody, the percentage of each subset within total gated lymphocytes was measured. A) Survival of CD8+ T cells in unstimulating culture conditions appears to be when derived from tumor-bearing mice and to an even greater extent when these mice received anti-CTLA-4 treatment. B) B cells is the predominant subset in splenocyte cultures from naïve and tumor-bearing mice following the anti-CD3 treatment period. In contrast CD4+ helper T cells is the dominant population in cultures derived from tumor-bearing mice treated with anti-CTLA-4.

Figure 1. Gating Strategy to Identify Lymphoid Subsets.Representative profile from anti-CD3 stimulated splenocytes from tumor-bearing mice treated with anti-CTLA-4 antibody.

Figure 2. Ex Vivo Proliferative Response of Splenic Subsets to Stimulation Measured by CFSE Dilution. In vivo anti-CTLA-4 treatment enhances the ex vivo proliferative response to TCR stimulation in all subsets analysis. In vivo anti-CTLA-4 treatment enhances the proliferative response is several subsets in the absence of ex vivo stimulation. A) Histogram overlays that display proliferative profiles of splenic immune subsets derived from each treatment group. The values on each histogram represent percent proliferating splenocytes derived from animals following anti-CTLA-4 treatment (red histogram). Values for naive and untreated CT26.WT groups not shown. B) Proliferating cells (within the proliferating cells gate; panel A) measured as a percent of total subset (mean ± standard deviation).

B Cells CD8+ T Cells CD4+ Helper T Cells Tregs NK Cells

CFSE

Nor

mal

ized

to M

ode

Naive

CT26

CT26+ anti-CTLA-4

Nor

mal

ized

to M

ode

Nor

mal

ized

to M

ode

MediaOnly

IsotypeControl Treated

Anti-CD3 Treated

GROUP

% P

rolif

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ing

% P

rolif

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ing

% P

rolif

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MediaOnly

IsotypeControl Treated

Anti-CD3 Treated

A

B

B Cells CD8+ T Cells CD4+ Helper T Cells Tregs NK Cells

CFSE

Nor

mal

ized

to M

ode

Naive

CT26

CT26+ anti-CTLA-4

Nor

mal

ized

to M

ode

Nor

mal

ized

to M

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MediaOnly

IsotypeControl Treated

Anti-CD3 Treated

GROUP

% P

rolif

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% P

rolif

erat

ing

% P

rolif

erat

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MediaOnly

IsotypeControl Treated

Anti-CD3 Treated

B CellsCD8+ T Cells CD4+ Helper T Cells Tregs NK Cells

Isotype ControlTreated

Anti-CD3Treated

16.4

3.4

18.8

18.7

27.4

27.4

7.6

23.2

11.5

16.9

57.9

12.4

16.8

5.2 0.8

70.5

7.1

4.33.9

5.9

77.0

7.0

4.92.5 2.4 20.3

18.9

49.5

2.3 1.1

B Cells

CD8+ T Cells

CD4+ Helper T Cells

Tregs

NK Cells

Naive

CT26

CT26 + Anti-CTLA-4

Dosing GroupIsotype Control Anti-CD3A B

Results & Conclusions▶ In this study we developed an application that measures ex vivo proliferation of five

splenic lymphocyte subsets simultaneously in culture with or without stimulation.

▶ We show that T cell receptor stimulation triggers various rates of proliferation in T cell, NK cells and B cells.

▶ Furthermore, we show that proliferation rates increased in cultures derived from CT26 tumor-bearing mice dosed with the anti-CTLA-4 immune checkpoint inhibitor antibody.

▶ This application can be used as a platform with which to investigate new drug candidates and their effects on lymphocyte anti-tumor responses.