Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

Summary

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Abstract

It was reported that breast cancer patients have pre-existing immune responses against their tumors^1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses³. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively⁴. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Protocol

1. Isolation of Lymphocytes⁵

1. Isolate tumor-draining lymph nodes or spleens from tumor-bearing FVBN202 transgenic mice and prepare single cell suspension in ice-cold RPMI1640 supplemented with 10% FBS. B/I activation in 50-ml polypropylene conical tubes results in a greater T cell yield compared to polystyrene tubes. Ketamine and Xylazine are injected i.p. for anesthesia. Cervical dislocation is used as a method of euthanasia.
2. Culture the cells (10⁶ cells/ mL) in complete medium containing 15% FBS with bryostatin-1 (5 nM) and ionomycin (1 μM) along with 80 U/ mL of IL-2 (Peprotech) for 16 h.
3. Wash the cells three times with warm medium (37°C) and culture at 10⁶ cells/ mL in complete medium with IL-7 (10 ng/ mL) and IL-15 (10 ng/ mL) (Peprotech) for 24 h.
4. Pulse the cells with IL-2 (40 U/ mL) for 24 h.
5. Split the cells and culture them with IL-7 and IL-15 (10 ng/ mL) for 4 more days. Change medium and split the cells if needed every 2 days.

2. Determine Fold Expansion of T Cells by Cell Counts and Flow Cytometry Analysis⁵

1. Cell counts by light microscopy
   1. Prepare appropriate cell dilution (1:100) in trypan blue and add few μL onto hemocytometer
   2. Count 9 squares and determine total cell number by dividing cell counts to the number of chambers multiplied by the dilution factor. The results will present number x 10⁴ cells/ mL.
2. Determine proportion of CD8+ and CD4+ T cells in the expanded cells by flow cytometry
   1. Block non-specific binding of antibodies to Fc receptors by culturing the cells with anti-CD16/CD32 antibody (Biolegend) for 20 min on ice and then wash the cells two times with 2 mL of ice-cold PBS supplemented with 1% sodium azide.
   2. Stain the cells by culturing with FITC-CD4 and PE-CD8 antibodies for 20 min on ice and then wash the cells two times with 2 mL of ice-cold PBS supplemented with 1% FBS and 0.1% sodium azide.
   3. Fix the cells with 1% paraformaldehyde and run samples on a Beckman Coulter FC 500 and analyze using Summit version 4.3 software.

3. Determine Tumor-specificity of the ex Vivo Expanded T Cells

1. Culture the ex vivo expanded lymphocytes in complete medium at a 10:1 ratio with irradiated neu positive MMC tumor cells (15,000 rad) for 24 h. ⁵
2. Harvest supernatants and store at -80°C until used. ^5,6
3. Detect IFN-γ using a Mouse IFN-γ ELISA Set (BD Pharmingen) according to the manufacturer's protocol. ^5,6

4. Determine Anti-tumor Function of the ex Vivo Expanded T Cells^5,6

1. Incubate T cells with tumor cells in a 10:1 effector:target ratios for 48 hours in complete medium at 3 mL complete medium (RPMI-1640 supplemented with 100U/ mL of penicillin, 100μg/ mL streptomycin, 10% FBS, glutamine and β- mercaptoethanol) and 20U/ mL of IL-2 (Peprotech) in 6 well culture dishes 37°C /5% CO₂.
2. Perform three color antibody staining for neu (anti-c-Erb2/c-neu, clone-4, Calbiochem) followed by PE- anti mouse IgG, Annexin V-FITC and Propidium Iodide (PI) according to manufacturer's protocol (BD Pharmingen)
3. Gate on neu positive tumor cells and analyze viability (Annexin V-/PI-) of the tumor cells

5. Mouse Model of Breast Cancer

FVBN202 transgenic female mice (Charles River Laboratories) can be used for the source of tumor-reactive T cells. These mice overexpress an unactivated rat neu transgene under the regulation of MMTV promoter and as a result develop spontaneous mammary carcinoma between 4-10 months of age⁷. These mice develop premalignant mammary hyperplasia similar to ductal carcinoma in situ (DCIS) prior to the development of spontaneous carcinoma8. Spontaneous tumor-bearing mice are used as donors of T cells.

6. Representative Results:

Activation of T cells with B/I for 16 hours results in killing of naíve T cells that are not sensitized with the tumor in vivo. After the B/I selectivity of tumor-reactive T cells they expand up to 2.8-fold within a 6-day culture with the gamma chain cytokines (Figure 1). Both CD8+ and CD4+ T cells are equally expanded with the gamma chain cytokines (Figure 2). The ex vivo-expanded T cells show high responsiveness against the tumors that donor mice were sensitized to, as evaluated by the production of IFN-γ in the presence of neu positive mouse mammary carcinoma (MMC) tumor cells (Figure 3). The ex vivo expanded T cells can induce apoptosis in the neu positive MMC tumor cells such that viability of the tumor cells drops from 92% to 61% within 48 hours (Figure 4).

Figure 1. Fold expansion of lymphocytes at different time points following B/I activation (day 1) and ex vivo expansion with the gamma chain cytokines (days 3, 5, and 7)

Figure 2. Total percentage of CD4+ and CD8+ T cells before and after a 7-day expansion with the gamma chain cytokines.

Figure 3. Tumor-stimulated IFN-γ production by T cells isolated from tumor-bearing mice prior to and after a 7-day expansion with the gamma chain cytokines, using IFN-γ ELISA

Figure 4. Cytotoxic function of the ex vivo expanded T cells with the gamma chain cytokines against neu positive mouse mammary carcinoma (MMC) tumor cells

Discussion

Selective expansion of tumor-reactive T cells with effector anti- tumor function can be achieved by the proposed protocol using B/I activation and ex vivo expansion with the gamma chain cytokines IL-2, IL-7 and IL-15. While IL-2 is a T cell growth factor that can support the differentiation and expansion of antigen-specific T cells, IL-7 can inhibit apoptosis of T cells and support their viability during expansion. IL-15 can support memory T cells that are important for generating long-term anti-tumor responses ...

Materials

Name	Company	Catalog Number	Comments
Bryostatin 1	Sigma-Aldrich	B7431-10ug
Ionomycin	Calbiochem	407950
Mouse IL-7	PeproTech Inc	217-17
Mouse IL-15	PeproTech Inc	210-15
Human IL-2	PeproTech Inc	200-02
RPMI1640	Invitrogen	11875
FBS	Gemini Bio Products	100-106
Penicillin/Streptomycin	Cellgro	30-002-CI
L- glutamine	Invitrogen	25030081
β- mercapt–thanol	Sigma-Aldrich	M7522
anti-CD16/32 antibody	Biolegend	101302
Annexin V-FITC Apoptosis Detection Kit	BD Biosciences	556547
FITC-CD4	Biolegend	100406
PE-CD8	Biolegend	100708
anti-c-Erb2/c–Neu	Calbiochem	OP16
PE- anti mouse IgG	Biolegend	405307
formaldehyde	Polysciences, Inc.	04018
Hemocytometer	Hycor	87144
Light microscope	VWR international	V200073
Mouse IFN-γ ELISA set	BD Biosciences	555138
Cell culture flasks	Greiner Bio-One	658175