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.
It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. 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.
1. Isolation of Lymphocytes5
2. Determine Fold Expansion of T Cells by Cell Counts and Flow Cytometry Analysis5
3. Determine Tumor-specificity of the ex Vivo Expanded T Cells
4. Determine Anti-tumor Function of the ex Vivo Expanded T Cells5,6
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
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 upon adoptive T cell therapy9-11. Changing the order and combination of these cytokines could affect differentiation of the expanded T cells which in turn may improve or reduce their anti-tumor efficacy12. The proposed protocol will not require the identification of tumor antigens. Selective expansion of tumor-reactive T cells results in the production of high numbers of anti-tumor T cells that can be used for adoptive T cell therapy of cancer patients. We have previously shown that the ex vivo expanded T cells protected animals against breast cancer following adoptive T cell therapy.
The authors have nothing to disclose.
This work was supported by NIH R01 CA104757 Grant (M. H. Manjili). We gratefully acknowledge the support of VCU Massey Cancer Center and the Commonwealth Foundation for Cancer Research.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Bryostatin 1 | Sigma | B7431-10ug | ||
Ionomycin | Calbiochem | 407950 | ||
Mouse IL-7 | PeproTech | 217-17 | ||
Mouse IL-15 | PeproTech | 210-15 | ||
Human IL-2 | PeproTech | 200-02 | ||
RPMI1640 | Invitrogen | 11875 | ||
FBS | Gemini | 100-106 | ||
Penicillin/Streptomycin | Cellgro | 30-002-CI | ||
L- glutamine | Invitrogen | 25030081 | ||
β- mercaptoethanol | Sigma | M7522 | ||
anti-CD16/32 antibody | Biolegend | 101302 | ||
Annexin V-FITC Apoptosis Detection Kit | BD Pharmingen | 556547 | ||
FITC-CD4 | Biolegend | 100406 | ||
PE-CD8 | Biolegend | 100708 | ||
anti-c-Erb2/c–Neu | Calbiochem | OP16 | ||
PE- anti mouse IgG | Biolegend | 405307 | ||
formaldehyde | Polysciences | 04018 | ||
Hemocytometer | Hycor | 87144 | ||
Light microscope | VWR | V200073 | ||
Mouse IFN-γ ELISA set | BD Pharmingen | 555138 | ||
Cell culture flasks | Greiner | 658175 |